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JAK2 and SRC Family Tyrosine Kinase Signaling via the Angiotensin II Type 1 Receptor

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 C-SRC/YES/FYN tyrosine kinases...
 SRC kinase-independent ERK ½ activation...
 ERK ½ regulates angiotensin II-dependent...
 The N-terminal SH2 domain of the...
 Conclusions and implications
 References
 Biographical sketch
 

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JAK2 AND SRC FAMILY TYROSI NE KINASE SIGNALING VIA THE ANGIOTENSIN II TYPE 1 RECEPTOR By MICHAEL D. GODENY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Michael D. Godeny

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This dissertation is dedicated to my famil y, for their constant love, support and guidance

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iv ACKNOWLEDGMENTS I would like to first thank my mentor, Dr Peter Sayeski. His guidance, patience and support have enabled me to successfully navigate my way through graduate school. Peter’s passion for research coupled to his enthusiasm for mentoring always kept me excited about scientific research. Furtherm ore, Peter has gone above and beyond the role of a graduate studies mentor by always bei ng readily available to answer any questions about my research, my career path and life in ge neral. In short, Peter has been the perfect role model, and I consider myself fortunate to have worked with such an outstanding individual. I would also like to thank my committee members for their in sight and guidance: Dr. Hideko Kasahara, Dr. Colin Sumners, a nd Dr. Lei Xiao. These individuals have played an invaluable role in my success in graduate school. Additionally, I would like acknowledge a few individuals who have provided technical assistance and reagents. I w ould first like to thank Tim Vaught (MBI Microscopy Facility) and Michae l Poulos (Swanson Lab) for th eir technical assistance, as I would not have been able to obtain any microscopy images without their help. In addition, I would like to thanks Dr. Phillip Soriano (University of Washington), Dr. Kenneth Bernstein (Emory University), Dr. Brad Berk (University of Rochester), Dr. Phillip J. Stork (Oregon Health Science Cent er) and Dr. Jessica Schw artz (University of

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v Michigan) for donating various plasmids and ce ll lines which have been essential for the completion of this work. Finally, I would like to thank all of the me mbers of the Sayeski lab, both past and present, with whom I have been fortunate enough to work: Dr. Ma Xianyue, Dr. Tiffany Wallace, Dr. Eric Sandberg, Issam McDoom, Jacqueline Sayyah, Dannielle VonDerLinden, Melissa Johns, B obby Blair and Andrew Magis. Each one of them has made the laboratory environment a joy to be i n. I thank them for sh aring their insight, ideas and reagents. I will miss all of them! Finally, I would like to thank my family and friends for being there when times were rough, and for being supportive and keep ing me sane during those times. In addition, I am indebted to Rick Swenson and Dana Moser, organizers of the Gainesville Arthritis Foundation’s Marathon Training Team. These individuals provided me with a new life-long hobby and helped to keep my f itness and stress in check while I was a graduate student.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xi v 1 INTRODUCTION........................................................................................................1 Overview....................................................................................................................... 1 Angiotensin II...............................................................................................................2 History of the Renin Angiotensin System.............................................................2 Physiological Effects of Angiotensin II Associated With Binding of the AT1 Receptor.............................................................................................................2 Angiotensin II Signaling........................................................................................4 The JAK Family of Tyrosine Kinases..........................................................................6 Structure................................................................................................................6 Jak2/STAT Signaling............................................................................................7 The Src Family of Tyrosine Kinases............................................................................8 Structure................................................................................................................8 c-Src/Yes/Fyn Knockout Mice..............................................................................8 AT1 Receptor-induced Signaling.........................................................................10 ERK1 and ERK2 MAP Kinases.................................................................................11 The MAP Kinase Superfamily............................................................................11 ERK1/2 Signaling................................................................................................12 Ribosomal S6 kinase...................................................................................................12 The RSK Family of Proteins...............................................................................12 Structure and Function of RSK Proteins.............................................................13 The Angiotensin II Signaling Paradigm.....................................................................15 Summary and Rationale..............................................................................................16 2 c-SRC/YES/FYN TYROSINE KI NASES MEDIATE A PORTION OF ANGIOTENSIN II-INDUCED ERK1/2 ACTIVATION AND CELLULAR PROLIFERATION.....................................................................................................18 Introduction.................................................................................................................18 Materials and Methods...............................................................................................21 Creation of WT/AT1 and SYF/AT1 Stable Cell Lines.........................................21

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vii Cell Culture and Reagents...................................................................................21 Immunoprecipitation and Western Blotting........................................................22 Antibodies............................................................................................................23 Immunofluorescence...........................................................................................23 Measurement of Cellular ATP Levels.................................................................24 Measurement of Formazan Production...............................................................24 Cell Count............................................................................................................24 Densitometric Analysis.......................................................................................25 Statystical Analysis.............................................................................................25 Results........................................................................................................................ .25 Characterization of WT/AT1 and SYF/AT1 Cells...............................................25 Angiotensin II-induced ERK1/2 Activati on Is Reduced By About 50% in Src Kinase Deficient Cells.....................................................................................26 Angiotensin II-induced ERK1/2 Nuclea r Translocation Is Not Dependent Upon Src Kinases.............................................................................................29 Angiotensin II Induced Cell Proliferation Is Reduced in Src Kinase Deficient Cells.................................................................................................................30 Discussion...................................................................................................................34 3 SRC KINASE-INDEPENDENT ERK1/2 ACTIVATION AND CELL PROLIFERATION IS MEDIATED BY HETEROTRIMERIC G PROTEINS AND PKC -DEPENDENT SIGNALING..................................................................36 Introduction.................................................................................................................36 Materials and Methods...............................................................................................38 Cell Culture.........................................................................................................38 Pharmacological Inhibitors..................................................................................39 siRNA Treatment of WT/AT1 Cells....................................................................39 Immunoprecipitation, Western Blotti ng and Densitometric Analysis................40 Immunofluorescence and Quantif ication of Fluorescence..................................40 Cell Count............................................................................................................40 Statistical Analysis..............................................................................................40 Results........................................................................................................................ .41 Src Kinase Independent ERK1/2 Activation Does Not Require EGF Receptor, PDGF Receptor or PI3K Activity....................................................41 Src Kinase Independent ERK1/2 Activ ation Is Dependent on MEK1/2, But Not Raf1...........................................................................................................43 Heterotrimeric G Proteins Mediate A Portion of ERK1/2 Activation In A Src Kinase-independent Manner............................................................................45 Protein Kinase C Mediates ERK1/2 Activation In A Src Kinaseindependent Manner.........................................................................................48 PKC Mediates MEK1/2 Activation Independent of Src Kinases......................52 ERK1/2 Nuclear Transloca tion Is Dependent Upon PKC In Response to Angiotensin II..................................................................................................53 Cell Proliferation Is Attenua ted Through Inhibition of PKC Signaling In Response to Angiotensin II..............................................................................55 Discussion...................................................................................................................57

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viii 4 ERK1/2 REGULATES ANGIO TENSIN II-DEPENDENT CELL PROLIFERATION VIA THE CYTOPLAS MIC ACTIVATION OF RSK2 AND NUCLEAR ACTIVATION OF ELK1.......................................................................60 Introduction.................................................................................................................60 Materials and Methods...............................................................................................62 Antibodies and Pharmacological Inhibitors........................................................62 Cell lines and Cell Culture..................................................................................63 Cell Lysate Preparation, Immunopreci pitation and Western Blotting................63 Densitometric Analysis.......................................................................................63 Immunofluorescence...........................................................................................63 Quantification of nuclear and cytoplasmic fluorescence.............................64 c-fos transcriptional activity.........................................................................64 Cell Migration Assay...........................................................................................65 Cell Count............................................................................................................65 Statistical Analysis..............................................................................................66 Results........................................................................................................................ .66 RSK Phosphorylation and ERK1/2-R SK Co-association Are Dependent Upon Src Kinases in Response to Angiotensin II............................................66 RSK Nuclear Translocation Is Src Kina se Dependent, While ERK1/2 Nuclear Translocation Is PKC Dependent in Respons e to Angiotensin II..................69 SRF and TCF Binding Within the c-fos Promoter Are Mediated in A RSK And A ERK1/2-dependent Manner, Respectively...........................................71 c-fos Protein Expression Is Dependen t Upon Src Kinase Signaling And PKC Signaling..........................................................................................................74 c-fos Phosphorylation Is Dependent Upon Src Kinase-RSK Signaling..............75 Angiotensin II-induced Cell Prol iferation Requires RSK And PKC Activity...77 Angiotensin II-induced ERK1/2 Activati on Is Mediated By Both Src Kinases and PKC in Vascular Smooth Muscle Cells...................................................81 Angiotensin II-induced Cell Migration Is Attenuated in VSMCs Treated With SL0101.............................................................................................................82 Discussion...................................................................................................................83 5 THE N-TERMINAL SH2 DOMAIN OF THE TYROSINE PHOSPHATASE, SHP-2, IS ESSENTIAL FOR JAK2-D EPENDENT SIGNALING VIA THE ANGIOTENSIN II TYPE 1 RECEPTOR..................................................................89 Introduction.................................................................................................................89 Materials and Methods...............................................................................................91 Cell Culture.........................................................................................................91 Immunoprecipitation...........................................................................................91 Western Blotting..................................................................................................91 GST Pull Down Assays.......................................................................................92 Luciferase Assay.................................................................................................92 Molecular Model of Jak2.....................................................................................92 Statistical Analysis..............................................................................................93 Results........................................................................................................................ .93

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ix SHP-2 46-110 and SHP-2 WT Cells.......................................................................93 Jak2 Phosphorylation Is Not Influenced by the N-terminal SH2 Domain of SHP-2...............................................................................................................93 STAT1 and STAT3 Phosphorylation and STAT-mediated Gene Transcription Require the N-terminal SH2 Domain of SHP-2..............................................95 Jak2-AT1 Receptor Co-association Is Mediated by SHP-2.................................97 Transfecting Wild Type SHP-2 back into SHP-2 46-110 Cells Restores STAT1 and STAT3 Phosphorylation and STAT -mediated Gene Transcription........100 Jak2 Tyrosine 201 Mediates Jak2-SHP2 Interactions.......................................101 Jak2 Tyrosine 201 Mediates AT1 Receptor-Jak2 Co-association, STAT1 and STAT3 Activation and STAT-mediated Gene Transcription........................106 Discussion.................................................................................................................108 6 CONCLUSIONS AND IMPLICATIONS...............................................................113 Summary of Results..................................................................................................113 Src Kinases and Angiotensin II -induced Cell Proliferation.....................................114 The role of SHP-2 in angiot ensin II, Jak2/STAT signaling......................................125 Angiotensin II signaling and disease........................................................................126 LIST OF REFERENCES.................................................................................................128 BIOGRAPHICAL SKETCH...........................................................................................143

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x LIST OF TABLES Table page 1-1 Known RSK substrates.............................................................................................15 1-2 Pharmacological inhibiti on of Src kinase-independe nt ERK1/2 inhibition.............41

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xi LIST OF FIGURES Figure page 1-1 Summary of the reni n-angiotensin system.................................................................3 1-2 Diagram of the rat AT1a receptor................................................................................5 1-3 Jak2 structural domains..............................................................................................7 1-4 The Jak/STAT signaling paradigm............................................................................9 1-5 Structure/function of conserved domain s within Src family tyrosine kinases.........10 1-6 Structure and signaling of ribosmal S6 kinase.........................................................14 2-1 Characterization of WT/AT1 and SYF/AT1 cells.....................................................27 2-2 Quantification of ERK1/2 activation in response to Ang II in WT/AT1 and SYF/AT1 cells..........................................................................................................28 2-3 Nuclear translocation of active ERK2 is unaffected by the loss of cSrc/Yes/Fyn..............................................................................................................31 2-4 Ang II-induced cell proliferation is reduced in Ang II-stimulated SYF/AT1 cells.......................................................................................................................... .33 3-1 ERK1/2 activation in SYF/AT1 cells does not require transactivation of the PDGFR or the EGFR or activation of PI3K.............................................................42 3-2 ERK1/2 activation in SYF/AT1 cells requires MEK1/2 activation, but not Raf1 activation..................................................................................................................44 3-3 Ang II-induced ERK1/2 activation is pa rtially dependent upon heterotrimeric G proteins.....................................................................................................................47 3-4 Ang II-induced ERK1/2 activation is partially dependent upon PKC.....................49 3-5 PKC mediates Ang II-induced ERK1 /2 activation independent of cSrc/Yes/Fyn..............................................................................................................51 3-6 PKC -specific siRNA attenuated Ang II-i nduced ERK1/2 activation in WT/AT1 cells.......................................................................................................................... .52

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xii 3-7 MEK phosphorylation is dependent upon PKC .....................................................53 3-8 Nuclear translocation of ac tive ERK2 is controlled by PKC -dependent signaling...................................................................................................................56 3-9 Ang II-induced cell proliferation is completely attenuated by blocking cSrc/Yes/Fyn and PKC -dependent signaling...........................................................57 4-1 RSK2 phosphorylation and RSK2-ERK 1/2 co-association are decreased in SYF/AT1 cells..........................................................................................................67 4-2 RSK2 and ERK1/2 nuclear phosphorylat ion in response to Ang II in WT/AT1 and SYF/AT1 cells....................................................................................................70 4-3 Ang II-induced SRF and elk1 nuclear phosphorylation in WT/AT1 and SYF/AT1 cells.......................................................................................................................... .73 4-4 c-fos transcriptional activity in WT/AT1 and SYF/AT1 cells in response to Ang II............................................................................................................................. ..75 4-5 c-fos protein levels in response to Ang II in WT/AT1 and SYF/AT1 cells..............76 4-6 Ang II-induced c-fos phosphorylation in WT/AT1 and SYF/AT1 cells..................78 4-7 Ang II-induced cell proliferation in response to RSK and PKC inhibition...........80 4-8 Ang II-induced ERK1/2 activation is mediated by c-Src/Yes/Fyn and PKC dependent signaling in VSMC.................................................................................82 4-9 Angiotensin II-induced cell migra tion is attenuated th rough selective RSK inhibition..................................................................................................................84 4-10 Mechanistic diagram illustrating how Src kinase and PKC -dependent ERK1/2 activation pathways dually regulate Ang II-induced cell proliferation....................85 5-1 Jak2 tyrosine phosphorylati on in SHP-2 WT or SHP-2 46-110 fibroblast cells........94 5-2 STAT1/3 phosphorylation and STAT-indu ced luciferase activity in SHP-2 WT or SHP-2 46-110 transfected fibroblast cells..............................................................96 5-3 The recruitment of Jak2 to the AT1 receptor is dependent upon SHP-2..................99 5-4 AT1/Jak2 co-association, STAT1 phosphoryl ation and STAT-induced luciferase activity are restored in SHP-2 46-110 fibroblasts transfected with wild type SHP2.............................................................................................................................. 102 5-5 SHP-2/Jak2 co-association occurs ma inly through interaction of Jak2 amino acids 1-294 and the N terminal SH2 domain of SHP-2.........................................103

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xiii 5-6 Jak2 tyrosine 201 is critical for Jak2/SHP-2 interaction........................................105 5-7 Mutation of Jak2 tyrosine 201 reduces Ja k2-dependent signaling in response to angiotensin II..........................................................................................................107 5-8 Proposed mechanism for Jak2/SHP-2 interactions upon stimulation of the AT1 receptor...................................................................................................................112 6-1 Possible CRM-1-dependent mechanism influencing ERK1/2 localization. .......118 6-2 Possible mechanism whereby phosphoryl ation of tyrosine 314 affects ERK1/2 subcellular localization...........................................................................................119 6-3 Proposed mechanism whereby substr ate recognition influences ERK1/2 subcellular localization...........................................................................................120 6-4 Preliminary data supporting the hypothe sis that ERK1/2 subcellular distribution effects the mechanism of upstream activation.......................................................122

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xiv Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy JAK2 AND SRC FAMILY TYROSI NE KINASE SIGNALING VIA THE ANGIOTENSIN II TYPE 1 RECEPTOR By Michael D. Godeny December 2006 Chair: Peter P. Sayeski Major Department: Medical SciencesPhysiology and Pharmacology The AT1 receptor is a prototypical G prot ein-coupled receptor activated through high affinity binding of the hormone, angiot ensin II (Ang II). More recent work has demonstrated that the AT1 receptor can activate tyrosine kinases independent of heterotrimeric G proteins. This disser tation focuses on tyrosi ne kinase-mediated signaling events downstream of the angiotensin II type 1 (AT1) receptor. Specifically, the involvement of Src family tyrosine kinases in angiotensi n II-induced ERK1/2 activation and cell proliferation is explored. In addition, an giotensin II, Jak2 signaling is explored in detail with resp ect to Jak2’s interaction w ith the phosphatase, SHP-2. As such, this work provides valuable in sight into angiotensin II signaling. Src family tyrosine kinases mediate as much as 50% of angiotensin II-induced ERK1/2 activation and cell proliferati on. The remaining 50% is mediated by heterotrimeric G protein and PKC signaling. In addition, th ese two signaling cascades activate ERK1/2 and initiate cell proliferation independent of one anot her. Interestingly,

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xv the cellular consequence associated with ERK1 /2 activation by each of these two distinct pathways is different. When ERK1/2 is activated by heterotrimeric G protein and PKC dependent signaling, it translocates into the nucleus and initiates cellular proliferation through the activation of the tr anscription factor, elk1. When ERK1/2 is activated by Src kinase-dependent signaling, it remains in the cytoplasm and phophorylates ribosomal S6 kinase (RSK). Ultimately, RSK translocat es into the nucleus and modulates cell proliferation via the activation of the se rum response factor (SRF), another known transcription factor. Thus, the cell mediat es angiotensin II-indu ced cell proliferation through the activation of ERK1/2 via two independent signaling pathways. Jak2 is another tyrosine kinase phosphorylat ed by angiotensin II. Here, it is demonstrated that angiotensin II, Jak2-dep endent signaling requires SHP-2. SHP-2 acts as an adaptor molecule (at the site of the N terminal SH2 domain), serving to recruit Jak2 to the AT1 receptor via interactions w ith the Jak2 tyrosine 201 residue. Jak2 then recruits STAT1 and STAT3 proteins, which dimerize an d translocate into the nucleus. STAT nuclear translocation in itiates the transcription of STAT responsive genes. As such, SHP-2 positively regulates Jak2 si gnaling in response to Ang II. Collectively, this work helps to redefine the angiotensin II si gnaling paradigm, and may aid in the future treatment Ang II-associated diseases.

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1 CHAPTER 1 INTRODUCTION Overview Angiotensin II (Ang II) is responsible for a wide array of biol ogical effects which are primarily mediated by th e angiotensin II type 1 (AT1) receptor. The intracellular signaling pathways associated with AT1 receptor activation relay an Ang II-induced signal from the cell surface to the appropriate intracellular proteins, resulting in a desired cellular outcome. Since the AT1 receptor is a prototypical G protein-coupled receptor (GPCR), many of these signaling events ar e dependent upon heterotrimeric G proteinmediated signaling. However, in the 1990’s a paradigm shift emerged in the field of angiotensin II signaling when it was discovered that the AT1 receptor could also activate tyrosine kinases and induce signaling independe nt of heterotrimeric G proteins. Since then, ongoing work has focused upon the char acterization of these tyrosine kinasemediated signaling events. This dissertati on will emphasize two specific tyrosine kinase families activated by the AT1 receptor: the Janus kinases (JAK) and Src kinases. The following chapter will serve as an introducti on to angiotensin II si gnaling as well as important signaling molecules involved in AT1 receptor-induced processes, including Janus and Src kinases. The remaining chapters will examine two specific JAK and Src kinase-dependent signaling events First, the role of Src fa mily tyrosine kinases will be explored in Ang II-induced cell proliferation, and a mechanism will be proposed for this process. Second, the activity of an important Janus kinase family member, Jak2, will be explored with respect to its interactions with a well-known phosphatase, SHP-2. As such,

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2 the following chapters provide informati on about the signaling mechanisms of two important tyrosine kinase families activated by angiotensin II. Angiotensin II History of the Renin Angiotensin System Angiotensin II was initially identified as a product resulting from the direct cleavage of a plasma substrate (later na med angiotensinogen) by the kidney-produced enzyme renin (11, 94). The groups of Braun-Menendez and Page and Helmer independently made this discovery, a nd named the peptide “hypertensin” and “angiotonin” respectively. Both groups se ttled on the name “angiotensin” and demonstrated that this peptide was a remarkab le inducer of vasoconstriction. Leonard T. Skeggs and colleagues later purified the im mediate precursor of Ang II, the 10 amino acid peptide angiotensin I, from hog renin a nd horse plasma (127). In one study, Skeggs accidentally purified angiotensin I in the pres ence of 0.15M NaCl, and noticed that an 8 amino acid variant of this protein was form ed. This octapeptide turned out to be angiotensin II, which was subsequently show n by Skeggs and colleagues to be produced from the cleavage of angiotensin I by a ngiotensin-converting enzyme (ACE) (126). Finally, the lung was identified as the tissue-source for ACE (88, 107), and the biochemical pathway known today as the “ren in-angiotensin” syst em was established (Figure 1-1). To this day, angiotensin II re mains the primary effector molecule of the renin-angiotensin system. Physiological Effects of Angiotensin II Associated With Binding of the AT1 Receptor The angiotensin II type 1 (AT1) receptor is regarded as the receptor which mediates the majority of the physiologica l responses associated with Ang II. It is expressed in a number of tissues, including the vasculature, heart, kidney, lung, ad renal gland, intestine

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3 and brain. Due to the presence of the AT1 receptor in a number of distinct tissue beds, Ang II is implicated in an array of physiol ogical responses. In the kidney, for example, Ang II increases glomerular filtration rate by stimulating constriction of the efferent arteriole (130). AT1 receptor stimulation in the adre nal cortex initiates aldosterone synthesis, resulting in the reab sorption of sodium in the distal convoluted tubule of the kidney (92). Ang II also induces sodium reab sorption in the intestine (68), while in the brain Ang II triggers a thir st response by directly stim ulating regions of the Figure 1-1. Summary of the reni n-angiotensin system. Angi otensinogen is cleaved by renin to yield angiotensin I. Angi otensin I is then further cleaved by angiotensin-converting enzyme (ACE) to produce Ang II. Ang II binds to either the AT1 or AT2 receptors, or is degraded to angiotensin III and other inactive metabolites by various peptidases. hypothalamus (99). Finally, Ang II acts as a pote nt vasoconstrictor w ithin the vasculature (48). These Ang II-induced effects within di fferent tissues collectively help maintain mammalian blood pressure and fluid electrol yte amounts at homeostatic levels. In Angiotensinogen (453 AA) D-R-V-Y-I-H-P-F-H-L-V-I-H--------------NH2COOH Renin D-R-V-Y-I-H-P-F-H-LAngiotensin I (10 AA) ACE D-R-V-Y-I-H-P-F*Angiotensin II (8 AA)AT1R AT2R Aminopeptidase R-V-Y-I-H-P-FAngiotensin III (7 AA) Inactive Metabolites Various Peptidases

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4 addition to its role as a regulator of blood pressure and fluid osmolality, angiotensin II also acts as a potent growth factor. These Ang II-induced cellular growth and proliferative responses are associated with di sease states. For example, Ang II has been linked to cardiac hypertrophy. Cardiac hypertrop hy is classified as a thickening of the muscles in the wall of the left ventricle to compensate for increases in preload volume, and Ang II has been directly implicated in this condition (25). In addition, Ang II contributes to aberrant vasc ular smooth muscle cell proliferation during neointimal formation as well as followi ng balloon-injury from angioplas ty (55, 93). Finally, Ang II has been linked to angiogenesis during cancer, a process by which new blood vessels form and grow in order to feed rapidly-prolif erating tumor cells (24). As such, Ang II is a major contributor to maladaptive growth a nd proliferative respons es associated with cardiovascular diseases and cancer. Angiotensin II Signaling The diversity of systemic effects attributed to angiotensin II is in part due to the multitude of intracellular signaling pathways activated by the AT1 receptor (Figure 1-2). For example, the AT1 receptor couples to and activate s heterotrimeric G proteins, and many downstream signaling events are dependent upon these events. Upon AT1 receptor activation, the G subunit binds GTP and dissociates from the G subunits, allowing each subunit to interact with other signaling mo lecules. Specifically, angiotensin II can couple to G q, which stimulates Phospholipase C beta (PLC ) to convert Phosphatidylinositol 4,5-bisphosphate (PIP 2) to Inositol 1, 4,5 Triphosphate (IP3) and Diacylglycerol (DAG) (113, 151). DAG activates some isoforms of Protein Kinase C (PKC), a serine/threonine kinase that can phosphorylate a number of substrates, while IP3

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5 1 7 319YIPP 312Y219W IP3release 309-359 Src kinase Activation 1-309 Jak2 co-association 319-322 Heterotrimeric G protein activation 312-314 Heterotrimeric G protein activation 219-225 Function Amino Acid Residue IP3release 309-359 Src kinase Activation 1-309 Jak2 co-association 319-322 Heterotrimeric G protein activation 312-314 Heterotrimeric G protein activation 219-225 Function Amino Acid Residue Bilipidmembrane Figure 1-2. Diagram of the rat AT1a receptor. The seven transmembrane spanning domains are drawn from left to right as indicated. The posit ions and functions of amino acid residues important for angiotensi n II signaling are also indicated. binds IP3 receptors on the endoplasmic reticulum and causes a calcium efflux into the cytoplasm. In addition, the AT1 receptor can couple to G and, in combination with G 12, activate phospholipase D in vascular smoo th muscle cells (147). A final example of AT1 receptor, heterotrimeric G protein-dependent signaling occurs when G s stimulates adenylate cyclase, which conve rts ATP to cyclic adenosine monophosphate (cAMP) (135). cAMP then stimulates P KA, an AGC kinase capable of phosphorylating a variety of cellular s ubstrates. Thus, the AT1 receptor can activate numerous secondary messengers via heterotrimeric G protein-depende nt signaling. However, there appears to

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6 be disparity in heterotrimeric G protein-medi ated signaling between cell types, which can be explained by cell-specific di fferences in the expression of and subtypes (118). In addition to being a prototypical GPCR, the AT1 receptor can also activate a variety of non-receptor associat ed tyrosine kinases. These include c-Src, Yes, Fyn, Pyk2, Jak2, and FAK (118). The mechanisms of tyrosine kinase activation by the AT1 receptor are poorly defined in many cases; however, some of the specific amino acid residues on the receptor necessary for tyrosine kinase activ ation have been identified. Interestingly, many of these residues are different from re sidues linked to heterotrimeric G protein activation. For example, Jak2 tyrosine kinase has been shown to co-associate with the AT1 receptor through a specific 319YIPP motif on the carboxyl te rminal tail (3). In contrast, heterotrimeric G protein activation has been linked to W219-A225 and Y312L314 motifs on the third intr acellular loop and C terminal tail (113, 151). Thus, a number of tyrosine kinases ar e activated downstream of the AT1 receptor, and often signal independently of hete rotrimeric G proteins. The JAK Family of Tyrosine Kinases Structure The Janus kinase (JAK) family of tyrosine ki nases is activated by a number of receptors, including the AT1 receptor (79). Each member of the Janus family shares the distinct structural feature of having a kinase domain directly adjace nt to a pseudokinase domain, and therefore cleverly receive d their namesake after “Ja nus,” the Roman god of two opposing faces. Members of the JAK family include Jak1, Jak2, Jak3, and Tyk2. Each of these proteins is approximately 130 kDa in mass, and are structurally similar since they contain seven conserved JAK homology domains. The structure of Jak2 is illustrated as an example of JAK structure (Figure 1-3).

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7 COOH NH2 JH1 JH2 JH3 JH4 JH5 JH6 JH7 Y201 Kinase Domain Pseudokinase Domain SH2-like FERM Domain Amino Acid JH738-122 JH6144-284 JH5288-309 JH4322-440 JH3451-538 JH2543-827 JH1836-1123 Y1007 Y1008 V617F Figure 1-3. Jak2 structural domains. Shown ar e the positions of the seven-conserved Jak homology (JH) domains as well as th e amino acid sequence for each domain within Jak2. Amino acid residues and mutations known to affect Jak2 activity are also indicated. Reproduced with permission from Current Medicinal Chemistry, in press, Copyright 2006 American Chemical Society. Jak2/STAT Signaling Of all the JAK family members, Jak2 has been perhaps implicated the most in angiotensin II signaling. Ev en though Jak2 lacks canonical SH2 and SH3 domains, it is still able to associate with the AT1 receptor and induce gene transcription. Jak2dependent gene transcription occurs th rough the phosphorylation of STAT (signal transducers and activators of transcription) proteins ( 8, 9, 79). When phosphorylated, STAT proteins form hetero/homodimers and migrate into the nucle us, where they bind STAT recognition sequences within gene pr omoters. These events trigger STATmediated transcription in a variety of early response ge nes. The Jak2/STAT signaling paradigm was originally identified in the c ontext of cytokine recep tors, but more recent

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8 work has established a mechanism by which GPCRs like AT1 signal through Jak2 (112). The specific events associated with Jak2/STAT signaling are described in detail in Figure 1-4. As such, Jak2 serves as a conduit betw een angiotensin II bind ing at the surface of the cell and gene transcription within the nucleus. The Src Family of Tyrosine Kinases Structure The Src family of tyrosine kinases was the fi rst tyrosine kinase family identified. This family of tyrosine kinases is comprised of fourteen different family members, including c-Src, Yes, Fyn, Yrk, Fgr, Lyn, Hck, Lck and Bl k. The fourteen different Src kinases are derived from nine separate genes, with a lternative splicing acc ounting for a portion of these gene products. All Src kinase family gene products are similar in size (55 62 kDa), and share common structural features including an SH2 do main, an SH3 domain and a tyrosine kinase domain (Figure 1-5). Three of the Src kinases—c-Src, Yes, Fyn— are ubiquitously expressed, while the expression of other family members is restricted to hematapoietic cells. Due to a common structur e and similar expressi on patterns, there is functional redundancy amongst certain family me mbers, namely c-Src, Yes and Fyn, as demonstrated through the generatio n of specific knockout mice (62, 131). c-Src/Yes/Fyn Knockout Mice Mutations in either the c-src or fyn genes were shown previously to lead to restricted nonoverlapping phenotypes only in a s ubset of cells in which these kinases are expressed, while a mutation in the yes gene does not lead to an overt phenotype (131). Except for brain, the level or distribution of re lated Src family kinases is not altered in major tissues, demonstrating that there ma y be functional redundancy among these Src

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9 P P P Jak2 Jak2Jak2 PStat StatLigandCytokine Receptor P P A Jak2 P Jak2 StatP LigandG Protein Coupled Receptor PStat P B Stat P Stat P Stat P Stat PSRE NucleusTranscription C D Figure 1-4. The Jak/STAT signaling paradigm. Ligand binding to th e receptor initiates receptor activation. For cytokine recept ors (A), receptor dimerization triggers Jak2 autophosphorylation, re sulting in the Jak2-depe ndent phosphorylation of STATs. In the case of the AT1 receptor (B), Jak2 becomes activated in the cytoplasm and is recruited to the rece ptor, where it phosphorylates the STATs. STAT molecules form homo/hetero dimers upon phosphorylation (C), and translocate into the nucleus. There, STATs bind STAT-responsive elements within gene promoters (D) and initia te transcription. Reproduced with permission from Current Medicinal Chemistry, in press, Copyright 2006 American Chemical Society. family members. Generation of c-src, yes, or fyn double mutants sought to provide more evidence for this redundancy of function. The src/fyn or src/yes double mutants die perinatally, while a substant ial proportion of fyn/yes doubl e mutants are viable but undergo degenerative renal cha nges leading to diffuse segm ental glomerulosclerosis (131). Finally, c-Src/Yes/Fyn triple knockout mice die during development (62). Taken

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10 NH2 Unique N Terminus M SH3 SH2 Activation Loop C Lobe N Lobe Y416 Y527 Catalytic Domain When phosphorylated, binds SH2 domain C terminal tail Phosphorylation regulated protein activity Activation loop Enzymatic activity; divided into two lobes Catalytic domain Binds phosphorylatedtyrosine residues SH2 domain Binds prolinerich sequences SH3 domain Anchors protein to membrane N terminal myristoylationsequence (M) Function Region When phosphorylated, binds SH2 domain C terminal tail Phosphorylation regulated protein activity Activation loop Enzymatic activity; divided into two lobes Catalytic domain Binds phosphorylatedtyrosine residues SH2 domain Binds prolinerich sequences SH3 domain Anchors protein to membrane N terminal myristoylationsequence (M) Function Region Figure 1-5. Structure/function of conserved domains within Src family tyrosine kinases. Shown above are the relative position s of conserved domains and regions characteristic of the Src family memb ers. Conserved phosphorylation sites are also indicated. The putative function of each domain/region is listed in the corresponding table. together, these data are consistent with the hypothesis that c-Src, Yes, and Fyn tyrosine kinases are able to compensate for the lo ss of one or more related Src kinases. AT1 Receptor-induced Signaling Like other tyrosine kinases, Src kinases ar e activated by a wide va riety of receptors, including the AT1 receptor. Work in vascular smooth muscle cells and cardiac myocytes has shown that Src kinases are phosphorylated in response to Ang II treatment (54, 109).

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11 Ang II-induced activation of Src kinases preced es the activation of important downstream signaling events, including the mitogen-ac tivated protein (MAP) kinase (53, 109, 115, 140) and PLC 1/IP3/Ca2+ (80) signaling pathways Work by the Sadoshima laboratory has shown that amino acid residues 1-309 ar e critical for Src kinase activation and downstream signaling, but not for other Src-independent events such as IP3 release (122). Thus, Src kinases must bind the C-terminal tail of the AT1 receptor in order to become activated. However, the mol ecular events describing how AT1 receptor activation leads to the activation of Src kinases are no t well understood. ERK1 and ERK2 MAP Kinases The MAP Kinase Superfamily MAP kinases are evolutionary conser ved enzymes that phosphorylate their substrates on serine/threonine residues. MAP kinase family members are grouped into three sub-families based on their activation sequences: the c-Jun NH2-terminal kinases (JNKs), the p38 MAP kinases, and the extracellu lar signal-regulated kinases (ERKs). All of these MAP kinases mediate an intracellular effect in response to extracellular stimuli, although different types of stimuli preferentia lly activate certain MAP kinases (98). For example, JNKs and p38 MAP kinases are ofte n activated in response to extracellular stress, including UV irradiation, heat shoc k, osmotic stress and inflammatory kinase stimulation. ERKs are often activated by growth factors and hormones, and mediate different cellular responses, including ce ll growth, differentiation, proliferation and growth arrest. In addition, the cellular outcome associated with MAP kinase activation will also depend upon the duration and magnitude of activation. For example, transient or sustained patterns of ERK1 /2 activation have been shown to differentially affect developmental and adult mammalian cell pr ocesses (1, 13, 21, 64, 76, 138).

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12 ERK1/2 Signaling A general three-tiered signa ling cascade describes th e activation of all MAP kinases. This starts with the activation of a MAP kinase kinase kinase by a receptor, which phosphorylates a MAP kinase kinase, wh ich in turn phosphorylates a MAP kinase in order to achieve the desired cellular eff ect. In the case of ERK1/2, the MAP kinase kinase kinase activated is Ra s, which phosphorylates Raf (MAP kinase kinase). Raf then phosphorylates MEK1/2, which is the immedi ate upstream activator of ERK1/2. MEKinduced ERK1/2 phosphorylation occurs via du al threonine and tyrosine phosphorylation at a conserved TEY motif within the ac tivation loop. Activated ERK1/2 then phosphorylates substrates at proline-directed serine and threonine residues. The substrates acted upon by ERK1/2 are often dependent upon the scaffolding proteins bringing the MAP kinase signa ling complex together, the lig and-receptor interaction responsible for ERK1/2 activa tion, the cell type and the pres ence of substrate within the cell (19, 20). Finally, ERK1/2 signaling is terminated via dephosphorylation by phosphatases. For example, MAP kinase phos phatase 1 dephosphorylates ERK1/2 in a Jak2-dependent manner, effectively shutting off ERK1/2 signaling (111). As such, ERK1/2 signaling occurs in a coordinate ma nner and is controlled by many proteins. The regulation of ERK1/2 signaling is required in order to cont rol such processes as cell growth, proliferation, differentiation, a nd growth arrest (19, 105). Ribosomal S6 kinase The RSK Family of Proteins Ribosomal S6 kinase (RSK) is a member of the AGC family of serine/threonine kinases, which also includes protein kinases A, G and C. RSK is the 90 kDa protein within this family, and was originally discovered in Xenopus laevis oocytes by Erikson

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13 and Maller in 1985 (35). To date, three isofor ms of RSK have been identified (RSK1-3), and each of these proteins are products of separate genes (37). RSK1-3 are approximately 90% identical, and exhibit functi onal redundancy in ti ssues where each is expressed, including the brain. Furthermore, RSK homologues are present in a variety of organisms, including human, mouse, rat, chicken, Drosophila and C. elegans As such, RSK proteins appear to be physiologically im portant, which is directly demonstrated by the fact that RSK defects have been linke d to human Coffin–Lowry syndrome, a disease characterized by mental retard ation, facial and digital dys morphologies and progressive skeletal malformations (143). RSK was originally identified for its ab ility to phosphorylat e the 40S ribosomal subunit protein, S6 (36). The phosphorylation of S6 promotes the translation of selected mRNAs important for cell growth. In 1990, however, a 70 kDa S6 kinase (p70S6K) of 60% sequence homology to RSK was identified, and this protein was found to be the primary protein responsible for S6 phosphorylat ion (6, 7, 17, 63). It is now believed that RSK phosphorylates S6 only unde r special circumstances, leaving the main function of RSK unknown (37, 38). What is agreed upon is that RSK is a substrate for ERK1/2, although the function of ERK1/2-induced RSK activation is still unknown in most cases, including during AT1 receptor activation. Structure and Function of RSK Proteins The structure of RSK is undoubtedly tied to its function. RSK proteins are unique since they contain both an N terminal a nd a C terminal kinase domain, connected by a linker region (Figure 1-6). The N terminal ki nase domain is responsible for the activation of other kinases, and recognizes the follo wing consensus motifs: Arg/Lys-X-Arg-X-X-

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14 Ser/Thr or Arg-Arg-X-Ser/Thr (66). The C terminal kinase domain is most closely associated with the calcium/ calmodulin-dependent group of kinases, and phosphorylation of this domain precedes the activation of the N terminal kinase domain. In addition, phosphorylation of amino acid residues within the linker region has also been shown to play a role in the activation of RSK proteins (37). As such, RSK activation is regulated by ERK1/2 and phosphorylates a variety of s ubstrates containing specific consensus motifs, including transcription factors, cy toplasmic proteins, steroid receptors and ribosomal proteins (Table 1-1). NH2COOH NTK CTK P P P P P ERK1/2 EB A B C C D Substrate Substrate P Region Amino Acid N terminal kinase (NTK)68-323 Linker323-422 C terminal kinase (CTK)422-675 ERK binding (EB)727-728, 730-731 Figure 1-6. Structure and signaling of ribosmal S6 kinase. Shown above are the positions of the N terminal kinase (NTK) domai n, the C terminal kinase (CTK) domain, and the ERK-binding (EB) domain. U pon ligand stimulation, ERK1/2 bind to the EB domain (A), which initiates p hosphorylation of residues within the CTK and linker region (B). In addi tion, the CTK phosphorylates the linker region (C). These events are necessary for the activation of the NTK (C), which phosphorylates RSK substrates (D) at specific consensus sequences. Specific amino acid sequences for RSK2 domains and the linker region are indicated.

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15 Table 1-1. Known RSK substrates. RSK Substrate Category Reference Glycogen synthase kinase 3 Cytoplasmic protein (34, 139) Sos Cytoplasmic protein (29) L1 cell adhesion molecule Cell Adhesion (155) Polyribosomes Translation (5) I /NF Transcription factor (39, 119) Estrogen receptor Steroid receptor (59) CREB Transcription factor (23, 40, 156) c-fos Transcription factor (14, 15) CBP Transcription co-activator (87) The Angiotensin II Signaling Paradigm A number of proteins, incl uding those just listed, are activated in response to angiotensin II. It was origin ally thought that the ability of angiotensin II to produce either a pressor response or a growth and proliferativ e response was dependent upon whether the signaling downstream of the AT1 receptor was mediated by tyrosine kinases or heterotrimeric G proteins. For example, increases in A ng II-induced vasoconstriction have been shown to be dependent upon Ang II-induced calcium release, which occurs in part through heterotrim eric G protein, PLC signaling (113, 151). On the other hand, many growth and proliferative responses ha ve been linked to Ang II-induced tyrosine kinase activation, primarily during cardiovasc ular and cancer disease states (24, 47, 65, 82, 100, 134, 140, 141). As such, heterotrimeric G protein and tyrosine kinase-mediated signaling have often been viewed as inde pendent signaling events, determining the cellular outcome associated with AT1 receptor activation in a given tissue. More recent work, however, has shown that heterotrimeric G protein and tyrosine kinase signaling events are often interwoven with respect to the AT1 receptor. For example, work from our group has shown that Jak2 tyrosine kinase regulates intracellular

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16 calcium release (150), a process which is also highly dependent upon heterotrimeric G proteins (51). Furthermore, Ang II-induced IP3 production has been shown to be dependent on both heterotrim eric G proteins (through PLC 1) as well as Src tyrosine kinases (via PLC 1) (80, 113, 151). Finally, this disse rtation will focus extensively on the mechanisms of Ang II-induced cell prolif eration, which (as described later) is both heterotrimeric G protein-dependent and tyrosine kinase-dependent. T hus, the portrayal of AT1 receptor signaling as occurring through two types of linear cascades with no interaction seems oversimplified, since so me signaling events are facilitated by interaction between both heterotr imeric G protein and tyrosine kinase signaling. Clearly, more work needs to be done in order to unde rstand the complexity behind angiotensin II signaling. Summary and Rationale Angiotensin II signaling occurs in part through heterotrimeric G protein as well as tyrosine kinase-mediated signaling events. Of the kinase-induced signaling pathways, many are reliant upon members of the Janus and Src families of tyrosine kinases. Since Ang II initiates a multitude of tissue-specific e ffects, it is important to understand how these tyrosine kinases regulate the signaling associated with su ch processes. In addition, it is also important to unde rstand how JAK and Src kinase activity is regulated since these proteins essentially serve as the “on” switch for many AT1 receptor-driven signaling processes. Angiotensin II has already been linked to both cardiovascular disease and cancer, and the AT1 receptor is currently a target for the tr eatment of these disease states (24, 31, 49, 60, 137). Not surprisingly, Jak2 and Src kina ses have also been implicated in many of these same disease states, and are al so promising candidates for therapeutic

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17 intervention (47, 65, 82, 100, 134, 140, 141). The goal of this study is to provide a better understanding of Src family tyrosine kinase and Jak2 tyrosine ki nase function with respect to the AT1 receptor. Specifically, the role of c-Src/Yes/Fyn in angiotensin IIinduced cell prolifera tion will be examined. In add ition, the role of SHP-2 in Jak2 signaling will also be explored. Since Src and Janus kinases play such a pivotal role in angiotensin II signaling, it is the hope that the knowledge ge nerated from this work will aid in the treatment of Ang II-associated diseases.

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18 CHAPTER 2 C-SRC/YES/FYN TYROSINE KINASES MEDIATE A PORTION OF ANGIOTENSIN II-INDUCED ERK1/2 ACTIVATION AND CELLULAR PROLIFERATION Introduction Src family tyrosine kinases mediate an ar ray of signaling processes in response to high affinity binding of angiotensin II to the AT1 receptor (118). Included in this list is the activation of a pro-mito tic MAP kinase signaling ca scade, resulting in the phosphorylation of intracellular ERK1/2. Berk et al have demonstrated that Ang IIinduced ERK1/2 activation is critically me diated by c-Src in vascular smooth muscle cells (53). The authors claim that Ang II-induced ERK1/2 activation was blocked by each of the following: pharmacological inhibiti on using broad tyrosine kinase inhibitors (CP-188,556 or Genistein), c-Src knockout VSMCs or retrovira l transduction of dominant-negative c-Src into rat VSMCs. Thus they conclude that ERK1/2 activation is completely dependent upon c-Src. While it is clear from these studies that c-Src is indeed im plicated in Ang IIinduced ERK1/2 activation, an essential role for c-Src in these signaling events may be over-interpreted. This is demonstrated by the fact that complete i nhibition of ERK1/2 activation was only achieved when cells were pretreated with 100 M of pharmacological inhibitor, while only partial inhibition of ERK1/2 activation occurred in c-Src -/mouse VSMCs or in rat VSMCs transduced with dom inant-negative c-Src. These findings also raise speculation as to whether the comple te inhibition of Ang II-induced ERK1/2 activation was achieved through the simulta neous, non-specific inhibition of other

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19 signaling proteins via the pharmaco logical inhibitors utilized in these experiments. In addition, it is not clear if the remaining ERK1 /2 activation present in the c-Src dominantnegative transduced VSMCs or c-Src -/VSMCs was due to functional redundancy by other Src kinases expressed with in these cells, incomplete i nhibition of c-Src function or activation of ERK1/2 by c-Src-independent si gnaling. Therefore, the dependency of Ang II-induced ERK1/2 activation on Src family tyrosine kinases is still in question. Other work by Schiffrin and colleagues has de monstrated a critical role for c-Src in growth signaling by angiotensin II in VSMC s from arteries of hypertensive patients (140). This growth response occurred thr ough the c-Src-dependent activation of ERK1/2 in response to Ang II treatment, and thus im plicated c-Src in grow th and proliferative effects associated with Ang II. Work done by our group identified a mechanism whereby c-Src activates ERK1/2 via coupling to the Shc/Grb2/Sos signaling cascade, leading to the activation of RAS in res ponse to Ang II (115). However, it should be noted that in each of these reports, ERK1/2 activation was not completely achieved though c-Src inhibition, again raising the question that Ang II-induced ERK1/2 activation is also occurring through other Src kinases or through Src kina se-independent signaling. Additional work has shown that other Sr c family tyrosine kinases are also implicated in Ang II-induced ERK1/2 activation. Sadoshima and Izumo demonstrated that in cardiac myocytes, Ang II activates Fyn and stimulates the association of Shc with Fyn and the subsequent formation of a Sh c-Grb-Sos complex (109). These authors further demonstrated that Fyn activates Ras through the formation of this complex, thus leading to ERK1/2 activation. As such, F yn can also activate ERK1/2 in response to angiotensin II. In addition, the Src kinase Ye s cannot be ruled out as a mediator of Ang

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20 II-induced ERK1/2 activation due to a simila r structure, expressi on pattern, and already proven ability to compensate for a loss of re lated Src kinase functi on (131). Thus, it is apparent that c-Src is not the only Src family tyrosine kinase mediating ERK1/2 activation in response to Ang II, as suggested from previous studies. Clearly, more work needs to be done in or der to determine the contribution that all Src family tyrosine kinases have on Ang II-induced ERK1/2 activation since previous reports have generated conflicti ng results. This is likely due to the fact that ERK1/2 activation was not examined in a completely Src kinase-deficient background. Here, cSrc/Yes/Fyn-deficient mouse em bryonic fibroblast (MEF) cells stably transfected with the AT1 receptor were utilized. These cells will be described in detail later in this chapter, and provide a powerful system fo r identifying the dependency of Ang II-induced ERK1/2 activation on Src family tyrosine kinases because they completely lack functional c-Src/Yes/Fyn (62). Therefore, th ese cells lack the ubiquitously expressed Src kinases present in mammalian cells, and also lack the remainder of Src family kinases since the expression of these prot eins is restricted to cells of hematopoietic lineage. As such, the possibility of compensation for the loss of one Src kinase by another is eliminated by the use of the cSrc/Yes/Fyn-deficient cells. From these studies, it was found that Src kinases mediate approximately 50% of angiotensin II-dependent ERK1/2 activation. It appears that other signaling processes mediate the remainder of ERK1/2 activation inde pendent of Src kinases. Interestingly, a loss of c-Src/Yes/Fyn did not affect the ability of ERK1/2 to transl ocate into the nucleus, but did cause a reduction in A ng II-induced cell proliferation. As such, c-Src/Yes/Fynactivated ERK1/2 mediates cell proliferation independent of nuclear translocation. These

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21 findings therefore provide valuable insight into signaling via the AT1 receptor and the intracellular events associated with A ng II-induced cell proliferation. Materials and Methods Creation of WT/AT1 and SYF/AT1 Stable Cell Lines Immortalized SYF and WT MEF cells were a gift from Dr. Philippe Soriano, and were previously isolated fr om c-Src/Yes/Fyn triple knockout and WT mice respectively at E9.5 (62). Both cell lines lack endogenous AT1 receptor expression, and were therefore stably transfected by our group with 20 g of an HA-tagged AT1 receptor wild type cDNA plasmid as previous ly described (111). The AT1 receptor used for transfection has an HA-tag pres ent after the methionine initi ation sequence. Two days after transfection, the cells were switched to medium supplemented with 500 g/ml Zeocin (Invitrogen) to select for stable tr ansfectants. Surviving colonies were ring cloned, and AT1 receptor binding assays were performed using [125I-Sar1,Ile8] angiotensin II (PerkinElmer Li fe Sciences) as described pr eviously (3). Nonspecific binding was defined as binding in the presen ce of 1.0 M unlabelled angiotensin II. Scatchard analysis was used to identify respective WT/AT1 and SYF/AT1 clones in which the binding parameters were similar. Cell Culture and Reagents WT/AT1 and SYF/AT1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum (Hyclone), 1 mM sodium pyruvate, 10 units /mL penicillin, 10 g/mL streptomyocin, 2 mM L-glutamine, 10 mM HEPES and 100 g/mL Zeocin. WT/AT1 and SYF/AT1 cells were growth arrested in serum-free DMEM for 48 hours prior to experiments. PP2 was obtained from Calbiochem and used at concen trations found to have maximum inhibitory

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22 effect (122). Cells were pretreated with in hibitor for the indicated time and stimulated with 100 nM Ang II as described. All cel l culture reagents were obtained from Invitrogen. All other reagents were obtained from SIGMA or Fisher. Immunoprecipitation and Western Blotting Immunoprecipitation and Wester n blot analysis were performed in order to assess protein expression and phosphorylation in th e indicated cells. To prepare whole cell protein lysates, cells were washed in tw o volumes of ice-cold PBS containing 1 mM Na3VO4 and lysed in 0.8 ml ice-cold RIPA buf fer (20 mM Tris [pH 7.5], 10% glycerol, 1% Triton X-100, 1% deocycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 4 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4 and 10 g/mL aprotinin). The samples were then sonicated at a medium power setting and incubated on ice for 30 min. Samples were centrifuged at 13,200 rpm for 5 min at 4oC. Supernatants were normalized for protei n content using the Bio-Rad Dc assay. Normalized lysates were then either dire ctly resuspended in SDS sample buffer and separated by SDS-PAGE for Western blot analysis or immunoprecipitated. Immunoprecipitations were pe rformed for 2 hrs at 4oC using 2 g of the indicated antibody and 20 L of Protein A/G Plus ag arose beads (Santa Cruz Biotechnology). Following immunoprecipitation, samples were centrifuged for 2 min at 7,000 x g. Protein bound A/G beads were then were then washed in wash buffer (25 mM Tris [pH 7.5], 150 mM NaCl and 0.1% Trit on X-100). Samples were washed a total of three times, and resuspended in SDS sample buffer. Protein-A/G bead complexes were boiled for 10 min in order to separate bound protei ns. Samples were se parated by SDS-PAGE, and transferred onto nitrocellulose membranes.

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23 Whole cell lysates or immunoprecipitates we re Western blotted with the indicated antibody for each experiment. Antibodies were used at a final concentration of 1:1000 in 5% milk/TBST plus sodium azide. Membrane s were subsequently stripped for 18 min, and then reprobed with the indicated antibody to confirm equal prot ein loading of all samples. Proteins were detected usi ng enhanced chemiluminescence following the manufacturer’s instruct ions (Amersham). Antibodies The cocktail of anti-ERK1/2(P) antibodies were from Promega and Santa Cruz Biotechnology. Note that these antibodies employed in the cocktail recognize the same phospho-tyrosine residues, and were used in order to increase si gnal:noise ratio. The anti-ERK1/2, the anti-MEK1/2, the anti-MEK1/2(P) and the anti-PKC antibodies were from Santa Cruz Biotechnology. The anti -phosphotyrosine antibody (PY20) was from BD Transduction Laboratories. The immunoprecipitating anti-PLC antibody was from Santa Cruz Biotechnology. The immunoprecipitating anti-GFP antibody was from Cell Signaling Technology. Immunofluorescence GFP-ERK2 plasmid was kindly provided by Philip J.S. Stork (50). WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (Lab-Tek) and grown to 50% confluency. The cells were washed one time w ith PBS (pH 7.4) to remove dead cells and debris. Cells were transfected for 5 hr s with 10 g of GFP-ERK2 plasmid using Lipofectin (Invitrogen) and following the manuf acturer’s instructions. The medium was replaced with serum-containing medium and incubated at 37oC for two days. Cells were washed twice in PBS and starved for 48 hr in serum-free medium. Following starvation, all cells were ligand-treated with 100 nM A ng II for 0, 5 or 10 min. Cells were rinsed

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24 once in PBS and fixed in 4% paraformaldehyde for 10 min. Slide chambers were removed and the slides were dipped twice in to chilled PBS. Excess PBS was drained from each slide and a coverglass was mount ed to each slide using Vectashield + DAPI mounting medium (Vector Labs). The edges of the slide were sealed with nail polish (Maybelline LLC). Slides were viewed on a Zeiss Axioplan II Fluorescence microscope. Measurement of Cellular ATP Levels WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to 80% confluency. Cells were serum-starved fo r 48 hr and then treated with 100 nM Ang II. Cellular ATP levels were assessed using the ViaLight HS proliferation/cytotoxicity kit (Cambrex) following the manufacturer’s protocol. A luminometer (Monolight model 2030) was used to measure bioluminescence. Measurement of Formazan Production WT/AT1 and SYF/AT1 cells were plated onto 96 well plates and grown to 80% confluency. Cells were then starved for 48 hr in serum-free medium and treated with 100 nM Ang II as indicated. Formazan producti on was measured using the CellTiter 96 Aqueous One Solution Reagent (Promega) fo llowing the manufacturer’s instructions. Production of formazan was proportional to incr eased absorbance at 490 nm as measured by spectrophotometry. Cell Count WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to 80% confluency. The cells were then seru m-starved and treated with 100 nM Ang II as indicated. Both cell types were then counted using a hemacytometer as described (122).

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25 Densitometric Analysis Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk Scientific). The average pixel value minus background was obtained for each cell type and normalized to the average pixel value for the respective non-Ang II-treated cells. Statystical Analysis Data were analyzed by two-way ANOVA. All data passed a Normality Test as well as Equal Variance Test. Pairwise comparisons were made following the HolmSidak method. All data are expressed as mean +/SEM of replicates from three independent experiments. = p<0.05, ** = p<0.01. Results Characterization of WT/AT1 and SYF/AT1 Cells Previous reports have indicat ed that c-Src is a critical mediator of intracellular ERK1/2 activation (53, 115, 140). The role of Src kinases in intracellular ERK1/2 activation was first explored, specifically in response to an giotensin II using cSrc/Yes/Fyn-deficient (SYF) and wild type (WT) MEF cells. These SYF fibroblasts were previously isolated at E9.5 from a developing cSrc/Yes/Fyn-deficient mouse embryo and have been shown to be completely devoid of these protei ns (62). MEF cells containing functional c-Src/Yes/Fyn were also isolated from WT littermates and served as controls (WT cells). Both the SYF and WT cells do not endogenously express the AT1 receptor (data not shown). Therefore, the AT1 receptor was stably transfect ed into both cell types to constitute angiotensin II signaling. These AT1 receptor stable cell lin es have been named SYF/AT1 and WT/AT1 respectively. Saturation binding st udies were then performed in order to identify respective SYF/AT1 and WT/AT1 clones in which the binding

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26 parameters were similar. SYF/AT1 (clone #16) and WT/AT1 (clone #2) both had a KD of 0.4 nM and a Bmax of 140-150 fmol/mg protein (Figure 21A). To demonstrate that these two cell lines were similar in all aspects ot her than the levels of c-Src/Yes/Fyn, the expression levels of Jak2 and STAT3, tw o non-Src kinase dependent genes, were examined. It was found that these two genes we re expressed at similar levels in the two cell types (Figures 2-1B and 2-1C). Next, the ability of Ang II to activate PLC 1 was examined in WT/AT1 and SYF/AT1 cells. It was found that both cell types were capable of increasing PLC 1 tyrosine phosphorylation levels to roughly equal levels, an indication that the AT1 receptor can signal similarly in both cell types when examining signaling events independent of c-Src/Yes/F yn (Figure 2-1D). Collectively, these data suggest that the WT/AT1 and SYF/AT1 cells are similar in all aspects except for cSrc/Yes/Fyn expression and signaling pathways that ar e dependent on these three proteins. Angiotensin II-induced ERK1/2 Activation Is Reduced By About 50% in Src Kinase Deficient Cells Ang II-induced ERK1/2 activation was next assessed in WT/AT1 and SYF/AT1 cells. Cells were stimulated with 100 nM Ang II for 0, 5 and 10 min, and ERK1/2 activity assessed via Western blot. Ang II-d ependent ERK1/2 activation was decreased in SYF/AT1 cells when compared to WT/AT1 cells after 5 and 10 mi n of Ang II treatment (Figure 2-2A). Furthermore, Ang II-induced ERK1/2 activation was reduced in WT/AT1 cells pretreated with the Src family kinase i nhibitor, PP2, to levels comparative to Ang IIstimulated SYF/AT1 cells (Figure 2-2B). Finally, ERK1/2 activation in the SYF/AT1 cells was partially restored by transiently-transfecting these cells with c-Src (Figure 2-

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27 2C). Taken together, these results demonstr ate that Src kinases mediate a portion of Ang II-induced ERK1/2 activation. It was next determined whethe r reductions seen in SYF/AT1 cell ERK1/2 activation were indeed due to the loss of c-Src/Yes/Fyn and not due to cl onal artifact. Previous Free 125I-SarIle (nM) 0.00.20.40.60.81.01.2125I-SarIle bound (fmol/mg protein) 0 20 40 60 80 100 120 140 WT/AT1 SYF/AT1 A.B. 0 5 10 0 5 10 Ang II (min) IB: Jak2 pAbs Jak2 WT/AT1SYF/AT1 111 kDa 0 5 10 0 5 10 Ang II (min) WT/AT1SYF/AT1 79 kDa STAT3 IB: STAT3 pAbsC. WT/AT1SYF/AT10 5 10 0 5 10 Ang II (min) IP: PLC 1 -pAbs IB: Phospho-TyrmAbs IB: PLC 1-pAbs PLC 1(P) PLC 1 111 kDa 111 kDa D. Figure 2-1. Characterization of WT/AT1 and SYF/AT1 cells. A: Saturation binding curve for WT/AT1 and SYF/AT1 cells. B–D: WT/AT1 and SYF/AT1 cells were stimulated with 100 nM Ang II for 0, 5, and 10 min, and whole cell lysates were prepared. B & C: Cell lysates were Western blotted with the indicated antibodies. D: Cell lysates were i mmunoprecipitated (IP) and then Western blotted with the indicated antibodies (t op panel). Western blots were then stripped and reprobed with the indi cated antibody to demonstrate equal protein loading. This figure is used with permission from (42). work in MEF cells showed that PDGF activat es ERK1/2 in a c-Src/Yes/Fyn-independent manner (62). Therefore, both WT/AT1 and SYF/AT1 cells were stimulated with 30 ng/ml PDGF for 0, 5, and 10 min. All cells were then lysed, and total protein extract was immunoblotted with anti-active ERK1/2 pAbs. Contrary to Ang II treatment, stimulation with PDGF resulted in a similar ac tivation of ERK1/2 in both the WT/AT1 and SYF/AT1

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28 IB: ERK1/2(P)-pAbs IB: ERK1/2-Abs 0 5 10 0 5 10 PDGF (min) WT/AT1SYF/AT1 36 kDa 36 kDa ERK1(P) ERK2(P) ERK1 ERK2 A. WT/AT1SYF/AT10 5 10 0 5 10 Ang II (min) IB: ERK1/2(P)-pAbs 36 kDa IB: ERK1/2-Abs 36 kDa ERK1(P) ERK2(P) ERK1 ERK2 WT/AT1SYF/AT10 5 10 0 5 10 0 5 10 Ang II (min) ---+ + + --IB: ERK1/2(P)-pAbs IB: ERK1/2-Abs ERK2 ERK2(P) 36 kDa 36 kDaB.PP2 C. IB: ERK1/2(P)-pAbs WT/AT1SYF/AT10 5 10 0 5 10 Ang II (min) ---+ + + c-Src GFP-ERK2(P) GFP-ERK2 + + + + + + GFP-ERK2 IB: GFP-pAb IP: GFP-pAb 61 kDa 61 kDa 0 1 2 3 4 5 6 7 8 0 5 10 Ang II (min)Phospho-ERK2 (Stimulated/Unstimulated) ** WT/AT1 SYF/AT1 WT/AT1 SYF/AT1D. E. Figure 2-2. Quantification of ERK1/2 activa tion in response to Ang II in WT/AT1 and SYF/AT1 cells. A: WT/AT1 and SYF/AT1 cells were treated with 100 nM Ang II, and ERK1/2 activation assessed via Western blot analysis B: WT/AT1 and SYF/AT1 cells were pretreated with 30 M PP2 or DMSO for 60 min, and stimulated with 100 nM Ang II. Activ e ERK1/2 levels were assessed via Western blot analysis using the indicated antibodies. C: SYF/AT1 cells were co-transfected with a plasmid encoding GFP-ERK2 and a c-Src-encoding plasmid or empty vector control as indi cated. Cells were stimulated with 100 nM Ang II, and whole cell lysate s immunoprecipitated with GFP antibody. ERK1/2 activation was assessed via Wester n blot. D: Cells were pretreated with 30 ng/mL PDGF, and ERK1/2 activa tion assessed via We stern blot. E: Quantification of active ERK2 amounts from A. Fold changes in active ERK2 in response to Ang II treatment were calculated by dividing average ERK2 pixel density in Ang II-treated cells by average pixel density in nontreated controls. This figure is used with permission from (42). cells (Figure 2-2D). Thus, while SYF/AT1 cells are capable of activating ERK1/2 to the same degree as WT/AT1 cells in response to PDGF, the decreased ERK1/2 activation

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29 seen in SYF/AT1 cells in response to A ng II is in fact due to the specific absence of cSrc/Yes/Fyn-depe ndent signaling. Quantification of band densities from 22A revealed that decreased ERK1/2 phosphorylation in the SYF/AT1 cells was statistically si gnificant (Figure 2-2E). Furthermore, maximum Ang II-induced ERK1/2 activation in the SYF/AT1 cells was observed after 5 min of Ang II treatment, whereas maximum ERK1/2 activation occurred 10 min post-Ang II treatment in the WT/AT1 cells. Maximum ERK1/2 phosphorylation levels were reduced by about 50% in the SYF/AT1 cells when compared to peak levels of ERK1/2 phosphorylation in the WT/AT1 cells. As such, c-Src/ Yes/Fyn tyrosine kinases mediate at most 50% of Ang II-induced ERK1 /2 activation since r oughly half of Ang IIactivated ERK1/2 activation persists in their absence. Angiotensin II-induced ERK1/2 Nuclear Tr anslocation Is Not Dependent Upon Src Kinases Clearly, Src/Yes/Fyn-dependent signaling is responsible for a portion of Ang IIinduced ERK1/2 activation. The effect of a loss of c-Src/Yes/Fyn signaling on Ang IIinduced ERK1/2 nuclear translocation was ne xt assessed. Previous reports have shown that ERK1/2 translocates into the nucleus and initiates gene tr anscription of early response genes via the phosphorylation of specifi c transcription factor targets (98). Other work has shown that ERK1/2 nuclear transl ocation is dependent upon heterotrimeric G protein signaling in response to Ang II (122). Therefore, it was next examined whether the elimination of c-Src/Yes/Fyn and theref ore a loss of about 50% of Ang II-induced ERK1/2 activation would also aff ect ERK1/2 nuclear translocation. WT/AT1 and SYF/AT1 cells were transfected with a GFP-ERK2 plasmid in order to track ERK2 movement in response to Ang II treatment. Cells were then stimulated

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30 with 100 nM Ang II, fixed and DAPI stained to visualize the nucleus. In the absence of Ang II, GFP-ERK2 was distributed fairly ev enly between both the nucleus and cytoplasm in WT/AT1 and SYF/AT1 cells (Figures 2-3A, 2-3B a nd 2-3G). DAPI counterstain of these images and merging of the GFP a nd DAPI images confirmed these findings (Figures 2-3D, 2-3E and 2-3J). In contrast, ERK1/2 accumulation was markedly increased in the nucleus of both WT/AT1 and SYF/AT1 cells treated with Ang II (Figures 2-3C, 2-3 H and 2-3I), and this finding wa s confirmed by DAPI counterstain (Figures 23F, 2-3K and 2-3L). As such, it appeared th at ERK1/2 nuclear translocation was present in both Ang II-stimulated WT/AT1 and SYF/AT1 cells. However, there was no statistically significant difference in nuc lear fluorescence between Ang II-stimulated SYF/AT1 cells when compared to WT/AT1 controls. As such, it appears that cSrc/Yes/Fyn do not influence ERK1/2 nuclear translocation. Quantification of nuclear fluorescence relative to cytoplasmic fluores cence revealed a significant increase in ERK1/2 nuclear fluore scence in both WT/AT1 and SYF/AT1 cells stimulated with Ang II, indicative of increased nuclear transl ocation (Figures 2-3M and 2-3N). Angiotensin II Induced Cell Proliferation Is Reduced in Src Kinase Deficient Cells Ang II-induced ERK1/2 activation has been shown to initiate cell proliferation (33, 89, 90, 133). It has primarily been thought that this occu rs through the translocation of ERK1/2 into the nucleus and the subsequent initiation of growth response gene transcription (122). Here, it has been dem onstrated that Ang II-induced ERK2 nuclear translocation is unaffected by the loss of c-Src/Yes/Fyn-mediated ERK1/2 activation

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31 Figure 2-3. Nuclear translocati on of active ERK2 is unaffected by the loss of c-Src/Yes/Fyn. A C, G – I: WT/AT1 or SYF/AT1 cells were transfected with a GFP-ERK2 plasmid and then stimul ated with Ang II for 0 and 10 min. Nuclear translocation of ERK2 was assessed by fluorescent microscopy. A C: GFP-ERK2 images in non-treated and Ang II-treated WT/AT1 cells. G I: Merging of images A – C respectively w ith DAPI stained images. G I: GFPERK2 images in non-treate d and Ang II-treated SYF/AT1 cells. J L: Merging of images G I respectively with DAPI stained images. M: Nuclear fluorescence from A C was quantifie d and normalized to cytoplasmic fluorescence. N: Nuclear fluoresce nce from G I was quantified and normalized to cytoplasmic fluorescence. All images are representative of the entire field and were taken at 40X ma gnification. Bar represents 15 microns. Shown is one of two independent results. This figure is used with permission from (42).

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32 (Figure 2-3). It was next determined wh ether eliminating c-Src/Yes/Fyn effects Ang IIinduced cell proliferation, independent of the ability of ERK1/2 to translocate into the nucleus. Ang II-induced cell proliferation was asse ssed via three different methodologies. First, cellular ATP levels were measured since ATP amounts have previously been reported to be excellent indicators of cell number (22). WT/AT1 and SYF/AT1 cells were treated with 100 nM Ang II and intracellular AT P levels were measured. After 4 hours of Ang II treatment, ATP levels had alr eady increased over 3 fold in WT/AT1 cells (Figure 2-4A). ATP levels in SYF/AT1 cells increased slightly by about 1.75 fold, but were markedly reduced after Ang II treatment compared to WT/AT1 controls. These data suggest that cell number was decreased in Ang II-stimulated SYF/AT1 cells when compared to WT/AT1 controls and therefore that A ng II-induced cell pr oliferation was significantly reduced by the el imination of c-Src/Yes/Fyn. Next, Ang II-induced cell pr oliferation was assessed th rough the measurement of formazan levels, which has previously been repo rted to also be an excellent indicator of increased cell number (12). WT/AT1 and SYF/AT1 cells were treated with 100 nM Ang II and then formazan production was assessed. Ang II-induced formazan production was significantly increased in WT/AT1 cells after 5 hours of Ang II treatment (Figure 2-4B). However, formazan production barely increased in SYF/AT1 cells treated for 5 hours with Ang II. SYF/AT1 cell formazan production was significa ntly decreased relative to WT/AT1 controls. Lastly, Ang II-induced cell proliferation was analyzed via direct cell count. WT/AT1 and SYF/AT1 cells were treated with 100 nM A ng II for 0 and 24 hr. Cells were

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33 A 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5ATP (Stimulated/Unstimulated)Ang II-+ -+CFold Change in Cell Number WT/AT1 SYF/AT1Ang II -+ -+ 0 0.5 1 1.5 2 2.5 3 3.5 4 ** ** B 0.5 1 1.5 21 *Formazan (Stimulated/Unstimulated)Ang II-+ -+ 2.5 ** WT/AT1 SYF/AT1 WT/AT1 SYF/AT1* Figure 2-4. Ang II-induced cell proliferati on is reduced in Ang II-stimulated SYF/AT1 cells. A: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 4 hr. Cellular ATP levels were then m easured. Data are expressed as fold change in ATP in Ang II stimulated cells relative to non-trea ted controls. B: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 5 hr. Formazan production was then measured. Data are expressed as fold change in formazan in Ang II stimulated cells relative to non-treate d controls. C: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 24 hr. All cells were then detached and counted using a hemacytometer. All data are expressed as a fold change in Ang II-stimulated rela tive to non-stimulated cells. All data are representative of three independent experiments. WT/AT1 control cells. Thus, these data also suggest that Ang II-induced cell proliferation is markedly reduced in SYF/AT1 cells relative to WT controls. This figure is used with permission from (42). detached and counted using a hemacytometer. WT/AT1 cell number was increased by over 3 fold when treated with Ang II for 24 hours relative to non-trea ted controls (Figure 2-4C). SYF/AT1 cell number also increased, but this increase was significantly reduced when compared to Ang II-treated WT/AT1 cells. As such, these data show that Ang II-

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34 induced cell proliferation was markedly reduced in SYF/AT1 cells lacking cSrc/Yes/Fyn-depe ndent signaling. In summary, Ang II-induced cell proliferat ion is reduced when c-Src/Yes/Fyn are eliminated from the cell. Eliminating the ~50% of Ang II-induced ERK1/2 activation dependent upon c-Src/Yes/Fyn therefore alters th e ability of these cells to proliferate in response to Ang II. Interest ingly, the decrease in Ang II-induced cell proliferation observed in SYF/AT1 cells occurs independent of the ability of ERK1/2 to translocate into the nucleus. In addition, a portion of cell proliferation is dependent upon cSrc/Yes/Fyn-independent signaling. Discussion Here, MEF cells completely devoid of c-Sr c/Yes/Fyn were utilized. In doing so, all Src kinase function and the possibility that ER K1/2 activation can be mediated via any of these very similar family members has been completely eliminated. It was found that while c-Src/Yes/Fyn tyrosine kinases do play a role in the activation of ERK1/2 as previously reported, ERK1/2 activation is not completely dependent on these proteins and persists at reduced levels in their absence. c-Src/Yes/Fyn are capab le of activating only about 50% of intracellular ERK1/2. An explanat ion for these results is that the remaining 50% of intracellular ERK1/2 are activated by c-Src/Yes/Fyn-independent mechanisms. As such, Ang II-induced ERK1/2 activation occurs through two independent signaling cascades, and is not completely dependent upon Src kinases as previous work has shown (53). Interestingly, while the Src kinase de pendent signaling pathways appears to mediate as much as 50% of Ang II-induced ERK1/2 activation, these signaling events do not influence Ang II-induced ERK1/2 nuclear translocation. It had previously been

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35 thought that ERK1/2 must tran slocate into the nu cleus in order to initiate events necessary for the start of cell proliferation, including the tr anscription of early response genes such as c-fos (14, 15, 109). Interestingly, the lo ss c-Src/Yes/Fyn had no effect on the ability of ERK1/2 to translocate into the nucleus. ERK1/2 was able to enter the nucleus in the absence of c-Src/Yes/Fyn; however, cell proliferation was still markedly reduced. Even more striking is the finding that c-Src/Yes/Fyn still influence cell proliferation, independent of ERK1/2 nuclear translocation. An explanation for these findings is that ERK1/2 ac tivated via c-Src/Yes/Fyndependent signaling acts upon cytoplasmic proteins to mediate proliferati on. Previous work has already shown that ERK1/2 can phosphorylate a number of cytoplas mic substrates, including members of the RSK, MSK and MNK families of proteins (37) Furthermore, many of these proteins have been shown to regulate th e activity of transcription f actors. For example, RSK has been shown to modulate phosphorylation of bot h the serum response factor (SRF) as well as CREB, both of which have been shown to be pro-mitotic (37). Therefore, ERK1/2 may also modulate transcript ion through the phosphorylati on of cytoplasmic proteins, which themselves activate transcription fact ors initiating pro-grow th and proliferative events. In addition, ERK1/2 activated vi a c-Src/Yes/Fyn-independent signaling may translocate into the nucleus to directly mediate transcriptional events (122). These events will be investigated and described in detail in the following chapters.

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36 CHAPTER 3 SRC KINASE-INDEPENDENT ERK1/2 ACTIVATION AND CELL PROLIFERATION IS MEDIATED BY HETEROTRIMERIC G PROTEINS AND PKC -DEPENDENT SIGNALING Introduction In the previous chapter, it was demons trated that approximately 50% of Ang IIinduced ERK1/2 activation is mediated by Src fa mily tyrosine kinases. In addition, these proteins also mediate a por tion of angiotensin II-induced cell proliferation. The remaining 50% of intracellular ERK1/2 act ivation and portion of Ang II-induced cell proliferation must therefore be occurring through Src kinase-inde pendent signaling. In this chapter, the events unde rlying Src kinase-independent ERK1/2 activation through the AT1 receptor will be discussed. Previous work has implicated numerous pr oteins other than Src kinases in Ang IIinduced ERK1/2 activation. For example, the AT1 receptor is capable of transactivating other receptors with intrinsic tyrosine ki nase activity, including the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), which in turn activate ERK1/2 (32, 74, 85). In addi tion to receptor transactivation, numerous cytoplasmic kinases have been shown to me diate ERK1/2 activation. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) blocks ERK1/2 activation in EGF-treated preglomerular smooth muscle cells (4, 45). Furthermore, various isoforms of Protein Kinase C (PKC) have been shown to mediate intracellular ERK1/2 activation in response to different ligands (45, 46, 70, 86). It is not clear from these reports, though, if the mechanism of ERK1/2 activation differs depending upon the recepto r activated, or if

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37 ERK1/2 activation occurs simultaneously via multiple independent signaling mechanisms in response to treatment with the same ligand. Here, we sought to determine whether AT1 receptor-generated ERK1/2 activation occurs via Src kinase-independent signali ng in addition to Src kinase dependent signaling. Specifically, the eff ect of PDGFR, EGFR, PI3K, PKC, Raf or MEK inhibition on Ang II-induced ERK1/2 activation was firs t investigated in order to identify a mechanism whereby ERK1/2 activation occurs in a Src kinase-independent manner. In addition, the effect of hetero trimeric G protein and PKC inhibition on Ang II-induced ERK1/2 activation was examined. An attenu ation of Src kinase-i ndependent ERK1/2 activation only occurred when heterotrimeric G protein, PKC or MEK activities were inhibited, suggesting that these proteins mediate Ang II-induced ERK1/2 activation independent of Src family tyrosine kina ses. In addition, it was found that MEK phosphorylation was dependent upon PKC activity, identifying a mechanism whereby PKC activates ERK1/2 via the upstream ac tivation of MEK. As such, ERK1/2 activation occurs via hete rotrimeric G protein/PKC signaling independent of Src family tyrosine kinases in response to AT1 receptor activation. It was previously observed that Src kinases mediate about 50% of ERK1/2 activation and a portion of Ang II-induced cell proliferation (Chapter 2). More importantly, Src kinase-activated ERK1/2 me diates cell proliferation without direct translocation of ERK1/2 into the nucleus. Previous wo rk has shown that ERK1/2 translocates into the nucleus to modulate gene transcripti on in response to Ang II (122). As such, ERK1/2 nuclear translocation must therefore be influenced by Src kinaseindependent signaling events. Therefore, whether heterotrimeric G protein/PKC

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38 signaling mediates Ang II-induced ERK1/2 nuc lear translocation and cell proliferation was also assessed. The effect of inhibition of th e heterotrimeric G protein/PKC pathway on ERK1/2 nuclear translocation an d cell proliferation was determined using a PKC myristoylated pseudosubstrate. In contrast to Src kinases, the nuclear translocation of ERK1/2 is dependent upon PKC activity as pretreatment with the PKC MP abolished Ang IIinduced ERK1/2 nuclear tran slocation. In addition, PKC inhibition also reduced Ang IIinduced cell proliferation. Interestingly, PKC inhibition in combina tion with Src kinase inhibition completely attenuated Ang II-induced cell proliferation. Therefore, ERK1/2 activation and cell proliferation are c ontrolled through both heterotrimeric G protein/PKC -dependent and Src kinase-dependent signaling events in response to angiotensin II. Each of these pathways app ears to mediate a portion of this response, but only heterotrimeric G protein/PKC signaling causes ERK1/2 to directly translocate into the nucleus. As such, it appears that Ang II-induced ERK1/2 activation is mediated by two independent signaling cascades ope rating through distinct mechanisms. Materials and Methods Cell Culture WT/AT1 and SYF/AT1 cells were cultured as described in Chapter 2. CHO/AT1 and CHO/AT1-M5 cells were cultured in F-12 me dia supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 units /mL penicillin, 10 g/mL streptomyocin, 1 mM sodium pyruvate, and 10 mM HEPES. WT/AT1 and SYF/AT1 were growth arrested in serum-free DMEM for 48 h prior to experime nts. CHO cells were growth arrested in the same manner for 24 h. All cell culture r eagents were obtained from Invitrogen.

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39 Pharmacological Inhibitors AG1295, AG1478, GDPS, G6976, G6983, LY294002, PD98059, PP2, Raf1 kinase Inhibitor 1 and Rotlerrin were all obtained from Calbiochem and used at concentrations found to have maximum inhibitory effect (32, 46, 74, 85, 89, 117, 122, 140, 148). Sodium fluoride (SIGMA), chel erythrine (LKT Labs), and the PKC myristoylated pseudosubstrate (BioMol) were also used at previously determined concentrations (58, 124, 164). SYF/AT1 cells were permeabilized with 5 nM saponin (USB) before treatment with GDPS (103). All other reagents were obtained from SIGMA or Fisher. Cells were pretreated with inhibito r for the indicated time and stimulated with 100 nM Ang II as described. siRNA Treatment of WT/AT1 Cells siRNA reagents were purchased from Santa Cruz Biotechnology. Cells were grown in 100 mm culture plates (Corning) to 80% confluency. Adherent cells were trypsinized and resuspended in serum-containi ng medium without antibiotics. Cells and medium were centrifuged at 500 x g for 5 min, and pelleted cells were resuspended in fresh serum-containing medium without antibi otics. Cells were transferred to 6 well culture plates (Corning) and grown to 40–50% confluency. Transfection reagents were prepared as described in the online prot ocol (http://www.scbt.com/support/protocols) with the exception that the c oncentration of siRNA used was increased four fold. Cells were next transfected for 48 hrs at 37oC with either control siRNA or PKC -specific siRNA in serum-containing medium without anti biotics. Cells were serum-starved for 48 hours and treated with 100 nM Ang II for 0, 5 and 10 min. Cells were lysed and whole cell protein lysates were prep ared. Cell lysates were sepa rated on a 10% SDS-PAGE gel,

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40 transferred to a nitrocellulose membrane and Western blotted with the indicated antibodies. Immunoprecipitation, Western Blo tting and Densitometric Analysis Cells were immunoprecipitated as described in Chapter 2. Proteins were detected using enhanced chemiluminescence as described in Chapter 2. The anti-MEK1/2, the anti-MEK1/2(P) and the anti-PKC antibodies used for Wester n blotting were from Santa Cruz Biotechnology. Western bl ots were scanned and densitized using UnScanIt Gel Analysis (Silk Scientific) as described in Chapter 2. Immunofluorescence and Quantification of Fluorescence Cells were transfected with GFP-tagged ERK2 and ERK2 movement examined via immunofluorescence as described in Chapter 2. Cells were pretreated with either 1 M PKC myristoylated pseudosubstrate or DMSO fo r 1 hour prior to stimulation with 100 nM Ang II. Nuclear or cytoplasm fluorescen ce was quantified using the Image J Progam (NIH) as described in Chapter 2. Cell Count Cell counts were performed using a hemacy tometer as described in Chapter 2. Cells were pretreated with either 1 M PKC myristoylated pseudosubstrate or DMSO for 1 hour prior to stimula tion with 100 nM Ang II. Statistical Analysis Data were analyzed by two-way ANOVA. All data passed a Normality Test as well as Equal Variance Test. Pairwise comparisons were made following the HolmSidak method. All data are expressed as mean +/SEM. = p<0.05, ** = p<0.01.

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41 Results Src Kinase Independent ERK1/2 Activat ion Does Not Require EGF Receptor, PDGF Receptor or PI3K Activity A mechanism for c-Src/Yes/Fyn-independe nt ERK1/2 activation in response to Ang II was first identif ied through pharmacological inhibi tion of candidate proteins. A brief literature search identified proteins which have previously been implicated in ERK1/2 activation in response to vari ous activating ligands (Table 3-1). Pharmacological inhibitors for each of thes e proteins were then obtained, and SYF/AT1 cells were treated for the indicated time with c oncentrations of these inhibitors previously found to suppress protein function, in order to identify proteins th at mediate ERK1/2 activation independent of c-Src/ Yes/Fyn. Cells were stimulat ed with 100 nM Ang II and whole cell lysates were Western blotted wi th phospho-ERK1/2 antibodies to identify changes in active ERK1/2 levels. Table 3-1. Pharmacological i nhibition of Src kinase-indep endent ERK1/2 inhibition. Inhibitor Treatment Protein Inhibitor Final Concentration Time (min) EGFR AG1478 30 M 30 Heterotrimeric G proteins GDP S 2 mM 20 MEK PD98059 50 M 60 Raf1 Raf1 kinase 1 inhibitor 30 M 60 PDGFR AG1295 30 M 60 PKC Chelerythrine 30 M 60 PI3K LY294002 30 M 60 The involvement of growth factor recep tor transactivation on c-Src/Yes/Fynindependent ERK1/2 activation was first examined. SYF/AT1 cells were pretreated with either PDGFR (AG1295) or EGFR (AG1478) se lective inhibitors, and then stimulated with 100 nM Ang II. No reduction in Ang II-induced ERK1/2 activation was observed

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42 A B 0 5 10 0 5 10 SYF/AT1 ---+ + + Ang II (min) AG1478 SYF/AT1 0 5 0 5 --+ + AG1295 Ang II (min)CIB: ERK1/2(P)-pAbs IB: ERK1/2-Abs IB: ERK1/2(P)-pAbs IB: ERK1/2-Abs 0 5 10 0 5 10 SYF/AT1 ---+ + + Ang II (min) LY294002 IB: ERK1/2(P)-pAbs IB: ERK1/2A bs 36 kDa 36 kDa ERK2(P) ERK2 ERK2(P) ERK1(P) ERK2 36 kDa 36 kDa ERK2(P) ERK1(P) ERK2 36 kDa 36 kDa Figure 3-1. ERK1/2 activation in SYF/AT1 cells does not require transactivation of the PDGFR or the EGFR or activa tion of PI3K. A – C: SYF/AT1 cells were pretreated with the indica ted pharmacological inhibitors at the concentrations and for the treatment times listed in Ta ble 1. Cells were stimulated with 100 nM Ang II for indicated times, and active ERK1/2 levels were assessed by Western blotting with anti-ERK1/2(P) antibodies (top panels). Total ERK protein loading was demonstrated by stripping the membrane and reprobing with anti-ERK1/2 antibodies (bottom panels). All West erns are representative of three independent experiments. This figure is used with permission from (42).

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43 in the PDGFR inhibitor-treated SYF/AT1 cells compared to non-tr eated controls (3-1A). Similarly, treatment of SYF/AT1 cells with the EGFR selective inhibitor had no effect on Ang II-induced ERK1/2 activation (Figure 3-1B). Collectively, these data indicate that Ang II-induced ERK1/2 activation occurring through c-Src/Yes/Fyn-independent mechanisms does not require EG FR or PDGFR transactivation. ERK1/2 activation has also previously been reported to occur through PI3Kdependent signaling mechanisms (4, 45, 86). The effect of PI3K inhibition on Ang IIinduced ERK1/2 activation was next determined in SYF/AT1 cells using the PI3K selective inhibitor, LY294002. Control treated SYF/AT1 cells exhibited the typical ERK1/2 activation response after 5 min of Ang II treatment and no decrease in ERK1/2 activation was observed in LY294002-treated cel ls (Figure 3-1C). In fact, ERK1/2 activation increased slightly when LY294002 was added to SYF/AT1 cells. Thus, PI3K does not regulate Ang II-induced ERK1/2 ac tivation independent of c-Src/Yes/Fyn. Src Kinase Independent ERK1/2 Activation Is Dependent on MEK1/2, But Not Raf1 ERK1/2 activation typically occurs th rough the activation of a MAPK signaling cascade, in which a MAPK-kinase-kinase (MAPKKK) phosphorylates a MAPK-kinase (MAPKK), which in turn phosphorylate and activate MAPKs such as ERK1/2. The typical MAPKKK involved in ERK1/2 activ ation is Raf1, while MEK1/2 are the MAPKK which dually phosphoryl ate ERK1/2. It was next investigated whether pharmacological inhibition of either Raf1 or MEK1/2 would effect Ang II-induced ERK1/2 activation in SYF/AT1 cells. SYF/AT1 cells pre-treated with the Raf1 kinase inhibitor exhibited no decrease in ERK1/2 ac tivation when compared to untreated cells (Figure 3-2A). In contrast, Ang II-induced ERK1/2 activation was completely absent from WT/AT1 cells and SYF/AT1 treated with the MEK1/2 inhibitor (Figure 3-2B).

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44 Therefore, Ang II-induced ERK1/2 activation occurring via c-Src/Yes/Fyn-independent mechanisms (i.e., in the SYF/AT1 cells) does not require Raf1. However, all Ang IIinduced ERK1/2 activation is dependent upon MEK activation. ---+ + + 0 5 10 0 5 10 SYF/AT1 Ang II (min) Raf1I IB: ERK1/2(P)-pAbs IB: ERK1/2-Abs ERK2(P) ERK1(P) ERK2 36 kDa 36 kDaA WT/AT1SYF/AT10 5 10 0 5 10 0 5 10 0 5 10 Ang II (min) ---+ + + ---+ + + PD98059 36 kDa IB: ERK1/2(P)-pAbs IB: ERK1/2-Abs ERK1(P) 36 kDa ERK1 ERK2(P) ERK2B Figure 3-2. ERK1/2 activation in SYF/AT1 cells requires MEK1/2 activation, but not Raf1 activation. A: WT/AT1 or SYF/AT1 cells were pretreated with the indicated pharmacological inhibitors at the concentrations and for the treatment times listed Table 1. Cells we re stimulated with 100 nM Ang II for the indicated times, and active ERK1/2 levels were assessed by Western blotting with anti-ERK1/2(P) antibodie s (top panels). Total ERK protein loading was demonstrated by stripping the membrane and reprobing with antiERK1/2 antibodies (bottom panels). All Westerns are representative of three independent experiments. This figure is used with permission from (42). In summary, ERK1/2 activation in Ang II-treated SYF/AT1 cells was not affected when cells were treated with EGFR, PDGFR, PI3K or Raf1 selective pharmacological inhibitors. However, Ang II-induced ER K1/2 activation was attenuated in SYF/AT1 cells

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45 treated with the MEK1/2 inhibitor. In tracellular ERK1/2 activation occurring independent of c-Src/Yes/Fyn requires upstr eam MEK1/2 activation in order to dually phosphorylate ERK1/2, but does not depend on AT1 receptor transactivation of the EGF and PDGF receptors or the phosphorylation of PI3K and Raf1. As such, other proteins must therefore be acting upstream of MEK1 /2 in order to activate ERK1/2 in a cSrc/Yes/Fyn-independent manner. Heterotrimeric G Proteins Mediate A Portion of ERK1/2 Activation In A Src Kinase-independent Manner Heterotrimeric G proteins have previous ly been shown to activate ERK1/2 in response to Ang II (122). Whether the re maining 50% of Ang II-induced ERK1/2 activation in the SYF/AT1 cells was mediated entirely by heterotrimeric G protein signaling was next examined. SYF/AT1 cells were permeabilized using saponin, and then pretreated with the heterotrim eric G protein inhibitor, GDPS (103). The -phosphate group of this compound has been replaced with a sulfate group, hindering the ability of heterotrimeric G proteins to exhange GDP fo r GTP and subsequently become activated. Permeabilized SYF/AT1 cells treated with vehicle control served as controls. Cells were then stimulated with 100 nM Ang II, and ER K1/2 activation assesse d via Western blot. SYF/AT1 cells permeabilized with saponin and treated with vehicle control still demonstrated ERK2 activation in response to Ang II (Figure 3-3A). However, ERK2 activation was attenuated in GDPS treated cells since ther e was no increase in ERK2 phosphorylation levels in Ang II-stimulated cells compared to untreated controls. These data suggest that Ang II-induced activati on of ERK1/2 occurring independent of cSrc/Yes/Fyn requires heterotrim eric G protein activation.

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46 The effect of ligand-independent activation of heterotrimeric G proteins on ERK1/2 activation in the SYF/AT1 cells was next tested. SYF/AT1 cells were pretreated with NaF, which causes heterotrimeric G protei ns to become constitutively activated independent of exogenous ligand addition (58). Cells were then stimulated with 100 nM Ang II, and ERK1/2 activation assessed via Western blot. Basal ERK1/2 activation was significantly increased in SYF/AT1 cells treated with NaF when compared to untreated cells (Figure 3-3B). Furthermore, the addi tion of Ang II did not fu rther increase ERK1/2 activation levels, suggesting that Ang II activates ERK1/2 in SYF/AT1 cells via a mechanism that is dependent upon heterotrim eric G proteins. The addition of EGF, which activates ERK1/2 in a c-Src/Yes/Fyn dependent manner (10), further increased ERK1/2 activation in NaF treated cells (data not shown). These data therefore suggest that Ang II-induced ERK1/2 activation occurr ing independent of c-Src/Yes/Fyn requires heterotrimeric G protein activation. To further demonstrate that heterotrimer ic G proteins mediate all c-Src/Yes/Fynindependent ERK1/2 activation in response to Ang II, a previously generated CHO cell line stably transfected with a mutant AT1 receptor was utilized. This mutant AT1 receptor contains specific mutations in the ca rboxyl terminus preventing heterotrimeric G proteins from coupling to the receptor and becoming activated (28). These cells are denoted as CHO/AT1-M5 cells here, and retain their ab ility to activate tyrosine kinases such as c-Src/Yes/Fyn. CHO cells stably expressing wild type AT1 receptor with similar affinity and abundance (dentoted as CHO/AT1) were used as controls. Both cell types were stimulated with 100 nM Ang II, and ER K1/2 activation assesse d via Western Blot. Ang II-induced ERK1/2 activation in CHO/AT1 cells reached maximum levels after 5

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47 A 0 5 10 0 5 10 SYF/AT1 ---+ + + Ang II (min) GDPSB 0 5 10 0 5 10 ---+ + + Ang II (min) NaF SYF/AT1CCHO/AT1CHO/AT1-M5 Ang II (min) WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs 36 kDa 36 kDa 0 5 10 0 5 10 ERK2(P) ERK2 36 kDa 36 kDa ERK2(P) ERK1 ERK2 ERK1 WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs 0 5 10 ERK2(P) ERK2 36 kDa 36 kDa PP2 -----+ + + Figure 3-3. Ang II-induced ERK1/2 activation is partially dependent upon heterotrimeric G proteins. A and B: SYF/AT1 cells were pretreated with either 2 mM GDPS for 20 min or 2 mM NaF for 1 hr, re spectively, and stimulated with 100 nM Ang II for 0, 5, and 10 min. C: CHO/AT1 and CHO/AT1-M5 cells were first pretreated with either 30 M PP2 or DMSO for 1 hour before Ang II treatment. ERK2 phosphorylation was de termined via Western blot using the indicated antibodies. A-C: Total ERK protein loading was demonstrated in each case by stripping the membrane and reprobing with the anti-ERK1/2 antibodies (bottom panels). All West erns are representative of three independent experiments. This figure is used with permission from (42).

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48 min of Ang II treatment (Fi gure 3-3C). ERK1/2 activati on also occurred in CHO/AT1M5 cells in response to Ang II, but maxi mum ERK1/2 activation was significantly reduced compared to CHO/AT1 cells. Therefore, Ang II-induced ERK1/2 activation is partially dependent upon heterotrimeric G prot ein activation. Furthermore, pretreatment of CHO/AT1-M5 cells with PP2 completely bloc ked Ang II-induced ERK1/2 activation, indicating that the remaining 50% of ERK1/2 activation is depe ndent upon Src kinases. Protein Kinase C Mediates ERK1/2 Activation In A Src Kinase-independent Manner Protein kinase C isoforms have been shown to be activated downstream of heterotrimeric G proteins and mediate ERK1 /2 activation (45, 46, 70, 86). The specific PKC isoforms responsible for activating ERK1 /2 in a c-Src/Yes/Fyn independent manner were next identified. SYF/AT1 cells were first treated with the broad range PKC inhibitor, chelerythrine. Ce lls were then stimulated w ith 100 nM Ang II, and ERK1/2 activation assessed by Western blot. ERK1/2 activation was eliminated in SYF/AT1 cells treated with chelerythrine relative to DMSO-t reated controls (Figure 3-4A). As such, these data confirm that Src kinase-independe nt ERK1/2 activation in response to Ang II specifically requires PKC. Over twelve different isotypes of PKC have been identified to date, and a number of PKC isoforms have already been linke d to ERK1/2 activation in response to stimulation by various ligands (46, 70, 86). Therefore, the specific PKC isoforms mediating Ang II-induced ERK1/2 activation in the SYF/AT1 cells were next identified. SYF/AT1 cells were pretreated with pharmacological inhibitors specific for a number of different PKC isoforms (Figures 3-4B, 3-4C a nd 3-4D). Cells were then stimulated with

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49 B0 5 10 0 5 10 ---+ + + Ang II (min) G6983 SYF/AT1C 0 5 10 0 5 10 ---+ + + Ang II (min) G6976 SYF/AT1 0 5 10 0 5 10 ---+ + + Ang II (min) Rottlerin SYF/AT1WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK1(P) ERK2(P) ERK2 36 kDaD 36 kDa ERK2 ERK2(P) ERK1(P) 36 kDa 36 kDa WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK2(P) ERK1(P) ERK2 ERK1 36 kDa 36 kDaA0 5 10 0 5 10 ---+ + + Ang II (min) Chelerythrine SYF/AT1WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK1(P) ERK2(P) ERK2D 1 2 S1 S2 S3 S4 0% 50% 100%Chelerythrine: Broad PKC inhibition G6983: , , G6976: , Rottlerin: + 1 2 S1 S2 S3 S4 0% 50% 100%Chelerythrine: Broad PKC inhibition G6983: , , G6976: , Rottlerin: + 36 kDa 36 kDaPercent SYF/AT1Cell ERK2 Activation E B0 5 10 0 5 10 ---+ + + Ang II (min) G6983 SYF/AT1C 0 5 10 0 5 10 ---+ + + Ang II (min) G6976 SYF/AT1 0 5 10 0 5 10 ---+ + + Ang II (min) Rottlerin SYF/AT1WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK1(P) ERK2(P) ERK2 36 kDaD 36 kDa ERK2 ERK2(P) ERK1(P) 36 kDa 36 kDa WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK2(P) ERK1(P) ERK2 ERK1 36 kDa 36 kDaA0 5 10 0 5 10 ---+ + + Ang II (min) Chelerythrine SYF/AT1WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ERK1(P) ERK2(P) ERK2D 1 2 S1 S2 S3 S4 0% 50% 100%Chelerythrine: Broad PKC inhibition G6983: , , G6976: , Rottlerin: + 1 2 S1 S2 S3 S4 0% 50% 100%Chelerythrine: Broad PKC inhibition G6983: , , G6976: , Rottlerin: + 36 kDa 36 kDaPercent SYF/AT1Cell ERK2 Activation E Figure 3-4. Ang II-induced ERK1/2 activation is partially dependent upon PKC. A: SYF/AT1 cells were pretreated with eith er DMSO or 30 M Cheleryrthine (A), 30 M G6983 (B), 30 M G6979 (C) or 30 M Rottlerin (D) for 60 min, and then stimulated with 100 nM Ang II for 0, 5, and 10 min. ERK1/2 activation was assessed via Western blot ted using the indicated antibodies (top panel). Total ERK1/2 protein loadin g was demonstrated by stripping the membrane and reprobing with the indi cated antibodies (bottom panels). These data are representative of three i ndependent experiments. E: The effect of inhibitor treatment on the per cent maximum Ang II-induced ERK2 activation. Maximum Ang II-induced ERK2 activation was quantified from A – D in the presence (+) or absence ( -) of inhibitor. Phospho-ERK2 bands were densitized. Values were then no rmalized to ERK2 activation in Ang IIstimulated cells not pretreated with inhibitor, and multiplied by 100. G6983 and chelerythrine pretreatment signi ficantly reduced ERK1/2 activation compared to vehicle-treated controls. This figure is used with permission from (42).

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50 100 nM Ang II, and ERK1/2 activation assessed via Western blot. A common trend was observed in that Ang II-induced ERK1/2 ac tivation was only reduced by pretreatment with compounds that inhibited a subset of PKC isoforms that included PKC (Figures 34E). The effect of PKC inhibition on Ang II-induced ERK1/2 activation was next assessed using a PKC myristoylated pseudosubstrate (MP), a potent and specific inhibitor for PKC (164). WT/AT1 and SYF/AT1 cells were first pretreated with either PKC MP or vehicle control. Both cell type s were then stimulated with 100 nM Ang II, and ERK1/2 activation a ssessed via Western blot. ERK1/2 activation in WT/AT1 cells was significantly reduced w ith the addition of the PKC MP, while ERK1/2 activation in SYF/AT1 cells treated with PKC MP was significantly reduced to levels found in nonligand treated cells (Figures 3-5A and 3-5B). Intere stingly, treatment of WT/AT1 cells with PKC MP reduced Ang II-induced ERK1/2 activ ation to levels present in Ang IIstimulated SYF/AT1 cells. As such, PKC appears to mediate Ang II-induced ERK1/2 activation independent of Src kinases. In order to reconfirm that roughly half of Ang II-induced ER K1/2 activation is mediated by PKC PKC -specific siRNA was utilized. WT/AT1 cells were transfected with either a scrambled siRNA control or a PKC -specific siRNA. The cells were then stimulated with 100 nM Ang II, and ERK1/2 activation was then assessed via Western blot. It was found that ERK1/2 activati on occurred normally in response to Ang II stimulation in WT/AT1 cells transfected with control siRNA (Figure 3-6A). ERK1/2 activation was significantly reduced in PKC -siRNA transfected cells when compared to

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51 cells transfected with control siRNA. This reduction equated to about a 50% decrease in ERK1/2 activity (Figure 3-6B), and was th e maximum reduction in active ERK1/2 A 0 5 10 0 5 10 ---+ + + Ang II (min) PKC MP SYF/AT1WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs 0 5 10 0 5 10 ---+ + + WT/AT1 ERK1(P) ERK2(P) ERK1 ERK2 36 kDa 36 kDa WT/AT1 SYF/AT1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Ang II -+ + -+ + PKC MP ++Phospho-ERK2 (Stimulated/Unstimulated) NSB-Figure 3-5. PKC mediates Ang II-induced ERK1 /2 activation independent of cSrc/Yes/Fyn. A: WT/AT1 and SYF/AT1 cells were pretreated with 1 M PKC MP for 1 hr, and then stimulated with Ang II for 0, 5 and 10 min. ERK1/2 activation was then assessed via Western blot analysis with the indicated antibodies (top panel). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (bottom panel). B: Phosphor ylated ERK2 amounts from A (5 min Ang II treatment) were quantified via de nsitometric analysis and expressed as a fold change relative to unstimulated controls. This figure is used with permission from (42). capable of being observed without causing ce ll lethality (data not shown). Reprobing 36A with anti-ERK1/2-Abs conf irmed that total ERK1/2 protein levels were similar in control siRNA and PKC siRNA transfected cells. A dditionally, a knockdown of PKC was only observed in cells transfected with PKC -specific siRNA, and did not affect other PKC isoforms including PKC These data further demonstrate that PKC partially

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52 C0 5 10 0 5 10 Ang II (min) PKC siRNA WT/AT1---+ + + + + + --ControlsiRNADWB: ERK1/2(P)-pAbs WB: ERK1/2-Abs 61 kDa 36 kDa 36 kDa ERK2(P) ERK2 PKC % Maximum ERK2 PhosphorylationAng II ++ + PKC siRNA 0 20 40 60 80 100 120 WT/AT1+ControlsiRNA ** 79 kDa PKC WB: PKC -mAb WB: PKC -mAb A B C0 5 10 0 5 10 Ang II (min) PKC siRNA WT/AT1---+ + + + + + --ControlsiRNADWB: ERK1/2(P)-pAbs WB: ERK1/2-Abs 61 kDa 36 kDa 36 kDa ERK2(P) ERK2 PKC % Maximum ERK2 PhosphorylationAng II ++ + PKC siRNA 0 20 40 60 80 100 120 WT/AT1+ControlsiRNA ** 79 kDa PKC WB: PKC -mAb WB: PKC -mAb A B Figure 3-6. PKC -specific siRNA attenuated Ang IIinduced ERK1/2 activation in WT/AT1 cells. A: WT/AT1 cells were transfected with either PKC siRNA or control siRNA. All cells were then treated with 100 nM Ang II for 0, 5, 10 min, and ERK1/2 activation assessed vi a Western blot analysis with the indicated antibodies (first panel). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (second panel). PKC protein knockdown was confirmed by Western blotting with the indica ted antibody (third panel). PKC protein levels were demonstrated by reprobi ng with the indicated antibody (fourth panel). D: The percentage of maxi mum ERK2 phosphorylation from C was determined by dividing by ERK2 activ ation in Ang II and control siRNA treated cells, and multiplying by 100. This figure is used with permission from (42). mediates Ang II-induced ERK1/2 activation. PKC Mediates MEK1/2 Activation Independent of Src Kinases Finally, whether PKC is acting upstream of MEK1/2 in order to activate ERK1/2 independent of c-Src/Yes/F yn was investigated. SYF/AT1 cells were pretreated with PKC MP and then stimulated with Ang II fo r 0, 5 and 10 min. Whole cell lysates were

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53 then Western blotted with anti-phosphospeci fic MEK1/2 antibodies in order to assess MEK1/2 activation. SYF/AT1 cells not pre-treat ed with the PKC MP exhibited an Ang II-dependent increase in MEK2 activation (F igure 3-7). However, Ang II-dependent MEK2 activation was absent from SYF/AT1 cells treated with PKC pseudosubstrate. Thus, PKC appears to be acting upstream of ME K in order to activate ERK1/2 in SYF/AT1 cells. 0 5 10 0 5 10 ---+ + + Ang II (min) PKC MP SYF/AT1IB: MEK1/2(P)-pAbs IB: MEK1/2-pAbs MEK2(P) MEK2 49 kDa 49 kDa 0 5 10 0 5 10 ---+ + + Ang II (min) PKC MP SYF/AT1IB: MEK1/2(P)-pAbs IB: MEK1/2-pAbs MEK2(P) MEK2 49 kDa 49 kDa Figure 3-7. MEK phosphorylati on is dependent upon PKC Cells were pretreated with either 1 M PKC MP or DMSO for 1 hr, and then stimulated with 100 nM Ang II. Whole cell lysates were West ern blotted with anti-phospho-MEK1/2 antibodies. The membrane was then st ripped and reprobed with anti-MEK1/2 antibodies in order to de monstrate equal protein lo ading. This Western is representative of three i ndependent experiments. Th is figure is used with permission from (42). ERK1/2 Nuclear Translocat ion Is Dependent Upon PKC In Response to Angiotensin II Collectively, these data thus far indica te that Ang II-induced ERK1/2 activation occurs via c-Src/Yes/Fyn-dependent and heterotrimeric G protein/PKC -dependent signaling. Currently, the functional conseque nce of having two independent mechanisms of ERK1/2 activation in response to Ang II-induced activation of the AT1 receptor is not well understood. Previous reports have shown that ERK1/2 translocates into the nucleus and initiates gene transcri ption of early response gene s via the phosphorylation of specific transcription factor targets (98). Other work has shown that ERK1/2 nuclear

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54 translocation is dependent upon heterotrimeric G protein sign aling in response to Ang II (122). In Chapter 2, it is demonstrated that a loss of c-Src/Yes/Fyn does not affect Ang II-induced ERK1/2 nuclear translocation, but re sults in a reduction in Ang II-induced cell proliferation. ERK1/2 nuclear transl ocation may therefore be controlled by heterotrimeric G protein/PKC signaling, leading to the induc tion of cell proliferation. The effect of blocking heterotrimeric G protein/PKC signaling on both Ang II-induced ERK1/2 nuclear translocation and cell proliferatio n was next examined through inhibition of the PKC -dependent signaling pathway using the PKC MP. Ang II-dependent ERK1/2 nuclear tran slocation was examined in SYF/AT1 cells in the presence or absence of PKC MP. These cells are only able to activate ERK1/2 and induce cell proliferation in a Src kinase independent manner (Chapter 2), allowing one to test the effect of PKC inhibition on Src kinase-i ndependent ERK1/2 nuclear translocation and Ang II-induced cell proliferation. SYF/AT1 cells were transfected with a GFP-ERK2 plasmid in order to track ERK2 movement in response to Ang II treatment. Cells were pretreated with either DMSO or PKC MP for 1 hour, and then stimulated with 100 nM Ang II. Cells were then fixed, and DAPI stained to visualize the nucleus. In the absence of Ang II, GFP-ERK2 was dist ributed fairly evenly between the nucleus and cytoplasm of the SYF/AT1 cells (Figure 3-8A). DAPI c ounterstain of figures 3-8A and merging of the GFP and DAPI images conf irmed these findings (Figure 3-8F). In contrast, ERK1/2 accumulation was markedly increased in the nucleus of SYF/AT1 cells treated with Ang II (Figure 3-8B & 3-8C ), and this finding was confirmed by DAPI counterstain (Figure 3-8G a nd 3-8H). ERK2 nuclear tr anslocation was blocked in SYF/AT1 cells pretreated with PKC MP (Figure 3-8D and 3-8E), and merging of GFP

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55 and DAPI images confirmed this reduction in Ang II-induced ERK2 nuclear translocation (Figure 3-8I and 3-8J). Quan tification of nuclear fluoresce nce relative to cytoplasmic fluorescence revealed that Ang II-induced G FP-ERK2 nuclear translocation was blocked in the presence of PKC MP (Figure 3-8K). Thus, ER K1/2 nuclear translocation in response to Ang II is influenced by heterotrimeric G protein/PKC signaling, while cSrc/Yes/Fyn also mediate ERK1/2 activa tion but do not influence ERK1/2 nuclear translocation. Cell Proliferation Is Attenuated Through Inhibition of PKC Signaling In Response to Angiotensin II Ang II-induced ERK1/2 activation has been sh own to initiate cell proliferation (33, 89, 90, 133). It has primarily been thought that this occurs through the translocation of ERK1/2 into the nucleus and the subseque nt initiation of gr owth response gene transcription (122). It has been demons trated that Ang II-induced ERK2 nuclear translocation is unaffected by the loss of c-Src/Yes/Fyn-mediated ERK1/2 activation (Chapter 2). The effect of elimin ating heterotrimeric G protein/PKC signaling on Ang II-induced cell proliferation was next investig ated, independent of the ability of ERK1/2 to translocate into the nucleus. Both WT/AT1 and SYF/AT1 cells were pretreated with PKC MP and then stimulated with 100 nM Ang II. Changes in cell number were assessed via a direct cell count. Ang II-induced cell prolif eration was reduced in WT/AT1 cells pretreated with PKC MP when compared to DMSO-treated cont rols (Figure 3-9). Interestingly, Ang IIinduced cell proliferation was not significantly different in PKC MP pretreated WT/AT1 cells and SYF/AT1 cells stimulated with A ng II, suggesting that PKC -dependent signaling mediates the portion of Ang II-indu ced cell proliferation not controlled by Src

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56 kinases. Finally, Ang II-induced cell prolif eration was completely blocked in SYF/AT1 cells pretreated with PKC MP. These data collectiv ely suggest that both PKC and cSrc/Yes/Fyn mediate Ang II-induced cell prolif eration through the ac tivation of ERK1/2, though the mechanisms by which this occurs appear to be different since ERK1/2 Figure 3-8. Nuclear translocation of active ERK2 is controlled by PKC -dependent signaling. A F: SYF/AT1 cells were transfected with a GFP-ERK2 plasmid and then stimulated with 100 nM Ang II for 0 and 10 min. Nuclear translocation of ERK2 was assessed by fluorescent microscopy. A C: GFPERK2 images in cells treated with 1 00 nM Ang II for 0 and 10 min. D & E: GFP-ERK2 images in cells pretreated with 1 M PKC MP or DMSO (1 hr), then stimulated with 100 nM Ang II. F J: Merging of images A – E respectively with DAPI stained images. K: Nuclear fluorescence from A E was quantified and normalized to cytopl asmic fluorescence. All images are representative of the entire field a nd were taken at 40X magnification. Bar represents 15 microns. Shown is one of two independent results. This figure is used with permission from (42).

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57 Fold Change in Cell Number WT/AT1 SYF/AT1Ang II -+ -+ -+ -+ 0 0.5 1 1.5 2 2.5 3 3.5 4 ** NS PKC MP --+ + --+ + ** Figure 3-9. Ang II-induced cell proliferati on is completely attenuated by blocking cSrc/Yes/Fyn and PKC -dependent signaling. WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 24 hr. Some cells were pretreated with 1 M PKC MP for 1 hr. All cells were th en detached and counted using a hemacytometer. All data are the mean of three independent experiments. This figure is used with permission from (42). translocates into the nucleus in response to activation by PKC and remains in the cytoplasm when activated by c-Src/Yes/Fyn. Discussion A diverse set of signaling pathways have been implicated in ERK1/2 activation, but the precise mechanisms of ERK1/2 activa tion in response to Ang II are not fully understood (4, 45, 69, 70, 85, 86, 89, 109, 115, 117, 140, 153). It appears that ERK1/2 activation occurs via multiple signaling mech anisms. However, the cellular outcome associated with the activation of ERK1/2 via different signaling cascades is in question. Are these signaling pathways functionally re dundant, or does the activation of ERK1/2 by one pathway result in a different cellular outcome than when ERK1/2 is activated by another signaling cascade?

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58 Here, SYF/AT1 MEF cells were utilized in order to identify the mechanism underlying Src kinase-independent ERK1/2 activation in response to Ang II as well as a functional consequence for activa ting ERK1/2 in this manner. The advantage of utilizing these cells is that ERK1/2 ac tivation was examined in a Sr c kinase-deficient background, eliminating all Src kinase function and the possibility that ERK1/2 activation can be mediated via any of these very similar fa mily members. It was found that while cSrc/Yes/Fyn tyrosine kinases do play a role in the activation of ERK1/2 as previously reported (54, 115, 140), ERK1/2 activation is not completely dependent on these proteins and persists at reduced levels in their absence. Interest ingly, c-Src/Yes/Fyn are capable of activating only about 50% of intracellular ERK1/2. This seems to be a generalized phenomenon in other cells types as we ll, including CHO and RASM cells. An explanation for these results is that the remaining 50% of intracellular ERK1/2 are activated by c-Src/Yes/Fyn-independent mechan isms. This was subsequently confirmed since it was found that about 50% of A ng II-induced ERK1/2 activation involved heterotrimeric G protein and PKC signaling. In summary, Ang II-induced ERK1/2 activation occurs via two specific mechanisms th at work independent of one another. While both pathways activate an equal por tion of ERK1/2 and contribute to cell proliferation, the mechanism whereby each pa thway independently mediates this effect appears to be different. It ha d previously been thought that ERK1/2 must translocate into the nucleus in order to initiate events necessa ry for cell proliferation to occur, including the transcription of early response genes such as c-fos (14, 15, 109). Interestingly, the results in this Chapter indicate that the loss c-Src/Yes/Fyn had no eff ect on the ability of ERK1/2 to translocate into the nucleus. ER K1/2 was able to enter the nucleus in the

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59 absence of c-Src/Yes/Fyn; however, cell prol iferation was still markedly reduced. An explanation for these findings is that ERK1/2 activated via c-Src/Yes/Fyn-dependent signaling acts upon cytoplasmic proteins to mediate proliferation, while ERK1/2 activated via PKC -dependent signaling tran slocates into the nucleus to directly mediate transcriptional events (14). A mechanism describing how c-Src/Yes/Fyn-activated ERK1/2 initiates Ang II-induced cell prolifera tion is described in the next chapter. The utility of having two mechanisms that dually activate ERK1/2 in response to stimulation of the AT1 receptor by Ang II is nonetheless intriguing. Both c-Src/Yes/Fyn and heterotrimeric G protein-dependent si gnaling appear to occur simultaneously in response to Ang II, and exhibi t an additive effect on ERK1/2 activation and subsequent cell proliferation in respons e to Ang II. Within adult mammalian systems, Ang IIinduced cell proliferation is associated with abnormal cell proliferation during cardiovascular diseases and can cer, and to date has not been implicated in cell growth and proliferation during a nondisease state (24, 140, 145, 157-159). As such, this study may have therapeutic merit since local inhi bition of both heterotr imeric G protein/PKC signaling as well as c-Src/Yes/ Fyn-dependent signaling may be necessary in order to completely block Ang II-induced cell proliferation duri ng disease states.

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60 CHAPTER 4 ERK1/2 REGULATES ANGIOTENSIN II-D EPENDENT CELL PROLIFERATION VIA THE CYTOPLASMIC ACTIVATION OF RSK2 AND NUCLEAR ACTIVATION OF ELK1 Introduction In the previous chapters, ERK1/2 activati on is shown to be mediated independently by either Src family tyrosine kinase-dep endent signaling or heterotrimeric G protein/PKC -dependent signaling in tw o different cell types. Both of these signaling mechanisms accounted for roughly 50% of Ang II-induced ERK1/2 activation. Interestingly, heterotrimeric G protein/PKC signaling regulates ERK1/2 nuclear translocation, while c-Src/Yes/Fyn-dependent signaling influences cytoplasmic ERK1/2 activation in response to Ang II. Furthermore, these two pathways were both implicated in Ang II-induced cell prolifer ation, with inhibition of both signaling cascades necessary in order to achieve complete attenuation of Ang II-induced cell proliferation. These data were very striking since prev ious reports had shown that ERK1/2 nuclear translocation was a critical step in initiating the transc ription of early response genes such as c-fos which promotes cell prolifera tion (109). However, it appears here that cytoplasmic ERK1/2, under the control of c-Src/Ye s/Fyn, also mediates Ang II-induced cell proliferation independent of nuclear ERK1/2. One explanation for how cytoplasmic ERK1 /2 can influence early response gene transcription is that it phosphorylates cytopl asmic substrates, which in turn translocate into the nucleus and regulate transcriptional activity. Members of the Ribosomal S6 kinase (RSK) family of proteins are we ll-known cytoplasmic targets of ERK1/2, and

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61 have been shown to promote the transcri ption and translation of selected mRNAs important for cell growth (57). Previous reports have show n that ERK1/2 activates RSK family proteins, however it is not clear if these proteins represen t a possible pathway by which Ang II-activated ERK can initiate the events leading to cell proliferation (110, 128, 132). In this chapter, the mechanisms whereby ERK1/2 activated by either c-Src/Yes/Fyn or heterotrimeric G proteins/PKC -dependent signaling gene rates the proliferative response associated with AT1 receptor activation will be defined. It was hypothesized that c-Src/Yes/Fyn-activated ERK1/2 medi ates Ang II-induced cell proliferation through RSK, whereas heterotrimeric G protein/PKC signaling regulates cell proliferation through control of ERK1/2 nuclear transloc ation and subsequent elk1 activation. To examine this, WT/AT1 and SYF/AT1 cells or CHO/AT1 and CHO/AT1-M5 cells were utilized. Each of these cell lines have been stably transfected with the AT1 receptor and were the same as those utilized in previous chapters. It was found that ERK1/2, activated via c-Src/Yes/Fyn-dependent signaling, phosphor ylates ribosomal S6 kinase 2 (RSK2), which subsequently translocat es into the nucleus and modul ates c-fos activity at the transcriptional and post-translational levels. These events partially mediate cell proliferation since pretreatment with SL 0101, a potent and specific inhibitor of RSK, significantly attenuated Ang II-induced cel l proliferation. ERK1/2 activated by heterotrimeric G protein/PKC signaling localizes to the nucleus, where it phosphorylates the transcription factor elk1 and regulates c-fos transcription. Together with ERK1/2RSK signaling, these events mediate Ang II-induced cell proliferation. As such, this study demonstrates that the AT1 receptor coordinately uti lizes both hetrotrimeric G

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62 protein and Src family tyrosine kinase si gnaling to achieve a co mmon cellular outcome via two different mechanisms acting in distinct cellular compartments. Materials and Methods Antibodies and Pharmacological Inhibitors A cocktail of phospho-specific ERK1/2 antibodies (Promega and Santa Cruz Biotechnologies) were used in order to incr ease signal to noise ratio. Both of these antibodies are specific for phospho-threonine 202 and phospho-tyrosine 204 within the conserved TEY motif. The phosphospecific RSK polyclonal antibody ( RSK(P)-pAb) was purchased from Cell Signaling Technologi es and recognizes the phospho-threonine 356/phospho-serine 360 motif. The phospho-sp ecific SRF antibody [SRF(P)] recognizes the phosphorylated Ser 103 residue; the phos pho-specific elk1 [e lk1(P)] monoclonal antibody recognizes phospho-serine 383. Both of these antibodies were purchased from Cell Signaling Technologies. The RSK1 antibody ( RSK1-pAb) and the RSK2 antibody ( RSK2-pAb) were obtained from Santa Cruz Biotechnology. A cocktail of ERK1/2 antibodies ( ERK1/2-Abs) were used to measure total ERK1/2 protein levels. This cocktail consisted of ERK1/2 monoclonal a nd polyclonal antibodies from Santa Cruz Biotechnology. The ERK1/2 monoclonal antibody was used separately for immunofluorescence. The Tyr(P) mAb (PY20) was from BD Transduction Laboratories. The c-fos polyclonal antibody ( cfos-pAb) was from Santa Cruz Biotechnology. The phospho-se rine polyclonal antibody [ Ser(P)-pAb)] was purchased from AnaSpec, Inc. The SL0101 compound was purchased from Toronto Research Pharmaceuticals. The PKC myristoylated pseudosubstrate (PKC MP) was purchased from Biomol Laboratories. PP2 a nd PD98059 compounds were obtained from Calbiochem. Leptomycin B (LMB) was purchased from Sigma.

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63 Cell lines and Cell Culture The Chinese hamster ovary cell lines (CHO/AT1-WT and CHO/AT1-M5 cells) were a gift from Dr. Kenneth Bernstein (Emory University), and have an equal abundance of AT1 receptor as well as affinity for Ang II (28). The WT/AT1 and SYF/AT1 mouse embryonic fibroblast cells are described in Chap ter 2. VSMC cells were cultured in the same media, but without Zeocin. WT/AT1, SYF/AT1, and VSMC were growth arrested in serum-free DMEM fo r 48 h prior to experiments. Cell Lysate Preparation, Immunop recipitation and Western Blotting For Westerns, cells were lysed in radi oimmune precipitation assay (RIPA) buffer containing protease inhibitors as descri bed in Chapter 2. Cell lysates were immunoprecipitated where indicated exactly as described in Chapter 2. Proteins were detected using enhanced chemiluminescence exactly as described in Chapter 2 (117). Densitometric Analysis Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk Scientific) as described in Chapter 2. The average pixel value minus background was obtained for each cell type and normalized to the average pixel value for the respective non-Ang II-treated cells. Immunofluorescence WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (Lab-Tek) and grown to 70% confluency. Cells were seru m-starved in serum free DMEM supplemented with BSA for 48 hr. Following starvation, all cells were ligand-treated with 100 nM Ang II. Slide chambers were removed from slid es, and cells were washed one time with PBS (pH 7.4). Cells were fixed in 4% para formaldehyde (Fisher) for 10 min at room temperature, rinsed in PBS and permeabilized for 3 min in acetone (Fisher) at -20oC.

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64 Cells were rinsed three times in PBS, and blocked for 15 min in 3% BSA/PBS at room temperature in a homemade hydration chambe r to prevent evaporation. Cells were incubated with the primary antibody indicat ed for each experiment (1:500 in 3% BSA/PBS) for 1 hr. All cells were next rinsed three times in PBS. Cells were incubated with the appropriate fluorochrome-conj ugated secondary antibody (1:100 in 3% BSA/PBS) for 1 hour at room temperature in a hydration chamber. In the case of the RSK2-pAb, the rabbit IgG-FITC secondary anti body (Sigma) was used. For the ERK2-mAb and elk1(P)-mAb, the mouse IgG-Texas Red secondary antibody (Sigma) was used, while Goat IgG-FITC secondary antibody (Santa Cruz) was utilized in conjunction with the SRF(P)-pAb. Upon completion of incubation with the appropriate secondary antibody, ce lls were washed three times in PBS. A coverglass was mounted to each slide using Vectashield + DAPI mounting medium (Vector Labs). The edges of the slides were sealed with nail polish sealant (Maybelline LLC) and allowed to dry. All dry slides were stored at -20oC until viewed. Slides were viewed on a Zeiss Axioplan II Fluorescence microscope. Quantification of nuclear and cytoplasmic fluorescence Fluorescence in the nucleus or cytoplasm was quantified using the Image J Progam (NIH) as described in Chapter 2. c-fos transcriptional activity SREw/Luc, mSRF/Luc, mTCF/Luc and TK/L uc plasmids were kindly provided by Dr. Jessica Schwartz (University of Michigan) (71). WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (LabTek) and transiently-transfected with each individual plasmid using 8 L lipofectin (I nvitrogen). All transfected cells were incubated for 5 hr at 37oC. The transfection was stopped by washing cells in PBS, and

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65 incubating the cells in serum-containing DMEM for 12-16 hrs. Cells were serum-starved in DMEM + BSA (0.5% wt/vol) for 12-16 hrs, and treated with 100 nM Ang II for the amount of time indicated in each experiment. Cells were placed in 1X Reporter Lysis Buffer (Promega), and exposed to one -80oC freeze/thaw cycle (30 mi n each) to aid in the disruption of cell membrane integrity. Cells were placed on a shaker at room temperature for one additional hour to ensure complete lysi s, and then transferred to a microcentrifuge tube. Cells were centrifuged at 12,000 x g for 2 min at 4oC, and 20 L of cell lysate was combined with 100 L luciferin substrate (Pro mega). Luciferase activity was measured in a Monolight 3010 Luminometer (PharMingen) at 10 sec intervals. Cell Migration Assay Cell migration assays were performed using a commercially available Cell Migration Kit (BioLabs). VSMCs were grown in 6 well cell culture plates. Cells were detached, and placed in the upper chamber of a migration apparatus in serum-free DMEM. The lower chamber of the apparatus was filled with serum-free DMEM. Cells were then stimulated with 100 nM Ang II in the presence of SL0101 or vehicle for 24 hr, and allowed to migrate from the upper chamber through a nylon membrane and into the lower chamber. The membrane was wash ed, stained and photographed to visualize migratory cells. The membrane was then pla ced in destaining solu tion and migratory cell number was quantified indirectly via spectrophotometry at absorp tion 595 nm. Cell Count WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to 80% confluency. Cells were serum-starved a nd treated with 100 nM Ang II as indicated. Both cell types were then counted usi ng a hemacytometer as described (122).

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66 Statistical Analysis Data were analyzed by two-way ANOVA. All data passed a Normality Test as well as Equal Variance Test. Pairwise comparisons were made following the HolmSidak method. All data are expressed as mean +/-SEM. = p<0.05, ** = p<0.01. Results RSK Phosphorylation and ERK1/2-RSK Co -association Are Dependent Upon Src Kinases in Response to Angiotensin II The data in Chapter 2 suggest that ER K1/2 activated by c-Src/Yes/Fyn acts upon cytosolic proteins to promote Ang II-dependent cell proliferation. It was first examined whether or not the phosphorylation of RS K, a well known cytoplasmic substrate of ERK1/2 (109, 128, 132), was dependent upon cSrc/Yes/Fyn and ERK1/2 signaling. WT/AT1 and SYF/AT1 cells were stimulated with Ang II, and RSK activation assessed via Western blot using a phos pho-specific RSK antibody that recognizes phosphorylated RSK1, RSK2 and RSK3. RSK activation occurred in response to Ang II in WT/AT1 cells and was maximal after 10 min of Ang II treatment and declined thereafter (Figure 4-1A and data not shown). However, Ang II-induced RSK activation was completely absent from SYF/AT1 cells, indicating that cSrc/Yes/Fyn are necessary for the activation of RSK in response to Ang II. These results were recapitulated by pretreating WT/AT1 cells with PP2, a Src kinase inhib itor (Figure 4-1B). In addition, Ang II-induced RSK phosphorylation in WT/AT1 cells was dependent upon ERK1/2 activity since pretreatment with the ERK activ ation inhibitor, PD98059, attenuated RSK activation in these cells (Figur e 4-1C). RSK activation in re sponse to Ang II is therefore mediated by Src kinases and ERK1/2.

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67 Figure 4-1. RSK2 phosphoryla tion and RSK2-ERK1/2 co-ass ociation are decreased in SYF/AT1 cells. A: WT/AT1 cells and SYF/AT1 cells were stimulated with 100 nM Ang II for 0, 5, and 10 min and total RSK phosphorylation assessed via Western blot (WB) using the indi cated antibody (top). Total protein loading was demonstrated by stripp ing the membrane and reprobing for ERK1/2 (bottom). WT/AT1 cells were pretreated with either 30 M PP2 (B) or 50 M PD98059 (C) for 30 min, and stimulated with 100 nM Ang II for 0, 5, and 10 min. Total RSK phosphorylat ion was assessed by Western blot using the indicated antibody (top). Tota l protein loading was demonstrated by stripping the membrane a nd reprobing for ERK1/2 (bottom). D: The presence or absence of RSK1 and RSK2 in cells was examined by Western blotting WT/AT1 and SYF/AT1 whole cell lysates with the indicated antibodies (top). Control MDCK and NIH3T3 whole cell lysa tes were also run on the same gel. Total protein loading was demonstrated by stripping the membrane and reprobing for ERK1/2 (bottom). E: Active RSK2-ERK co-association was assessed in WT/AT1 and SYF/AT1 whole cell lysates by immunoprecipitating (IP) and Western blotting w ith the indicated antibodies (top). Total protein loading was demonstrated by blotting whole cell lysates with the indicated antibodies (bottom). All Westerns are representative of at least three independent blots. This figure is used with permission from (43).

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68 The specific isoform(s) of RSK phosphoryl ated in response to Ang II within the WT/AT1 and SYF/AT1 cells was next determined. Of the three known RSK isoforms, only RSK1 and RSK2 are phosphorylated by ER K1/2 (37). Whole cell lysates from WT/AT1 and SYF/AT1 cells were prepared and Wester n blotted with RSK1 or RSK2 specific antibodies alongside positive control whole cell lysates. RSK1 was not expressed in WT/AT1 cells, but was expressed in SYF/AT1 cells (Figure 4-1D). These expression levels did not change with the a ddition of Ang II (data not shown). However, RSK2 was expressed equally in both cell type s. Therefore, RSK2 is most likely the isoform phosphorylated in the presence of cSrc/Yes/Fyn. As an aside, c-Src/Yes/Fyn may regulate RSK1 protein degradation si nce RSK1 was present in c-Src/Yes/Fyn deficient cells, but absent from cells containing these proteins. Previous reports have show n that active ERK1/2 bind RSK proteins via the ERK docking site and, once bound, modulate RSK activity (121, 128, 132). It was next determined whether RSK and ERK1/2 co-assoc iation is controlled by c-Src/Yes/Fyn, in order to determine if the ERK1/2 activated by c-Src/Yes/Fyn binds to RSK. WT/AT1 and SYF/AT1 whole cell lysates were immunoprecipi tated with a phospho-specific ERK1/2 antibody, and then Western blotted with th e phospho-specific RSK antibody. ERK1/2RSK co-association was evident after 5 min of Ang II treatment in WT/AT1 cells, but was completely absent from SYF/AT1 cells (Figure 4-1E). As such, these data demonstrate that ERK1/2 and RSK co-associate in respons e to Ang II, and this is c-Src/Yes/Fyndependent.

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69 RSK Nuclear Translocation Is Src Kinase Dependent, While ERK1/2 Nuclear Translocation Is PKC Dependent in Response to Angiotensin II Phosphorylated RSK has been shown to tr anslocate into the nucleus and modulate the transcriptional activity of target genes in response to stimulati on of various cytokine and growth factor receptors (16, 162). It was next determined whether RSK nuclear translocation occurs in response to Ang II, and whether this event is regulated by cSrc/Yes/Fyn. WT/AT1 and SYF/AT1 cells were pretreated with leptomycin B (LMB) to prevent the nuclear exportation of proteins and then stimulated with Ang II. RSK nuclear accumulation was then assessed by immu nofluorescence. Translocation of RSK2 into the nucleus occurred in res ponse to Ang II treatment in WT/AT1 cells, and the nuclear accumulation of RSK2 was confirme d by merging the RSK2 image with DAPI (Figures 4-2A & 4-2B, 4-2E & 4-2F). However, Ang II-induced RSK2 nuclear translocation did not occur in the absence of c-Src/Yes/Fyn (Figures 4-2C & 4-2D, 4-2G & 4-2H). Pretre atment of WT/AT1 cells with PP2, a Src family kinase inhibitor, also prevented Ang II-induced RSK2 nuclear accumu lation (Figures 4-2I & 4-2J, 4-2K & 42L). Quantification of nuclear and cyt oplasmic fluorescence confirmed that RSK2 nuclear translocati on occurred in WT/AT1 cells stimulated with Ang II, but was blocked in SYF/AT1 cells or in WT/AT1 cells pretreated with PP2 (Figure 4-2M). Collectively, these data show that Ang II-induced RSK nuclear transl ocation is regulated by cSrc/Yes/Fyn. Ang II-induced ERK1/2 nuclear transloca tion and RSK2 nuclear translocation patterns were next assessed in WT/AT1 and SYF/AT1 cells. All cells were pretreated

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70 WT/AT1SYF/AT1Ang II -+ -+ LMB + + + + PP2 + + WT/AT1SYF/AT1 Ang II -+ -+ LMB + + + + AB CD EF GH I J KL NOPQ RSTU V V WX RSK2 RSK2/DAPI Merged RSK2 RSK2/DAPI Merged ERK1/2 RSK2/ERK1&2/ DAPI Merged H 0 10 20 30 40 50 60 Ang II -+ -+ -+ PP2 ----+ +L 0 10 20 30 40 50 60 70 Ang II -+ -+ J Y RSK2ERK1/2 fluorescence (Nucleus/Cytoplasm) RSK2 fluorescence (Nucleus/Cytoplasm) ** ** ** NSM Z WT/AT1 SYF/AT1 WT/AT1 SYF/AT1 Figure 4-2. RSK2 and ERK1/2 nuclear phosphor ylation in response to Ang II in WT/AT1 and SYF/AT1 cells. A-L, N-Y: WT/AT1 and SYF/AT1 cells were pretreated with 0.005 g Leptomycin B for 5 min prior to stimulation with 100 nM Ang II for 10 min. All cells were incubate d with antibodies specific for the indicated proteins (right ) and respective fluorochrome-conjugated secondary antibody. E-H, K, L, V-X: Images were DAPI stained and merged with the respective fluorescent prot ein images. I-L: WT/AT1 cells were pretreated with 20 M PP2 for 1 hr prior to stimul ation with 100 nM Ang II. M: Nuclear fluorescence from A L was quantifie d and normalized to cytoplasmic fluorescence. Z: Nuclear fluorescence from R U was quantified and normalized to cytoplasmic fluorescence. All images are representative of the entire field and were taken at 40X ma gnification. Bar represents 15 microns. This figure is used with permission from (43). with LMB and then stimulated with Ang II. RSK2 and ERK1/2 nuclear translocation were then assessed via immunofluorescence. Bo th RSK2 and ERK1/2 translocated into the nucleus in response to Ang II in WT/AT1 cells (Figures 4-2N & 4-2O, 4-2R & 4-2S). Merging of RSK2 and ERK1/2 images c onfirmed that RSK2 and ERK1/2 nuclear

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71 translocation patterns were the same in these cells (Figures 4-2V and 4-2W). However, nuclear translocation of ER K1/2 persisted in SYF/AT1 cells while RSK2 nuclear translocation was attenuated (Figures 4-2P and 4-2Q, 4-2T and 4-2U). Merging of the ERK1/2 and RSK2 images confirmed that thes e two proteins exhibit different patterns of nuclear translocation in SYF/AT1 cells (Figures 4-2X and 4-2Y). Quantification of nuclear and cytoplasmic fluores cence revealed that GFP-ERK2 nuclear translocation was similar in WT/AT1 and SYF/AT1 cells (Figure 4-2Z). As such, these data demonstrate that ERK1/2 nuclear transl ocation is regulated by PKC -dependent signaling whereas RSK2 nuclear translocation is controlled by c-Src/Yes/Fyn. SRF and TCF Binding Within the c-fos Promoter Are Mediated in A RSK And A ERK1/2-dependent Manner, Respectively c-fos transcription is regulated via the bindi ng of specific transc ription factors to the serum response element (SRE) within the c-fos promoter, namely the serum response factor (SRF) and ternary complex fact or (TCF) (71, 78, 84, 162). RSK has been implicated in the phosphorylation of the SR F while ERK1/2 have shown to phosphorylate TCF proteins, thereby increas ing the activity of these tr anscription factors (78, 84, 142, 162). However, the roles of the SR F and the TCF during Ang II-induced c-fos transcription ar e still unknown. Whether SRF and TCF activity are regulated by either PKC -dependent or cSrc/Yes/Fyn-dependent signali ng in response to Ang II was next examined. WT/AT1 and SYF/AT1 cells were stimulated with Ang II, and SRF or TCF nuclear phosphorylation assessed via immunofluorescence. Nuclear SRF phosphorylati on occurred in response to Ang II treatment in WT/AT1 cells (Figures 4-3A and 4-3B, 4-3F and 4-3G). Ang IIinduced SRF phosphorylation was completely lost in SYF/AT1 cells stimulated with Ang

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72 II (Figures 4-3D and 4-3E, 4-3I and 4-3J ). In addition, pret reatment of WT/AT1 cells with PKC MP did not affect Ang II-induced SRF phosphorylation (Figures 3C and 3H). Quantification of nuclear and cytoplasmic fl uorescence revealed that Ang II-induced SRF phosphorylation in WT/AT1 cells was not affected by PKC MP pre-treatment, while Ang II-induced SRF phosphorylation did not occur in SYF/AT1 cells (Figure 4-3Q). Thus, SRF phosphorylation is dependent upon c-Src/Yes/Fyn-dependent signaling and not PKC The nuclear phosphorylation of elk, a TCF ac tivated in response to ERK1/2, was assessed in the same manner. Elk1 phosphoryl ation occurred in response to Ang II in WT/AT1 cells (Figures 4-3K and 4-3L, Figures 4-3N and 4-3O). In addition, Ang IIinduced elk1 phosphorylation persisted in SYF/AT1 cells (data not show n). Pretreatment with PKC MP attenuated elk1 nuc lear phosphorylation (Figur es 4-3M and 4-3P). Quantification of nuclear and cytoplasmic fl uorescence revealed that Ang II-induced elk1 phosphorylation in WT/AT1 cells was blocked by PKC MP pretreatment (Figure 4-3R). Thus, TCF phosphorylation app ears to be mediated by PKC -dependent signaling and not by c-Src/Yes/Fyn.

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73 Ang II -++ WT/AT1PKC MP --+ A ng II -++ -+ WT/AT1PKC MP --+ -SYF/AT1ABCD E F G HIJ KL M NOP SRF(P) SRF(P)/DAPI Merged elk1(P) elk1(P)/DAPI Merged 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 -10 **NS NSSRF(P) fluorescence (Nucleus/Cytoplasm) elk1(P) fluorescence (Nucleus/Cytoplasm)Ang II -+ + -+ PKC MP--+ -Ang II -+ + PKC MP--+QR ** ** WT/AT1 SYF/AT1 WT/AT1 Figure 4-3. Ang II-induced SRF and el k1 nuclear phosphorylation in WT/AT1 and SYF/AT1 cells. A-P: WT/AT1 or SYF/AT1 cells were stimulated with 100 nM Ang II for 10 min. All cells were inc ubated with antibodies specific for the indicated proteins (right) and thei r respective fluorochrome-conjugated secondary antibody. F-J, N-P: Protein fluorescence images were merged with DAPI stained images. C & H, M & P: Ce lls were pretreated with 1 M PKC MP for 1 hr prior to Ang II treatment. Q: Nuclear fluorescence from A E was quantified and normalized to cy toplasmic fluorescence. R: Nuclear fluorescence from K M was quantified and normalized to cytoplasmic fluorescence. These results are repr esentative of three independent experiments. All images are representati ve of the entire field and were taken at 40X magnification. Bar represents 15 microns. This figure is used with permission from (43). Both the SRF and TCF have been shown to modulate c-fos transcriptional activity in response to growth hormone treatment (71). Whether SRF and TCF binding of the c-

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74 fos SRE occurred in response to Ang II, and, if these events were a ltered by the absence of c-Src/Yes/Fyn, were next tested. WT/AT1 and SYF/AT1 cells were transiently transfected with wild-type or mutated SRE-luci ferase plasmids and then stimulated with Ang II. The wild-type SRE plasmid (SRE w/Luc) alone mediated Ang II-induced luciferase expression in both cell types (Figure 4-4). Ang II-induced luciferase activity was reduced by about 50% in SREw/Luc transfected SYF/AT1 cells relative to transfected WT/AT1 cells, indicating that c-fos transcriptional activity is in part dependent upon c-Src/Yes/Fyn. Furthermore, mutation of either the SRF or TCF binding sites only partially blocked Ang II-induced luciferase expression in WT/AT1 cells. In addition, Ang II-induced luciferase expre ssion was completely blocked in SYF/AT1 cells transfected with either the mSRF/Luc or mT CF/Luc plasmids. Note that the reporter plasmid alone (TK/Luc) consistently failed to respond to Ang II in both cell types. These data therefore indicate that the SRF and TCF transcription factors both partially modulate c-fos transcriptional activity in either a c-Src/Yes/Fyn or PKC -dependent manner, respectively. c-fos Protein Expression Is Dependen t Upon Src Kinase Signaling And PKC Signaling It was next determined if the inhibition of PKC -dependent and/or Src kinasedependent signaling reduced cfos protein levels. WT/AT1 and SYF/AT1 cells were stimulated with Ang II and c-fos protein leve ls assessed via Western blot. c-fos protein exhibited an Ang II-dependent increase in both cell types, and was maximal after 60 min of Ang II treatment (Figure 4-5A). Howe ver, Ang II-induced c-fos protein production was reduced in SYF/AT1 cells relative to WT/AT1 cells. Infact, there was about a 50% reduction in c-fos in SYF/AT1 cells compared to WT/AT1 cells (Figure 4-5B).

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75 0% 50% 100% 150% 200% SREw/Luc + WT/AT1SYF/AT1AngIILuciferase Activity (% of Control) mSRF/Luc + mTCF/LucTK/Luc + + SREw/Luc + mSRF/Luc + mTCF/LucTK/Luc + + * ** NS NS Figure 4-4. c-fos transcriptional activity in WT/AT1 and SYF/AT1 cells in response to Ang II. WT/AT1 or SYF/AT1 cells were transfected with the indicated plasmid and then stimulated with Ang II for 0 or 5 hr. Data are expressed as percentage of luciferase activity relative to unstimulated cells. These results are representative of three independent experiments. Th is figure is used with permission from (43). Pretreatment of WT/AT1 cells with PKC MP also reduced c-fos protein amounts by 50%, while PKC MP addition to Ang II-stimulated SYF/AT1 cells resulted in a complete loss of Ang II-induced c-fos protein producti on. These data demonstrate that Ang IIinduced c-fos protein synthesis is partially influenced by PKC and partially by Src kinase-dependent signaling. c-fos Phosphorylation Is Dependent Upon Src Kinase-RSK Signaling Previous reports have shown that c-fo s activity is not only regulated at the transcriptional level, but also post-transl ationally via specific phosphorylation events (16). Specifically, phosphorylation of c-fos by RSK at Ser residues w ithin the C-terminal tail increases the stability of c-fos a nd the subsequent growth-promoting effects associated with extended c-fos activity (16) Whether c-fos serine phosphorylation is

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76 dependent upon c-Src/Yes/Fyn mediated ERK/ RSK2 activation was next tested. WT/AT1 36 kDa 49 kDa 0 60 600 60 60 Ang II (min) SYF/AT1 WT/AT1WB: ERK2-pAb c-fos ERK2 % Maximum c-fos PhosphorylationAng II +-+ 0 20 40 60 80 100** WT/AT1 SYF/AT1PKC MP --+ --+ WB: cfos-pAb + + PKC MP --++ NSA B Figure 4-5. c-fos protei n levels in response to Ang II in WT/AT1 and SYF/AT1 cells. A: WT/AT1 or SYF/AT1 cells were stimulated with 100 nM Ang II for 0 or 60 min, and c-fos protein production and tota l protein levels assessed by Western blot using the indicated antibodies. So me cells were pretreated with 1 M PKC MP for 1 hr as indicated. B: These data are expressed as the percentage of maximum c-fos protein production in response to Ang II. Protein amounts were densitized and values were normali zed to the amount of c-fos protein in WT/AT1 cells treated with Ang II. These results are representative of three independent experiments. This figure is used with permission from (43). and SYF/AT1 cells were stimulated with Ang II and cell lysates were immunoprecipitated with anti-phosphoserine antibody and Western blotted with a c-fos specific antibody to assess for changes in c-fos phosphorylation at Se r residues. A marked four-fold increase in c-fos phosphorylation was observed in WT/AT1 cells after 60 min of Ang II treatment (Figure 4-6A). Phosphorylated c-fos levels remained elevated af ter 120 min of Ang II treatment, and began to dec line by 240 minutes. In cont rast, a comparatively small

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77 increase in c-fos phosphorylation wa s observed in Ang II-treated SYF/AT1 cells after 60 min, and phosphorylated c-fos levels declin ed to baseline amounts by 120 min. These results are displayed graphical ly, and were normalized to to tal c-fos protein amounts to account for differences in total c-fos protein (Figure 4-6B). Coll ectively, these data suggest that c-fos phosphorylation is reduced in the absence of c-Src/Yes/Fyn and subsequent ERK/RSK2 activati on in response to Ang II. Angiotensin II-induced Cell Prol iferation Requires RSK And PKC Activity The consequence of selective RSK inhi bition on Ang II-induced cell proliferation was next assessed since RSK activation (unde r the influence of Src kinase activated ERK1/2) appears to modulate c-fos levels and activity. Recently, a highly selective and potent RSK inhibitor, SL0101, was isolat ed by Lannigan and colleagues (129). Interestingly, this compound is a natural product derived from the foresta refracta plant found in the amazon rain forest. SL0101 has al ready been shown to selectively inhibit RSK without interfering with upstream activ ators of RSK like ERK, MEK, EGFR and PKC (77). Furthermore, this compound has be en shown to prevent cell proliferation in cancer cells and thus has established RSK as a target for therapeutic intervention and SL0101 as an anti-cancer agent (18). Howeve r, a role for RSK in Ang II-induced cell proliferation remains to be established. WT/AT1 and SYF/AT1 cells were stimulated with Ang II, and cell proliferation assessed via a direct cell count. A marked 3fold increase in cell number was observed in WT/AT1 cells treated with Ang II (Figure 47A). Ang II-induced increases in cell number were reduced by 1.5 fold in SYF/AT1 cells relative to WT/AT1 cells. Pretreatment with SL0101 reduced WT/AT1 cell number to levels found in SYF/AT1 cells treated with Ang II. However, SL0101 pr etreatment did not affect Ang II-induced

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78 Ang II (min) 0 60 120 240 0 60 120 240 IP: Ser(P)-mAb WT/AT1SYF/AT1 c-fos(P) 49 kDa WB: cfos-pAb Ang II (min)Fold change phosphorylatedc-fos (stimulated/total c-fos protein/ unstimulated) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 WT/AT1SYF/AT10 60 120 240** ** **A B Figure 4-6. Ang II-induced c-fos phosphorylation in WT/AT1 and SYF/AT1 cells. WT/AT1 or SYF/AT1 cells were stimulated with 100 nM Ang II for 0, 60, 120, or 240 min. A: c-fos phosphorylation was then assessed by immunoprecipitating and Western blotting with the indicated antibodies. B: Protein bands were densitized and values were expressed as a fold change in c-fos phosphorylation relative to unstimulat ed cells as a function of total c-fos protein amounts. These results are representative of three independent experiments. This figure is used with permission from (43). increases in SYF/AT1 cell number, suggesting that A ng II-induced cell proliferation occurring independent of c-Src/Yes/Fyn is not dependent upon RSK2 and also that SL0101 exhibits low toxicity. Finally, PKC MP pretreatment completely attenuated Ang II-induced increases in SYF/AT1 cell number but only partia lly attenuated Ang IIinduced increases in WT/AT1 cell number. Furthermore, the addition of PKC MP and SL0101 to WT/AT1 cells blocked all Ang II-induced increases in cell number. These data therefore demonstrate that PKC partially regulates Ang II-induced cell proliferation

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79 independent of RSK2. Furthermore, RSK2 mediates Ang II-induced cell proliferation downstream of c-Src/Yes/Fyn sin ce the addition of SL0101 to SYF/AT1 cells did not further lower the already reduced amount of Ang II-induced cell pro liferation exhibited by these cells. To recapitulate these findings in anothe r cell type, the effect of SL0101 addition on cells devoid of heterotrimeric G protein activation (CHO/AT1-M5 cells) was examined. A marked 3-fold increase in cell number was observed in CHO/AT1 cells treated with Ang II (Figure 4-7B). Ang II-induced incr eases in cell number were reduced by about 0.5 fold in CHO/AT1-M5 cells relative to CHO/AT1 cells. Pretreatment of CHO/AT1-M5 cells with SL0101 completely blocked Ang II-i nduced increases in cell number, whereas SL0101 partially attenuated cell number in CHO/AT1 cells stimulated with Ang II to levels found in Ang II-treated CHO/AT1-M5 cells. Thus, RSK2 does not regulate Ang IIinduced cell proliferation thr ough heterotrimeric G protein-dependent mechanisms. The pretreatment of CHO/AT1 cells with PKC MP partially reduced Ang II-induced cell proliferation; however, CHO/AT1-M5 cell number was not aff ected by the addition of PKC MP. Therefore, PKC appears to partially me diate Ang II-induced cell proliferation, and it does so dow nstream of heterotrimeric G proteins. Finally, all Ang IIinduced cell proliferation in CHO/AT1 cells was blocked by pretreatment with PKC MP and SL0101. Collectively, these data furt her demonstrate that Ang II-induced cell proliferation is regulated by heterotrimeric G protein/PKC and c-Src/Yes/Fyn/RSKdependent signaling

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80 0 0.5 1 1.5 2 2.5 3 3.512345678 WT/AT1 SYF/AT1Fold Increase in Cell NumberB A -+ -+--++--++--++ Ang II ---++++----++++ SL0101 -------++++++++ PKC MP 0 0.5 1 1.5 2 2.5 3 3.512345678 0 0.5 1 1.5 2 2.5 3 3.512345678 c-Src/Yes/Fyn ERK1/2 c-Src/Yes/Fyn ERK1/2 c-Src/Yes/Fyn ERK1/2 -Src/Yes/Fyn ERK1/2 c-Src/Yes/Fyn ERK1/2 c-Src/Yes/Fyn ERK1/2 c-Src/Yes/Fyn ERK1/2 AT1 -c-Src/Yes/Fyn ERK1/2 AT1 heterotrimeric G protein PKC ERK1/2 CHO/AT1 CHO/AT1M5 ** NS ** ** * *-+ -+--++--++--++ Ang II ---++++----++++ SL0101 -------++++++++ PKC MPFold Increase in Cell Number Figure 4-7. Ang II-induced cell prolifer ation in response to RSK and PKC inhibition. A: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 (-) or 24 (+) hr. Some cells were pretreated with either 30 M SL0101, 1 M PKC MP, or both inhibitors in combination for 1 hr as indicated. Cells were counted and fold changes in cell number plotted relative to unstimulated cells (left). The drawing on the right illustrates the intact endogenous ERK1/2 activation pathway in the SYF/AT1 cells. B: CHO/AT1 and CHO/AT1-M5 cells were stimulated with Ang II for 0 (-) or 24 (+) hr. Some cells were pretreated with either 30 M SL0101, 1 M PKC MP, or both inhibitors in combination for 1 hr as indicated. Cells were counted a nd fold changes in cell number plotted relative to unstimulated cells (left). The drawing on the right illustrates the intact endogenous ERK1/2 act ivation pathway in CHO/AT1-M5 cells. These results are representative of three inde pendent experiments. This figure is used with permission from (43).

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81 Angiotensin II-induced ERK1/2 Activatio n Is Mediated By Both Src Kinases and PKC in Vascular Smooth Muscle Cells Thus far, it has been demonstrated th at Ang II-induced ERK1/2 activation is mediated by Src kinase and he terotrimeric G protein/PKC signaling in both MEF and CHO cells. It was next determined if Ang II-i nduced ERK1/2 activation is also mediated by both c-Src/Yes/Fyn and hete rotrimeric G protein/PKC signaling in cells which endogenously express the AT1 receptor. Here, primary cultures of VSMCs isolated from rat aortas were utilized. c-Src/Yes/Fyn or PKC activity was then blocked using pharmacological inhibitors. VSMC were pretreated with either PP2, PKC MP or PKC MP and PP2 in combination. Cells were then stimulated with 100 nM Ang II for 0, 5 and 10 min. Whole cell lysates we re prepared and Western blotted with phospho-specific ERK1/2 antibodies to identify changes in ERK1/2 activation. Ang II-induced ERK1/2 activation occurred after 5 mi n of Ang II treatment in VSMC (Figure 4-8A). ERK1/2 activation was significantly reduced in VSMC treated with either PP2 or the PKC pseudosubstrate alone, and treatm ent with either of these i nhibitors alone resulted in about a 50% reduction in ERK2 phosphorylation (Figure 4-8B). Furthermore, ERK1/2 activation was completely reduced in cel ls treated with both PP2 and the PKC pseudosubstrate in combination. Collectively, th ese data confirm that the mechanisms of intracellular ERK1/2 activation are the same in the AT1 receptor-transfected MEF and CHO cells as in VSMCs which endogenously express the AT1 receptor. VSMC ERK1/2 activation occurs via c-Src/Yes/Fyn or heterotrimeric G protein/PKC -dependent signaling in response to angiotensin II.

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82 VSMC 0 5 10 0 5 10 0 5 10 0 5 10 Ang II (min) WB: ERK1/2(P)-pAbs WB: ERK1/2-Abs ---+ + + ---+ + + ------+ + + + + + PP2 PKC MP 36 kDa 36 kDa ERK2 ERK1(P) ERK2(P) % Maximum ERK2 PhosphorylationAng II ++++ + + PP2 PKC MP ++ 0 20 40 60 80 100 120 ** NS A B Figure 4-8. Ang II-induced ERK1/2 activation is mediated by c-Src/Yes/Fyn and PKC dependent signaling in VSMC. A: VS MCs were pretreated with either DMSO, 30 M PP2, 1 M PKC myristoylated pseudosbstrate, or both PP2 and PKC MP in combination. All inhibi tor treatment times were 1 hr. ERK1/2 activation was then measured via Western blot analysis using the indicated antibodies (top panel). Total ERK1/2 protein loading was demonstrated by stripping the membrane and reprobing with the indicated antibodies (bottom panel). B: Three re presentative Wester n blots of A were scanned and densitized and the perc ent maximum ERK2 phosphorylation was calculated by dividing by active ERK2 amounts in non-inhibitor treated cells after 5 min of Ang II stimulation and mu ltiplying by 100. This figure is used with permission from (43). Angiotensin II-induced Cell Migration Is Attenuated in VSMCs Treated With SL0101 Previous work has demonstrated that ab errant migration and proliferation of VSMCs is triggered by angiotensin II duri ng cardiovascular diseases such as atherosclerosis (72). VSMC migration pr ecedes proliferation, and results in the

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83 formation of a fibrous plaque. It was next tested whether Ang II-induced VSMC migration could be attenuat ed by pretreating with SL0101. VSMCs were pretreated for 1 hour with the indicated concentration of SL0101 or vehicle control, and then stim ulated with 100 nM angiotensin II for 24 hours. Cells were allowed to migrate across a nylon membrane where they were then stained, photographed and counted via quantification of the stain using a spectropho tometer. Little to no cell migration occurred in the absence of Ang II; however, cell migration did occur in response to Ang II (Figure 4-9). Furthe rmore, Ang II-induced cell migration was attenuated in a dose-dependent manner through the addition of SL0101. Maximum reductions in Ang II-induced cell migration were observed with the addition of 100 M SL0101. As such, some Ang II-induced cell migration is RSK-dependent. Discussion In Chapters 2 and 3, intracellular ERK1/2 activation is shown to be mediated by both c-Src/Yes/Fyn-dependent and heterotrimeric G protein/PKC -dependent signaling, and both of these signaling pathways contribut e equally to cell prol iferation in response to Ang II (42). Here, these findings are ex tended by defining the mechanism as to how this occurs (summarized in Figure 4-10). The key to these findings is that heterotrimeric G protein/PKC -dependent signaling dictates whet her ERK1/2 translocates into the nucleus and phosphorylates specific transcripti on factors like elk1, leading to increased cfos transcriptional activity. c-Src/Yes/F yn-signaling, on the other hand, phosphorylates ERK1/2 in the cytoplasm, where ERK1/2 remains and complexes with RSK2. RSK2 becomes activated, and then transloc ates into the nucleus to modulate c-fos transcription and c-fos protein activity. As such, thes e signaling events coordinately regulate proliferation in response to Ang II.

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84 OD 595 nm p = 0.01 30 x 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1234 -Ang + Ang + Ang + Ang + Vehicle + Vehicle 30 M SL0101 100 M SL0101 p = 0.01Effect of RSK inhibition on angiotensin II-induced cell migration Figure 4-9. Angiotensin II-induced cell migr ation is attenuated through selective RSK inhibition. Cells were pr etreated with the indicated concentration of SL0101 or vehicle control for 1 hour. Cells we re then stimulated with 100 nM Ang II for 24 hr, and allowed to migrate. Mi gratory cells (purple) were photographed after Ang II treatment, and ce ll number was assessed through spectrophotometry. These data are re presentative of three independent experiments. This figure is used with permission from (43). These findings support the idea that two separate pools of ERK1/2 exist within the cell: a pool of ERK1/2 which complexes with and activates RSK2 and a pool of ERK1/2 which translocates directly into the nucleus. However, ERK2 activation resulted in the dissociation of the ERK2-RSK complex within these cells. Other studies in COS7 cells ectopically expressing th e three RSK isoforms demonstrat ed that ERK-RSK complexes

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85 AT1 PKC MEK1/2 c-Src/Yes/Fyn MEK1/2 Cell membrane ERK1/2 ERK1/2(P) ERK1/2 ERK1/2(P) Nucleus ERK1/2(P) RSK2(P) RSK2(P) elk1 (P) SRF elk1 SRF (P) SRE c-fos c-fos RSK2(P) c-fos(P) Cell Proliferation Figure 4-10. Mechanistic diagram illustrating how Src kinase and PKC -dependent ERK1/2 activation pathways dually regul ate Ang II-induced ce ll proliferation. ERK1/2 activation is separately mediat ed by Src family tyrosine kinase and heterotrimeric G protein/PKC signaling in response to Ang II. With regards to PKC regulation, ERK1/2 translocates in to the nucleus up on stimulation of the AT1 receptor. Here, ERK1/2 phosphorylates elk1, whic h binds to the c-fos SRE and partially regulates c-fos transcriptional activity. c-fos transcriptional activity is also regulated by binding of the SRF. The SRF is phosphorylated in response to nuclear RSK2, which tran slocates into the nucleus after being phosphorylated by ERK1/2 in the cytoplasm. Cytoplasmic ERK1/2 phosphorylation is regulated by c-Sr c/Yes/Fyn-dependent signaling, independent of heterotr imeric G protein/PKC activity. Additionally, nuclear RSK2 directly phosphorylates c-fos and in creases the activity and stability of this protein. Thus, two independent pathways of ERK1/2 activation coordinately regulate An g II-induced cell prolifer ation by inducing c-fos transcription and increasing c-fos ac tivity through the post-translational modification of this protein. This figu re is used with permission from (43).

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86 did not dissociate when ERK1/2 was activated by EGF (161). Here, ERK1/2 and RSK2 rapidly co-associate in response to Ang II, with maximum co-associ ation occurring after 5 min of Ang II treatment. This co-asso ciation is completely dependent upon cSrc/Yes/Fyn signaling. Other pools of ERK1/2 do not co-associate with RSK since nuclear accumulation of ERK1/2 occurs well before RSK2 nuclear translocation, indicating that some ERK1/2 does not comple x with RSK2. Additi onally, this nuclear translocation of ERK1/2 occurs at the same time as a portion of ERK1/2 complexes with RSK2. Note that RSK2-ERK1/2 complexes do not translocate into the nucleus together since pretreatment of cells with PKC MP blocked all ERK1/2 nuclear translocation but did not affect RSK2 nuclear translocation (data not shown). Therefore, ERK1/2 and RSK2 nuclear translocation occur separately in response to Ang II; some ERK1/2 directly enters the nucleus whereas another portion co mplexes with RSK2. Previous studies have also shown that a pool of ERK complexe s with RSK, though RSK-ERK co-association patterns may differ depending upon the receptor activated (12). The duration as well as the magnitude of ERK1/2 activation has also been proposed to regulate gene expression and other sp ecific intracellular responses. Catt and colleagues have shown that the magnitude a nd duration of ERK1/2 activation in response to gonadotropin-releasing hormone (GnR H) depends upon the mechanism whereby ERK1/2 is activated, with a more sustaine d pattern of ERK activ ation occurring through G q/PKC-dependent signaling, and a more tran sient pattern of ERK activation caused by transactivation of the EGFR by the GnRH receptor (123). Sustained GnRH-induced ERK1/2 activation lead to ERK1/2 nucl ear accumulation, whereas ERK1/2 activated transiently failed to accumulate in the nucleus Other reports have shown that sustained

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87 vs. transient patterns of ERK1/2 lead to di fferent cellular responses including cell death or cell growth/proliferation (98). In Chapters 2 and 3, it was demonstrated that sustained patterns of Ang II-induced ERK1/2 activati on occurred in cells in which both cSrc/Yes/Fyn and heterotr imeric G protein/PKC signaling were intact (44). More transient patterns of ERK1/2 activation are evident when either of these pathways was disrupted. In addition, longer and more robust ERK1/2 activa tion pattern achieved through the simultaneous activ ation of both signaling cas cades resulted in greater amounts of Ang II-induced cell proliferation. Less robust and transient patterns of ERK1/2 activation achieved by the disruption of one pathway resulted in a diminished amount of cell proliferation in response to Ang II. Cell proliferation was found to be dependent upon the amount of c-fos transcribed and phosphoryl ated, with a greater amount of c-fos transcription occurring when bot h heterotrimeric G protein/PKC and cSrc/Yes/Fyn signaling pathways were dually activated. Furthermore, c-fos protein stability was extended when cSrc/Yes/Fyn-dependent signal ing was intact. Thus, the magnitude and duration of ERK1/2 activation affect Ang II-induced cell proliferation as well. Ang II-induced cell proliferation is associ ated with aberrant Ang II release and subsequent cell growth and prolifera tion during the progression of various cardiovascular diseases a nd cancers (24, 140, 145, 157, 159). Here, we show that both heterotrimeric G protein and Src family tyro sine kinase signaling must be blocked in order to completely attenuat e Ang II induced ERK1/2 activ ation. RSK2 can also be inhibited, as downstream target for ERK1 /2 activated by c-Src/Yes/Fyn-dependent signaling. Thus SL0101, a compound previously shown to be an anti-cancer agent, may

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88 also provide possible therapeutic benef its to patients suffering from certain cardiovascular diseases. In summary, Ang II-induced cell proliferat ion is mediated by two mechanistically different signaling pathways, both dependent on ERK1/2. Whether ERK1/2 activates RSK2 or translocates into the nucleus in orde r to mediate cell proliferation is determined by c-Src/Yes/Fyn or hetero trimeric G protein/PKC signaling, respectively. Interestingly, both of these pathways positiv ely regulate c-fos transcription and c-fos protein activity via the phosphor ylation of different transcri ption factors which initiate cell proliferation. Thus, this work demonstrates that the AT1 receptor, a prototypical GPCR, coordinately utilizes both heterotrimeric G protein and Src fam ily tyrosine kinasedependent signaling pathways in order to ach ieve angiotensin II-induced proliferation.

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89 CHAPTER 5 THE N-TERMINAL SH2 DOMAIN OF THE TYROSINE PHOSPHATASE, SHP-2, IS ESSENTIAL FOR JAK2-DEPENDENT SI GNALING VIA THE ANGIOTENSIN II TYPE 1 RECEPTOR Introduction Chapters 2-4 have focused on the role of Sr c family tyrosine ki nases in angiotensin II-dependent signaling. In addition to Src kinases, members of the Janus family of tyrosine kinases are also critical mediator s of angiotensin II-dependent signaling. Specifically, Jak2 has been shown to play a role in a number of signaling processes occurring downstream of the AT1 receptor (112). However, the cellular events underlying Jak2-dependent signali ng downstream of this prot otypical G protein-coupled receptor are not completely understood. It is clear that angiotensin II is a potent activator of Jak2, in vitro and in vivo (3, 79, 83). Once activated, Jak2 is recruited to the AT1 receptor signaling complex, where it mediates STAT activation and subsequent tr anscriptional regulation (2, 3, 116). Under certain conditions however, the signaling prope rties of angiotensin II can be maladaptive and lead to cardiovascular pa thologies (82, 95, 96, 100). Interestingly, Jak2 has also been linked to many of the same disease states that angiotensin II has b een linked (91, 95). Furthermore, blockage of either angiotensin II signaling with AT1 receptor antagonists or inhibition of Jak2 function using the Jak2 pharmacological inhib itor, AG490, has the same beneficial effect of ameliorating these cardiovascular diseases. Thus, some have suggested that the deleterious actions of angiotensin II may be mediated, in part, by the intracellular actions of Jak2.

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90 How Jak2 is activated by and recruited to the AT1 receptor is poorly understood. Previous work suggests that Ja k2 is first activated in the cytoplasm and then recruited to the AT1 receptor (3). The amino acids on the AT1 receptor that facilitate this interaction are encoded by the 319YIPP motif (3). Once bound to the receptor, Jak2 then acts as a molecular bridge in recruiting STAT1 to th e receptor complex (2). STAT1 is then phosphorylated by Jak2 and translocates into the nucleus where it modulates gene transcription. Work by Marrero and colle agues suggest that the protein tyrosine phosphatase SHP-2 may act to facilitate Jak2-dependent signaling via the AT1 receptor (81). However, the mechanism by wh ich this occurs is not known. Here, it was hypothesized that the N-terminal SH2 domain of SHP-2 is required for angiotensin II-mediated, Jak2-dependent signali ng. To test this, angiotensin II-dependent signaling was measured in fibroblasts derive d from mice in which the N-terminal SH2 domain of SHP-2 was lacking (SHP-2 46-110). Fibroblasts from wild type litter mates served as controls (SHP-WT). SHP-2 was found to be constitu tively bound to the AT1 receptor, and this occurs independent of the N-terminal SH2 domain of SHP-2. While the SHP-2 46-110 cells were capable of activati ng Jak2 tyrosine kinase, they were unable to facilitate AT1 receptor/Jak2 co-association, STAT activation and subsequent Ang II-mediated gene transcription when compared to wild type controls. These data therefore suggested that the N-te rminal SH2 domain of SHP-2 was acting to recruit Jak2 to the AT1 receptor signaling complex. Furthermore, the N-terminal SH2 domain of SHP-2 bound Jak2 predominantly, but not exclusively at Ja k2 tyrosine residue 201. When this tyrosine was converted to phenylalanine, th e ability of Ja k2 to activate subsequent downstream signaling events wa s reduced. Finally, wild type SHP-2

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91 transfected back into the SHP-2 46-110 cells restored Ang II-mediated, Jak2-dependent signaling. Collectively, these data suggest that the N-terminal SH2 domain of SHP-2 is essential for Ang II-mediated, Jak2-dependent signaling. The functional consequence of this interaction is to recruit Jak2 to the AT1 receptor signaling complex and in turn promote downstream Jak2-dependent signaling. Materials and Methods Cell Culture SHP-2 46-110 and wild type litter mate control fibroblasts (SHP-2 WT) were generated as described (114). The cells were passaged weekly with DMEM media containing 4.5 g/L of glucose and supplemented with 10% fetal bovine serum. The cells were transfected with 10 g of an HA-tagged AT1 receptor cDNA expression plasmid (111) using 10 l Lipofectin, following the manu facturer’s instructi ons (Invitrogen). Cells were subsequently growth arrested in serum-free DMEM for 48 hrs prior to experimental use. All cell culture reagents were obtained from Invitrogen. All other reagents were purchased from Sigma Chemical or Fisher Scientific. Immunoprecipitation Normalized cellular lysates (~0.5 mg/m l) were immunoprecipitated exactly as described in Chapter 2 (117). The imm unoprecipitating anti-phosphotyrosine antibody (PY20) was from BD Transduction Laborator ies. The immunoprecipitating anti-HA (F7) and anti-Jak2 (HR758) antibodies were from Santa Cruz Biotechnology. Western Blotting Proteins were detected using enhanced chemiluminescence exactly as described in Chapter 2 (117). The anti-Stat1, anti-Stat 3, anti-SHP-2, anti-phosphot yrosine (PY99) and

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92 anti-GST blotting antibodies were from Sa nta Cruz Biotechnology. The anti-Jak2 (HR758) blotting antibody was fr om Upstate Biotechnology, Inc. GST Pull Down Assays The creation, expression and purification of the four GST/Jak2 fusion proteins has been described (163). Protein ly sates were prepared from SHP-2 46-110 and wild type litter mate control fibroblasts as described. Cell lysates were first precleared with 7.5 g of Sepharose-bound-GST for 1 h at 4oC. To each sample, 1.0 nmol of the indicated GST/Jak2 fusion protein was then adde d and incubated for 90-120 min at 4oC on a rocking platform. The beads were washed 45 times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100) and resuspended in sample buffer. Sample buffer containing proteins were boiled, se parated by SDS-PAGE, transferred onto nitrocellulose membranes, and West ern blotted as described above. Luciferase Assay Cells were transfected with 10 g HA-tagged AT1 receptor cDNA expression plasmid and 5 g of a luciferase reporter c onstruct containing four tandem repeats of the IFNActivating Sequence (GAS) element, upstream of a minimal TK promoter, in 10 l Lipofectin. The cells were subsequently seeded into 6-well plates at 5x105 cells per well and allowed to attach overnight. The cells were serum starved for 48 hrs and treated as indicated. Luciferase activity was measured fr om detergent extracts in the presence of ATP and luciferin using the Reporter Lysis Buffer System (Promega) and a luminometer (Monolight Model 3010). Molecular Model of Jak2 A molecular model encoding full length, muri ne Jak2 has been described (41). The coordinates of the model were kindly provided to us in PDB format and regenerated

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93 using the PDB software program (www.rcsb.or g/pdb). Side chain surface accessability of tyrosine 201 was calculated using the vol ume area dihedral angle reporter program, VADAR (154). Statistical Analysis Statistical comparisons were made by Student's t Test. Data are represented as the mean +/SEM. Results SHP-2 46-110 and SHP-2 WT Cells Figure 6-1A provides a brief overview of the cell lines used in these studies, which have previously been desc ribed (114). The SHP-2 46-110 cells are mouse embryonic fibroblasts which lack the N-te rminal SH2 domain (amino acids 46-110) on both alleles. The control cells are fibroblasts derived from wild type litter mates, and express the full length 64 kDa isoform of SHP-2. The SHP-2 46-110 cells express a 57 kDa truncated form of the protein at ~25% of wild type levels, presumably due to inefficient splicing of the mutant allele (114). We examined the cells and found that while they express detectable levels of Jak2, they express little to no AT1 receptor protein (data not shown). Thus, for the studies described below, the AT1 receptor was transiently transfected into both cell types in order to establish a viable signaling sy stem. Furthermore, both cell types were transfected with similar efficiency. Jak2 Phosphorylation Is Not Influenced by the N-terminal SH2 Domain of SHP-2 It was first tested whether there was a ny difference in how the two cell lines activated Jak2. For this, both sets of cells were transiently transfected with the AT1 receptor and then treated with 100 nM angiotensin II for the indicated times. Protein lysates were then prepared and subse quently immunoprecipita ted with anti-Jak2

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94 antibody. The precipitates were Western blotte d with anti-Tyr(P) an tibodies in order to measure Jak2 tyrosine phosphorylation levels (F igure 5-1B, top). These results show that angiotensin II was able to increase tyrosine phosphorylation levels of Jak2 in both cell types. To show that all lanes were pr ecipitated equally, the membrane was Western blotted with anti-Jak2 anti body (Figure 5-1B, bottom). SH2 SH2 PTP YY SH2 SH2 SH2 SH2 PTP YYASHP-2 WT SH2 SH2 PTP YY SHP-2 46-110C SHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IP: Tyr(P)-mAb IB: Jak2-pAb Jak2(P) 111BSHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IB: Tyr(P)-mAb Jak2(P) 111IP: Jak2-pAb IB: Jak2-pAb 111 Jak2NH2NH2COOH COOH SH2 SH2 SH2 SH2 PTP YY SH2 SH2 SH2 SH2 PTP YYASHP-2 WT SH2 SH2 PTP YY SHP-2 46-110C SHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IP: Tyr(P)-mAb IB: Jak2-pAb Jak2(P) 111BSHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IB: Tyr(P)-mAb Jak2(P) 111IP: Jak2-pAb IB: Jak2-pAb 111 Jak2NH2NH2COOH COOH Figure 5-1. Jak2 tyrosine phosphoryl ation in SHP-2 WT or SHP-2 46-110 fibroblast cells. A: Cartoon representing the structure of the full-length 64 kDa SHP-2 (SHP-2 WT) and a truncated 57 kDa isoform (SHP-2 46-110). SH2= phosphotyrosine binding SH2 domain, PTP= protein ty rosine phosphatase domain, YY= the YY regulatory domain. B: SHP-2 WT or SHP-2 46-110 cells were stimulated with 100 nM angiotensin II for 0, 3 and 6 min. Cellular lysates were immunoprecipitated (IP) with anti-Ja k2 pAb and immunoblotted (IB) with anti-Tyr(P) mAb in order to determine total Jak2 phosphorylation (top). The membrane was then stripped and reprobe d with anti-Jak2 pAb to demonstrate equal protein loading (bottom). C: Cellular lysates were immunoprecipitated with anti-phosphotyrosine mAb and immunoblotted with anti-Jak2 pAb to determine Jak2 tyrosine phosphoryla tion levels. All Westerns are representative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permission from Elsevier.

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95 To measure this in an alternate manner a reciprocal precipitation was performed whereby lysates were immunoprecipitated wi th anti-Tyr(P) antibody and then Western blotted with anti-Jak2 antibody (Figure 5-1C). These results were similar to those in Figure 6-1B as both cell types were able to increase the tyrosine phosphorylation levels of Jak2, in response to angiotensin II. Collect ively, the data in Figure 5-1 suggest that the N-terminal SH2 domain of SHP-2 is not required for angiotensin II-mediated Jak2 activation. STAT1 and STAT3 Phosphorylation and STAT-mediated Gene Transcription Require the N-terminal SH2 Domain of SHP-2 Distal to Jak2 activation is STAT activa tion and gene transcription. To determine whether the N-terminal SH2 do main of SHP-2 had any effect on these cellular events, the ability of angiotensin II to promote ST AT phosphorylation and STAT-mediated gene transcription was measured. Both cell t ypes were again transfected with the AT1 receptor and then treated with 100 nM angiotensin II for the indicated times. The lysates were subsequently immunoprecipiated with anti -Tyr(P) antibody and then Western blotted with anti-STAT1 antibody (Figure 5-2A). Thes e data showed that while the Control cells were able to markedly incr ease the tyrosine phos phorylation levels of STAT1, the SHP2 46-110 cells were not. When STAT3 levels were measured, a similar pattern was observed; the Control cells had markedly increased STAT3 tyrosine phosphorylation levels, while the SHP-2 46-110 cells did not. To determine whether the N-terminal SH2 domain had any effect on STAT mediated gene transcription, both sets of cells were transfected with the AT1 receptor plasmid and a luciferase reporter plasmid en coding a STAT responsive element. It has

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96 previously been shown that this plasmi d is a good indicator of Jak/STAT-mediated transcription (149). The cells were treated wi th 100 nM angiotensin II for the indicated A B SHP-2 WT SHP-2 46-110Ang II (min) 0 5 15 0 5 15 IP: Tyr(P)-mAb IB: Stat1-pAb STAT 1(P) 79 SHP-2 WTSHP-2 46-110Ang II (min) 0 5 15 0 5 15 IP: Tyr(P)-mAb IB: STAT 3-pAb STAT 3(P) 79 Ang II (hrs) 051015202530 0 1 2 3 4 5 ** **C SHP-2 WT SHP246-110 Luciferase: fold increase over unstimulated Figure 5-2. STAT1/3 phosphoryl ation and STAT-induced luci ferase activity in SHP-2 WT or SHP-2 46-110 transfected fibroblast cells A: SHP-2 WT or SHP-2 46110 transfected cells were stimulated with 100 nM angiotensin II for 0, 5 and 15 min. Cellular lysates were immunoprecipitated with anti-Tyr(P) mAb and immunoblotted with anti-STAT1 pAb in order to determine total STAT1 phosphorylation. B: SHP-2 WT or SHP-2 46-110 transfected cells were stimulated with 100 nM angiotensin II for 0, 5 and 15 min. Cellular lysates were immunoprecipitated with anti-Ty r(P) mAb and immunoblotted with antiSTAT3 pAb in order to determine ST AT3 tyrosine phosphorylation. C: SHP2 WT or SHP-2 46-110 transfected cells were co-tra nsfected with the luciferase reporter plasmid encoding a STAT responsive element. Cells were then stimulated with 100 nM angiotensin II fo r the indicated times and luciferase activity assessed. Data represent mean fold increase in luciferase activity relative to unstimulated cells. = p<0.05, ** = p<0.01. All data are representative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permission from Elsevier.

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97 times and luciferase activity was measured (Figure 5-2C). SHP-2 46-110 cells had a marked reduction in their ability to generate luciferase activity in response to angiotensin II when compared to the wild type control cells. Collectively, the data in Figure 5-2 suggest that the N-terminal SH 2 domain of SHP-2 is required for angiotensin II-mediated STAT1 and STAT3 tyrosine phosphorylati on and angiotensin II-mediated Jak/STATdependent gene transcription. Jak2-AT1 Receptor Co-association Is Mediated by SHP-2 Previous work has suggested that SHP2 can act as a positive mediator of angiotensin II-mediated, Jak2-dependent sign aling (81). Therefore, the relationship between the AT1 receptor, Jak2 and SHP-2 was next determined, within these two cell types. Here, both sets of cells we re transfected with an HA-tagged AT1 receptor plasmid and subsequently treated with 100 nM angi otensin II for the indicated times. The AT1 receptor was immunoprecipitated from the lysates via the addition of anti-HA antibody. First, the precipitates were Western blotte d with anti-SHP-2 antibody in order to access AT1/SHP-2 interactions (Figure 5-3A). In both cell types, SHP-2 was constitutively bound to the AT1 receptor and this did not change with ligand addition. It is worth noting however, that overall, there is less SHP-2 46-110 protein when compared to wild type SHP-2, but presumably this is due to the fact that the SHP-2 46-110 protein is expressed at ~25% the levels that of wild type. Thus, it appears that the N-terminal SH2 domain has no effect on AT1/SHP-2 interactions. HA immunoprecipitates were next Western blotted with anti-Jak2 antibody to access AT1/Jak2 co-association. While angiot ensin II was able to promote AT1/Jak2 coassociation in the wild type cells, it was unable to do this in the SHP-2 46-110 cells (Figure 5-3B). Furthermore, overnight ECL exposur e failed to yield any Jak2 bands in the SHP-

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98 2 46-110 cells (data not shown). T hus, it appears that the N-terminal SH2 domain of SHP2 is required for angi otensin II-dependent, AT1/Jak2 co-association. Cells were again transfected with the AT1 receptor and this time the lysa tes were immunoprecipitated with anti-Jak2 and then Western blotted with anti-SHP-2 an tibody in order to access Jak2/SHP-2 co-association (Figure 5-3C, top) While angiotensin II induced Jak2/SHP-2 co-association in the wild type cells, th is was completely lacking in the SHP-2 46-110 cells. Once again, overexposure of the film demons trated that no Jak2/SHP-2 co-association was present in SHP-2 46-110 cells (data not shown). To de monstrate that all lanes were loaded similarly, the same membrane wa s Western blotted with anti-Jak2 antibody (Figure 5-3C, bottom). Thus, these data s uggest that the N-terminal SH2 domain is required for angiotensin II-depende nt Jak2/SHP-2 co-association. Lastly, it has been previously demonstrat ed that the angiotensin II-dependent physical interaction of Jak2 with the AT1 receptor requires AT1 receptor amino acids 319-322, encoding the 319YIPP motif. When 319YIPP was mutated to 319FAAA, the AT1 receptor was unable to bind Jak2 in response to angiotensin II (3). Here, the role of the 319YIPP motif on AT1/SHP-2 interaction was determined. For this, the wild type cells were transfected with a plasmid enc oding either an HA-tagged, wild type AT1 receptor cDNA plasmid or an HA-tagged AT1 receptor cDNA in which the 319YIPP motif was converted to 319FAAA. The cells were treated with 100 nM angiotensin II for the indicated times and AT1 receptor protein was immunoprecipi tated from the lysates via the addition of anti-HA antibody. The precipitates were then Western blotted with anti-SHP2 antibody to access AT1/SHP-2 interactions (Figure 53D). SHP-2 was found to be constitutively bound to the AT1 receptor, and this did not change with ligand addition

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99 BSHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IP: HA-mAb IB: Jak2-pAb Jak2 111ASHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IP: HA-mAb IB: SHP-2-pAb 61 SHP-2 WT SHP-2 46-110 CSHP-2 WT SHP-2 46-110Ang II (min) 0 3 6 0 3 6 IB: SHP-2-pAb SHP-2 WT 61 IP: aJak2-pAb IB: Jak2-pAb Jak2 111DAT1-WTAT1-319FAAAAng II (min) 0 3 6 0 3 6 IP: HA-mAb IB: SHP-2-pAb SHP-2 61 Figure 5-3. The recruitment of Jak2 to the AT1 receptor is dependent upon SHP-2. A: AT1/SHP-2 co-association was examined in SHP-2 WT or SHP-2 46-110 transfected fibroblasts. Cells were s timulated with 100 nM angiotensin II for 0, 3 and 6 min. Cellular lysates were immunoprecipitated with anti-HA mAb and immunoblotted with anti-SHP-2 pAb. B: Cellular lysates were immunoprecipitated with anti-HA mAb and immunoblotted with anti-Jak2 pAb. C: Cells were stimulated with 100 nM angiotensin II for 0, 3 and 6 min. Cellular lysates were immunopreci pitated with anti-Jak2 pAb and immunoblotted with anti-SHP-2 pAb (top) The membrane was stripped and reprobed with anti-Jak2 pAb to demonstr ate equal protein loading (bottom). D: AT1/SHP-2 co-association was examined in AT1-WT and AT1-319FAAA transfected cells. Cellular lysates were immunoprecipitated with anti-HA mAb and immunoblotted with anti-SHP-2 pAb. All Westerns are representative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permission from Elsevier.

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100 (lanes 1-3). However, when cel ls were transfected with the 319FAAA mutant receptor, no AT1/SHP-2 co-association was observe d, thus demonstrating that the 319YIPP motif is critical for angiotensin II-mediated, AT1/SHP-2 co-association. Transfecting Wild Type SHP-2 back into SHP-2 46-110 Cells Restores STAT1 and STAT3 Phosphorylation and STAT -mediated Gene Transcription These data in Figures 6-2 and 63 demonstrate that in the SHP-2 46-110 cells, signaling distal to Jak2 is virtually lost. Previous work has shown that the 46-110 mutation is recessive in nature and therefor e does not act in a domi nant negative fashion (114). Therefore, it was hypothesized that tran sfecting wild type SHP-2 back into these cells would restore these angiotensin II-media ted, Jak2-dependent signaling events, via the introduction of the N-terminal SH2 domain of SHP-2. To test this hypothesis, SHPWT cells transfected with AT1 receptor, and SHP-2 46-110 cells co-transfected with AT1 receptor and wild type SHP-2, were utilized. The samples were immunoprecipitated with anti-HA antibody and then Western blotte d with anti-Jak2 antibody to measure AT1/Jak2 co-association (Figure 5-4A). These results indicate that addition of wild type SHP-2 into the SHP-2 46-110 cells restored the ability of Jak2 to bind the AT1 receptor in response to angiotensin II. Next, angiotensin II-dep endent STAT1 phosphorylation was examined (Figure 5-4B). These results similarly show that placement of wild type SHP-2 into the SHP-2 46-110 cells restores angiotensin II-depende nt STAT1 tyrosine phosphorylation. Finally, we again measured Jak2-dependent ge ne transcription via the GAS-luciferase construct (Figure 5-4C). Thes e data show that transfecti on of wild type SHP-2 cDNA into the SHP-2 46-110 cells restores angiotensin II -mediated, Jak2-dependent gene transcription when compared to simila rly treated wild type SHP-2 cells.

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101 In summary, these data in Figure 5-4 dem onstrate that when wild type SHP-2 is added back to the SHP-2 46-110 cells, the ability of Jak2 to signal distal to its autophosphorylation is fully restored. Collect ively, these data demonstrate that the Nterminal SH2 domain of SHP-2 is es sential for angiotensin II-mediated AT1/Jak2 coassociation, STAT1 tyrosine phos phorylation and Jak2-dependent gene transcription. Jak2 Tyrosine 201 Mediates Jak2-SHP2 Interactions In Figure 5-3, it was demonstrated that Jak2 and SHP-2 form a physical coassociation in response to angi otensin II and that th is interaction requires the N-terminal SH2 domain of SHP-2. It was next determin ed which regions(s) of Jak2 were mediating this interaction. For these expe riments, a series of GST/Jak2 fusion proteins were utilized, and GST pull down assays were performed in vitro Figure 5-5A is a cartoon showing each specific GST/Jak2 fusion protein used in this assa y. First, wild type cells were treated with angiotensin II for the indicated times and pr otein lysates were prepared. Equal molar amounts of each GST/Jak2 fusion protein were added as indicated. The pull downs were eventually separated by SDSPAGE and Western blotted w ith anti-SHP-2 antibody to detect SHP-2 binding (Figure 5-5B). It wa s found that the GST/Jak2 construct encoding amino acids 1-294 strongly bound SHP-2 in a ligand-dependent manner. Additionally, the second construct encoding amino acids 295-522 al so showed some, albeit reduced, SHP-2 binding capability. However, when the same pull down assay was performed on the SHP2 46-110 cells, no SHP-2 binding was observed (Figure 5-5C). These data therefore suggest that the physical co-association of SHP-2 with the GST/Jak2 fusion proteins is absolutely dependent on the N-terminal SH2 domain of SHP-2 as the GST/Jak2 fusion proteins bound wild type SHP-2, but failed to bind SHP-2 46-110.

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102 Tyrosine residue(s) on Jak2 that were binding SHP-2 were next examined. Phosphotyrosine motifs that bind SH2 domains have a general consensus sequence of ASHP-2 46-110+ SHP-2Ang II (min) 0 3 6 0 3 6 IP: HA-mAb IB: Jak2-pAb Jak2 111BSHP-2 WT SHP-2 46-110+ SHP-2Ang II (min) 0 5 15 0 5 15 IP: Tyr(P)-mAb IB: STAT 1-pAb STAT 1(P) 79 Ang II (hrs) 0510152025 0 1 2 3 4 5 6 7 SHP-2 WT SHP2-46-110+ SHP-2 WT CLuciferase: fold increase over unstimulatedSHP-2 WT Figure 5-4. AT1/Jak2 co-association, STAT1 pho sphorylation and STAT-induced luciferase activity are restored in SHP-2 46-110 fibroblasts transfected with wild type SHP-2. A: AT1/Jak2 co-association was examined in SHP-2 WT or SHP-2 46-110 + SHP-2 transfected fibroblasts. Cells were stimulated with 100 nM angiotensin II for 0, 3 and 6 min. Cellular lysates were immunoprecipitated with anti-HA mAb and immunoblotted with anti-Jak2 pAb. B: STAT1 phosphorylation was examined in SHP-2 WT or SHP46-110 + SHP-2 transfected fibroblasts. Cell ular lysates were immunoprecipitated with anti-Tyr(P) mAb and immunoblotte d with anti-STAT1 pAb. C: SHP-2 WT or SHP-2 46-110 + SHP-2 transfected cells were co-transfected with the luciferase reporter plasmid encoding a ST AT responsive element. Cells were then stimulated with 100 nM angiot ensin II for the indicated times and luciferase activity assessed. Data repres ent mean fold increase in luciferase activity relative to unstimulated cells. All data are representative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with perm ission from Elsevier.

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103 A Jak2 WT (aa1-1129) GST/Jak2-1 (aa1-294) GST/Jak2-2 (aa295-522) GST/Jak2-3 (aa523-746) GST-Jak2-4 (aa747-1129)Pseudo Kinase Kinase Unique N TerminusB Ang II (min) 0 3 6 0 3 6 0 3 6 0 3 6 Jak2-1 Jak2-2Jak2-3Jak2-4IB: SHP-2-pAbSHP-2 WT 61C Ang II (min) 0 3 6 0 3 6 0 3 6 0 3 6 Jak2-1 Jak2-2Jak2-3Jak2-4IB: SHP-2-pAbS H P 2 D4 6 1 1 0 W C L .SHP-2 46-110 61 IB: GST-pAb GST/Jak2-4 GST/Jak2-1 GST/Jak2-2 GST/Jak2-3 GST/Jak2-4 GST/Jak2-1 GST/Jak2-2 GST/Jak2-3 IB: GST-pAb GST/Jak2-4 GST/Jak2-1 GST/Jak2-2 GST/Jak2-3 49 49 49 49 Figure 5-5. SHP-2/Jak2 co-association occurs mainly through interaction of Jak2 amino acids 1-294 and the N terminal SH2 doma in of SHP-2. A: Cartoon illustrating the various GST/Jak2 fusi on proteins used in B and C. The white box indicates the portion of wild type Jak2 contained with in the fusion protein and the corresponding domains. Encompassing Jak2 amino acid sequences are listed for each GST fusion protein. B: GST pull down assays measuring the ability of each GST/Jak2 fusion protei n to co-associate with SHP-2 WT. SHP-2 WT transfected cells were stimula ted with 100 nM angiotensin II for 0, 3 and 6 min. Cellular lysates were se parately incubated with each GST/Jak2 fusion protein as indicated and immunobl otted with anti-SHP-2 pAb (top). The membrane was then stripped and reprobed with anti-GST pAb to demonstrate equal protein loading (b ottom). C: GST pull down assays measuring the ability of each GST/Jak2 fusion protein to co-associate with SHP-2 46-110. SHP-2 46-110 transfected cells were stimulated with 100 nM angiotensin II for 0, 3 and 6 min. Cellula r lysates were separately incubated with each GST/Jak2 fusion protein as i ndicated and immunoblotted with antiSHP-2 pAb (top). The membrane was th en stripped and reprobed with antiGST pAb to demonstrate equal protein loading (bottom). All Westerns are representative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permission from Elsevier.

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104 YXX(L/V/M). Two tyrosines in the first 294 ami no acids of Jak2 bear some similarity to this sequence. They are 152YLFV and 201YNSV. For these experiments, these two tyrosines were individually converted to phenyl alanine in the contex t of the GST fusion protein encoding the first 294 amino acids of Jak2. GST pull down assays were then performed similar to those described above usi ng wild type cells. It was found that while the Y152F mutant was able to bind SHP-2, the Y201F mutant was not (Figure 5-6A). As such, the data suggest that the binding occurring between the GST fusion protein encoding the initial 294 amino acids of Jak2 and SHP-2, is mediated by Jak2 tyrosine residue 201. While the data in Figure 5-6A indicate that SHP-2 is able to bind Jak2 residue 201 in the context of a GST fusion protein en coding the initial 294 amino acids of Jak2, whether tyrosine 201 was solven t accessible in the context of the full length protein was still in question. Kroemer and colleagues prev iously generated a molecular model of full length Jak2 (41, 73). A recent work describing the crystal structure encoding a portion of the Jak2 kinase domain suggests that Kroemer’s model is highly accurate (75). Hence, the PDB coordinates of the full length Ja k2 model were obtained, and it was found that tyrosine 201 was very much on the surface of the structure and presumably capable of binding SH2 domains (Figure 5-6B). The side chain surface accessability of tyrosine 201 was calculated to be 148.9 angstroms squared, wh en the criteria for designating a residue as being solvent accessible is 15-20 angstroms squared. Collectively, these data in Figures 5-5 a nd 5-6 suggest that Jak2 binds SHP-2. This interaction is critically dependent on th e N-terminal SH2 domain of SHP-2 as the GST/Jak2 fusion proteins can bind wild type SHP-2, but cannot bind SHP-2 46-110.

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105 A BJak2-1 WTAng II (min) 0 5 0 5 0 5 Jak2-1 Y152F Jak2-1 Y201F IB: SHP-2-pAbSHP-2 WT 61 61 GST/Jak2-1IB: GST-pAb 49 49 Figure. 5-6. Jak2 tyrosine 201 is critical fo r Jak2/SHP-2 interaction. A: GST pull down assays measuring the ability Jak2 to co-associate with SHP-2 WT when Jak2 tyrosine residues are mutated at eith er position 152 or 201. Cells were stimulated with 100 nM angiotensin II fo r 0 or 5 min. Cellular lysates were separately incubated with either Ja k2-1 WT, Jak2-1 Y152F or Jak2-1 Y201F GST fusion proteins, and immunoblotte d with anti-SHP-2 pAb (top). The membrane was then stripped and reprobe d with anti-GST pAb to demonstrate equal protein loading (bottom). These Westerns are representative of three independent experiments. B: The positi on of tyrosine residue 201 (shown in red) in the context of full length Jak2 Shown is a lower magnification (left) and higher magnification (right) of the structure. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permi ssion from Elsevier. Furthermore, Jak2 tyrosine residue 201 app ears to be the major mediator of this phosphotyrosine/SH2 interaction.

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106 Jak2 Tyrosine 201 Mediates AT1 Receptor-Jak2 Co-association, STAT1 and STAT3 Activation and STAT-mediated Gene Transcription The effect of tyrosine residue 201 on angiotensin II-mediated, Jak2-dependent signaling was next examined. Here, tyrosine 201 was converted to phenylalanine in the context of a full length, Jak2 cDNA expression plasmid. For these experiments, COS7 cells were transiently co-transfected with an HA-tagged AT1 receptor plasmid and a plasmid encoding either a full length, wild type Jak2 cDNA (Jak2-WT), or a full length Jak2 cDNA in which tyrosine 201 was conve rted to phenylalanine (Jak2-Y201F). AT1/Jak2 co-association, STAT1/3 phos phorylation and STAT-mediated gene transcription were then assessed. With regards to AT1/Jak2 co-association, cell lysates were immunoprecipitated with anti-HA antibody and then West ern blotted with anti-Jak2 antibody (Figure 5-7A). AT1/Jak2 co-association occurred in response to angiotensin II treatment of Jak2-WT-transfect ed cells, but was reduced in cells transfected with the Jak2-Y201F plasmid. These data suggest that the tyrosine 201 residue contained within the JH6-JH7 domains of Jak2 contributes to AT1/Jak2 co-association. It was next assessed whether the Jak2-Y 201F mutation also influenced STAT1/3 tyrosine phosphorylation. Lysates from cells transfected with either the Jak2-WT or Jak2-Y201F plasmids were immunoprecipita ted with anti-Tyr(P ) antibody and then Western blotted with either anti-STA T1 or anti-STAT3 antibodies. STAT1 phosphorylation occurred in response to angiotensin II treatment of JAK2-WTtransfected cells, but was attenuated in cel ls transfected with the Jak2-Y201F plasmid (Figure 5-7B). Likewise, STAT 3 phosphorylation was also atte nuated in cells transfected with Jak2-Y201F plasmid when compared to Ja k2-WT-transfected cells (Figure 5-7C).

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107 CAng II (min) 0 5 15 0 5 15Jak2-WT Jak2-Y201F IP: aTyr(P)-mAb IB: aSTAT3-pAb STAT 3-(P) 79BAng II (min) 0 5 15 0 5 15Jak2-WT Jak2-Y201F STAT 1-(P)IP: aTyr(P)-mAb IB: aSTAT1-pAb 79 0 1 2 3 4 5 6 7 8 9 0510152025 Jak2-WT Jak2-Y201FAng II (hrs)D* ** **AAng II (min) 0 3 6 0 3 6Jak2-WT Jak2-Y201F IP: HA-mAb IB: aJak2-pAb Jak2 111 Luciferase: fold increase over unstimulated Figure 5-7. Mutation of Jak2 tyrosine 201 reduc es Jak2-dependent signaling in response to angiotensin II. A: AT1/Jak2 co-association was measured in Jak2-WT or Jak2-Y201 transiently-transfected COS7 cells. Cells were stimulated with 100 nM angiotensin II for 0, 3 or 6 min. Cell lysates were immunoprecipitated with anti-HA mAb, and then immunoblotted anti-Jak2 pAb. B: STAT1 phosphorylation was m easured in Jak2-WT or Jak2-Y201 transiently-transfected COS7 cells. Cells were stimulated with 100 nM angiotensin II for 0, 5 or 15 min. Cell lysates were immunop recipitated with anti-Tyr(P) mAb and then immunoblott ed anti-STAT1 pAb. C: STAT 3 phosphorylation was measured in Ja k2-WT or Jak2-Y201 transientlytransfected COS7 cells as in B with the exception that cellular lysates were immunoblotted with anti-STAT3 pAb in stead. D: Jak2-WT or Jak2-Y201F transfected cells were co-transfected with the luciferase reporter plasmid encoding a STAT responsive element. Cells were then stimulated with 100 nM angiotensin II for the indicated times and luciferase activity assessed. Data represent mean fold increase in luciferase activity relative to unstimulated cells. All data are re presentative of three independent experiments. Reproduced from Cellular Signaling, in press article, Copyright 2006 with permission from Elsevier.

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108 As such, Jak2 tyrosine 201 contributes to angiotensin II-mediated, Jak2-dependent STAT1/3 activation in addition to AT1/Jak2 co-association. Lastly, the effect of the Jak2 Y 201F mutation on STAT-mediated gene transcription was assessed. COS7 cells were transfected with the AT1 receptor plasmid and a luciferase reporter pl asmid encoding a STAT responsive element. In addition, these same cells were co-transfected with either the Jak2-WT plasmid or the Jak2-Y201F plasmid. All cells were then treated with 100 nM angiotensin II for the indicated times and luciferase activity was measured. Th e Jak2-Y201F cells exhi bited a significant reduction in their ability to generate luciferase activity in response to angiotensin II, when compared to the wild type control cells (Figur e 5-7D). These data therefore suggest that Jak2 tyrosine 201 contributes to Jak2/STAT-de pendent gene transcription in addition to Jak2/AT1 co-association and STAT1/3 phosphorylation. Discussion Here, the functional role of the N-termin al SH2 domain of SHP-2 in angiotensin IImediated, Jak2-dependent signal transduction was analyzed. It was found that SHP-2 appears to constitutively bind the AT1 receptor and this is independent of the N-terminal SH2 domain. However, this SH2 domain appear s essential for the recruitment of Jak2 to the AT1 receptor and the signaling even ts that are distal to AT1/Jak2 co-association. Specifically, in cells that lack the N-termin al SH2 domain of SHP-2, angiotensin II was unable to promote AT1/Jak2 co-association, STAT tyrosine phosphorylation and Jak2dependent gene transcription. Thus, the N-te rminal SH2 domain of SHP-2 appears to be a rate limiting factor in angiotensin II-me diated, Jak2-dependent signal transduction. Region(s) of Jak2 that specifically bind SHP-2 were also identified. GST pull down assays indicated that th e initial 294 amino acids strongl y bind wild type SHP-2, but

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109 not a mutant form of SHP-2 lacking the Nterminal SH2 domain. Additionally, a GST fusion protein containing Jak2 amino acids 295-522 also bound wild type SHP-2, but not the mutant. However, this second fusi on protein bound SHP-2 with about 10% the efficiency of the construct containing amino acids 1-294. Site directed mutagenesis of the GST fusion protein containing Jak2 amino acids 1-294 indicated that Jak2 tyrosine residue 201 was the principal mediator of SH P-2 binding as conversion of this tyrosine residue to phenylalanine abolished this intera ction. Furthermore, analysis of a molecular model encoding full length Jak2 suggests that Y201 is on the surface of the full length protein. The data in Figure 6-7 demonstrate that mutation of Jak2 tyrosine residue 201 markedly reduces, but does not fully abolish si gnaling events distal to Jak2. There are several possible explanations for this. Fi rst, the GST fusion pr otein containing amino acids 295-522 was found to specifically bi nd the N-terminal SH 2 domain of SHP-2, although not as efficiently as the construct containing amino acids 1-294 (Figure 5-5). Thus, it is possible that, in addition to tyro sine 201, a tyrosine residue located between amino acids 295 and 522 may be binding the SH2 do main. Examination of this region of Jak2 identified nine tyrosine re sidues. However, none bear a reasonable similarity to the consensus SH2-binding motif, YXX(L/V/M). Thus if one or more of these tyrosines is binding the N-terminal SH2 do main of SHP-2, it is doing so through a less conserved binding motif. Second, it has been previously demonstr ated that the Jak2 tyrosine motif, 231YRFRR, is required for binding the AT1 receptor in transfected COS7 cells (116). When this sequence was converted to 231FAAAA, Jak2 was unable to bind the AT1

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110 receptor. It appears that the 231YRFRR motif provides a critical structural function rather than a phosphotyrosine inte raction as a single Y231F mutation does not prevent AT1/Jak2 co-association. The amino terminal half of Jak2 has nineteen “band 4.1” domains. These domains are characterized as having hydrophobic clusters among charged amino acids. Homology between domains is not determined by primary amino acid sequence, but rather by secondary structure prediction wh ich allows for the detection of hydrophobic clusters within domains of very low sequen ce identity. The doma ins are separated by intervening unrelated sequences of variable lengths. Analysis of the region around amino acids 200-235 indicates that tyrosine 201 resides between domains 8 and 9 while the 231YRFRR motif is in the middle of domain 10. Thus, one possible explanation for the inability of the Jak2-231FAAAA mutant to bind the AT1 receptor, even with an intact tyrosine at position 201, is that the Jak2-231FAAAA substitution muta tion alters the tertiary structure of domain 10, which in turn reduces the otherwise exposed nature of tyrosine 201. What is known, however, is th at the N-terminal SH2 doma in of SHP-2 is singularly essential for binding Jak2 and bringing Jak2 to the AT1 receptor signaling complex; when this single domain is deleted, the ability of Jak2 to bind the AT1 receptor is completely lost. In contrast however, there appears to be some redundancy in how Jak2 binds SHP-2 and in turn the AT1 receptor; areas showing some binding ability, either directly or via a tertiary structure, in clude tyrosine 201, the 231YRFRR motif and possibly a tyrosine located between amino acids 295-522. A pr oposed model for the role of SHP-2 in promoting AT1 receptor-induced JAK/STAT signaling is summarized in Figure 5-8.

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111 Whether the AT1-Jak2-SHP2 complex recruits othe r proteins still remains to be elucidated. In summary, this work provides novel insi ght into the proximal signaling elements that facilitate angiotensin II-mediated, Jak/STAT-dependent signaling. As such, these studies may provide insight in to the signaling associated wi th other GPCRs that activate Jak2.

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112 Jak2 YY201 Jak2 YY201 Ang II Ang II Jak2 YY201 Jak2 YY201 Adaptor Protein Adaptor Protein ?? STAT STAT STAT STAT STAT STAT STAT STAT STAT STAT-mediated gene transcriptionAng II Ang II A BSH2 SH2 SHP-2 SH2 SHP-2 SH2 Figure 5-8. Proposed mechanism for Jak2/SH P-2 interactions upon stimulation of the AT1 receptor. A: In the absence of ligand, SHP-2 is bound to the AT1 receptor while Jak2 and STATs remain within the cytoplasm. B: Upon binding of angiotensin II to the AT1 receptor, phosphorylated Jak2 binds SHP2 at the N-terminal SH2 domain. W ith respect to Jak2, this interaction predominately occurs at an exposed tyrosine 201 residue on the surface of Jak2. Other Jak2 tyrosine residues or ot her adaptor proteins may also mediate Jak2/SHP-2 interactions. Once the AT1/Jak2/SHP-2 complex is formed, Jak2 tyrosine phosphorylates the STATs, resulting in STAT-dependent gene transcription. Reproduced from Cellula r Signaling, in pre ss article, Copyright 2006 with permission from Elsevier.

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113 CHAPTER 6 CONCLUSIONS AND IMPLICATIONS Summary of Results Since the discovery in the 1990’s that the AT1 receptor—a prototypical G proteincoupled receptor—can activate ty rosine kinases, a wave of research has been aimed at elucidating the roles of these kinases in angiotensin II signaling as well as the mechanisms leading to their activation. Th is work expands upon the current knowledge of angiotensin II, tyrosine kinase-mediated signaling. First, the role of Src family tyrosine kinases in intracellular ERK1/2 activation and a mechanism for how these proteins mediate angiotensin II-i nduced cell prolifera tion is established. It appears that Src kinases activate as much as 50% of ERK1/2 within the ce ll, and Src kinase-activated ERK1/2 mediates cell proliferation through the cytoplasmic activation of RSK. In addition, it is demonstrated that the rema ining 50% of ERK1/2 activation and Ang IIinduced cell proliferation is mediat ed by heterotrimeric G protein/PKC signaling, and occurs in a Src kinase-independent manne r. ERK1/2 activated by these signaling proteins directly translocates into the nucleus and modulat es a portion of early response gene transcription and cell proliferation. Ne xt, RSK is for the first time implicated in angiotensin II-induced cell proliferation. Given the recent discovery of a potent and highly-selective naturally occu rring inhibitor of RSK (77), RSK inhibition represents a promising new area of therapeutic interven tion for the treatment of Ang II-associated diseases. Finally, this work also identif ies an SHP-2 dependent mechanism for the activation of another important tyrosine kinase, Jak2. As su ch, more insight is gained

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114 into how the AT1 receptor activates this kinase and initiates Jak2/STAT signaling inside the cell. Collectively, the work in this disse rtation defines important aspects of tyrosine kinase-mediated signaling downstream of the AT1 receptor, and helps to redefine the angiotensin II signali ng paradigm. Src Kinases and Angiotensin II-induced Cell Proliferation Previously published findings have had di screpancies with regards to the role of Src kinases in Ang II-induced ERK1/2 activation. Some reports claim that these events are entirely mediated by Src kinase-depe ndent signaling (53, 115, 140), whereas other work has shown that ERK1/2 activation can occur via other signaling pathways not traditionally thought to be Src-dependent (33, 44-46, 70, 86, 124, 153). Some caveats to the findings dealing specifically with Src kinases were that these studies relied on either non-specific pharmacological inhibi tors, or only inhibited c-Sr c activity and did not take into account other functionally redundant Src family members, such as Yes and Fyn. Therefore, a role for Src kinases in angiot ensin II-induced ERK1/2 activation had not yet firmly been established. Here, MEF cells isolated from c-Src, Yes, Fyn compound knockout mice were used to discern the role of Src kinases in Ang II-induced ERK1/2 activation and cell proliferation, providing th e advantage of studying these processes in a Src kinase-deficient background. These results from Chapters 2-3 indicat e that Ang II-induced ERK1/2 activation does not rely solely on Src kinase signaling. Roughly about 50% of intracellular ERK1/2 activation is mediated via Src kinase-depe ndent signaling. In re sponse to Ang II, Src kinases are most likely activated thr ough the Shc/Grb2/Sos signaling cascade as previously described (115). Sos then serves as a guanine exchange factor (GEF), and activates RAS by exchanging GDP for GTP. Once Ras becomes activated, it activates

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115 ERK1/2 via the traditional MAP kinase si gnaling cascade, consisting of Ras induced Raf1 phosphorylation, followed by Raf1-dep endent MEK1/2 activation and finally MEK1/2-induced ERK1/2 activation. These ev ents have all been pr eviously described, and provide a mechanism whereby Src kina ses activate ERK1/2 in response to AT1 receptor activation. Src kinases are a rate-limiting step in the activation of as much as 50% of total phosphorylated ERK1/2. Treatment of SYF/AT1 cells with Ang II resulted in a reduced amount of ERK1/2 activation when compared to WT/AT1 control cells. These results clearly show that a portion of Ang II-induced ERK1/2 activation is Src-dependent. In addition, inhibition of either he terotrimeric G protein or PKC activity completely attenuated the remaining portion of ERK1/2 activation. Thus, PKC and heterotrimeric G protein activation are rate-limiting steps in the activation of ERK1/2 independent of Src kinases. As such, ERK1/2 activation is controlled by two-independent signaling pathways. There is also a difference in what happen s to ERK1/2 when activated by either Src kinase or heterotrim eric G protein/PKC -dependent signaling as described in Chapter 4. In response to c-Src/Yes/Fyn signaling, ER K1/2 activates RSK2. This presumably occurs in the cytoplasm since GFP-ERK2 st udies found that ERK2 does not translocate into the nucleus when activated by Src kinases (Chapter 2). In response to heterotrimeric G protein/PKC signaling, ERK1/2 translocates into th e nucleus. Therefore, the ability of ERK1/2 to enter into the nucleus or rema in within the cytoplasm and phosphorylate substrates like RSK is determined by the upstream signaling pathwa y activating ERK1/2. A looming question is how exactly does intrace llular ERK1/2 distinguis h that it has been

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116 activated by either c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, and know to either enter the nucleus or phosph orylate RSK within the cytoplasm. The exact answer to this question is stil l unresolved; however, a number of possible mechanisms can explain how the cell distinguis hes between Src kinase or heterotrimeric G protein/PKC activated ERK1/2 (Figure 6-1). For example, the subcellular distribution of ERKs may be altered depending upon the upstream signaling initiating ERK1/2 activation. Previous work ha s suggested that ERK1/2 nucle ar localization is in part mediated by the nuclear exporti n protein, Crm-1 (122). Overe xpression of Crm-1 inhibits nuclear translocation of phospho-ERK1/2 as we ll as elk1 phosphorylation in response to Ang II, presumably by causing ERK1/2 to rapi dly exit the nucleus, thereby preventing nuclear accumulation (122). In addition, inhi bition of Crm-1 restor es the ability of ERK1/2 to translocate into the nucleus in ce lls lacking heterotrimer ic G protein signaling or treated with PKC inhibitor. Furthermore, it has been suggested that Crm-1 activity is negatively regulated by hetero trimeric G protein and PKC signaling (122). Therefore, Crm-1 protein appears to govern whether or not ERK1/2 enters the nucleus, and is regulated by heterotrimeric G protein/ PKC signaling. Perhaps heterotrimeric G protein/PKC activation shuts off a portion of Crm-1 nuclear export activity, causing some ERK1/2 to enter into the nucleus. In addition, a por tion of ERK1/2 would be activated by Src kinase depende nt signaling, but would be se questered in the cytoplasm by the remaining active Crm-1. As such, ER K1/2 subcellular localization would be regulated at the point of Crm-1 by the pr esence or absence of heterotrimeric G protein/PKC signaling. A more detailed study of Crm-1 function with respect to

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117 ERK1/2 distribution will need to be performe d in order to examine the validity of this hypothesis. This proposed mechanism is illustrated in Figure 6-1. ERK1/2 nuclear localization could al so be influenced via specific phosphorylation/dephosphorylation events dependent upon the upstream activation pathway. It has been well es tablished that ERK1/2 activat ion occurs via phosphorylation at a conserved T-E-Y motif within the activation loop by MEK. Perhaps other phosphorylation or dephosphorylation even ts mediate whether ERK1 and ERK2 translocate into the nucleus or remain with in the cytoplasm. For example, previous studies have demonstrated that conversi on of amino acid residue s 312-320 to alanine within GFP-ERK2 prevented ERK2 from being retained in the cytoplasm (106). Further work by Takishima and colleagues demonstrated that a specific Y314A mutation within the GFP-ERK2 fusion protein was sufficient to block the cytoplas mic accumulation of ERK2 (125). This mutation blocked the ability of ERK2 to be anchored in the cytoplasm by PTP-SL, a protein tyrosine phosphatas e which has been shown to retain phosphorylated ERK2 within the cytoplasm when overexpressed (165). Thus, perhaps the dephosphorylation of ERK2 at tyrosine 314 by PTP-SL is triggered by Src kinasedependent signaling, effectively anchoring this pr otein within the cytoplasm so that it can dock to and activate RSK. Alternatively, dephosphorylation of ERK1/2 by PTP-SL could prevent ERK1/2 dimerization, an event whic h is thought to be necessary for ERK1/2 nuclear translocation to occur. As such, ERK1 /2 would effectively be retained within the cytoplasm. This mechanism is illustrated in Figure 6-2. Another possibility is that the ability of phosphorylated ERK1/2 to either be targeted to the nucleus or cytoplasm is de pendent upon substrate-recognition. It has been

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118 shown that active ERK1/2 localize to effect ors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P) or D-domain (docki ng domain) motifs (97). Furthermore, ERK1/2 ERK1/2 ERK1/2 RSK ERK1/2 PKC Src kinase ERK1/2 Nucleus Cytoplasm Angiotensin II A ERK1/2 CRM-1 CRM-1 Figure 6-1. Possible CRM-1-dependent mech anism influencing ERK1/2 localization. ERK1/2 is activated in a Src kinase or PKC -dependent manner in response to Ang II. ERK1/2 activated by PKC translocates into the nucleus. When active, CRM-1 shuttles ERK1/2 back into the cytoplasm, where it sequesters ERK1/2 phosphorylated by Src kinases. Src kinase activated ERK is then free to bind RSK. A portion of CRM-1 activity would be shut-off by heterotrimeric G protein/PKC signaling, allowing some ERK1/2 to remain in the nucleus. Murphy and colleagues have shown that mutations in the DEF-domain binding pocket prevent activa tion of DEF-domain-containing e ffectors but not RSK, which contains a D domain (27). Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RS K activation but not DEF-domain signaling proteins. Additionally, ERK1/2 DEF mutants were deficient in elk1/TCF activity. As

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119 such, perhaps when ERK1/2 is activated within the cytoplasm, a portion these phosphorylated proteins are sequestered by RSK through binding of the ERK1/2 CD domain to the ERK1/2 ERK1/2 ERK1/2 RSK PKC Src kinase Nucleus Cytoplasm Angiotensin II B ERK1/2 PTP-SL P Y314 ERK1/2 P Y314 ERK1/2 P Y314 ERK1/2 P Y314 ERK1/2 Y314 Y314 Figure 6-2. Possible mechanism whereby phos phorylation of tyrosine 314 affects ERK1/2 subcellular loca lization. ERK1/2 activation occurs via PKC and Src kinase-dependent signaling. Once ac tivated, ERK1/2 is phosphorylated at tyrosine 314 through an undetermined mechanism, causing ERK1/2 to dimerize and translocate into the nucle us. Src kinases phosphorylate PTP-SL, which dephosphorylates nearby ERK1/2 at tyrosine 314. This disables ERK1/2 from dimerizing, and sequester s it to the cytoplasm. Cytoplasmic ERK1/2 then binds and activates RSK.

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120 D domain of RSK. This would prevent a por tion of ERK1/2 from di rectly translocating into the nucleus. There appears to be much more ERK1/2 than RSK inside the cell, as C ERK1/2 ERK1/2 ERK1/2 RSK PKC Src kinase ERK1/2 Nucleus Cytoplasm Angiotensin II ERK1/2 ERK1/2 ERK1/2 CD Domain ERK1/2 CD Domain D Domain DEF Domain DEF Domain DEF Domain DEF Domain elk1/TCF Figure 6-3. Proposed mechanism whereby s ubstrate recognition influences ERK1/2 subcellular localization. ERK1 /2 activation occurs via PKC and Src kinasedependent signaling. ERK1/2 activation via PKC signaling causes receptor dimerization, which sequesters the CD domain but exposes residues important for DEF domain recognition (DEF domain ). Dimerized ERK1/2 translocates into the nucleus, and activates elk1/ TCF via DEF domain interactions. ERK1/2 in the cytoplasm recognizes RSK via D domain interaction. was observed by Western blot as well as immunofluorescence in Chapters 2-4. This observation is in line with previous findings that show only about 50% of ERK1/2 colocalizes with RSK in Xenopus oocytes (52). Therefore, it is likely that some ERK1/2 does not come in contact with RSK and forms dimers, while at the same time a portion of ERK1/2 is binding to RSK. Dimerization may block the ability of ERK1/2 to interact

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121 with the D domains of RSK in the cytoplasm, while at the same time creating a favorable tertiary structure for DEF domain re cognition. Perhaps Src kinase and PKC upstream signaling affect the processes mentioned in the proceeding two paragraphs, which ultimately contributes to favored substrate recognition and ultimately an ERK1/2 effect within the nucleus or cytoplasm. Thes e events are illustrated in Figure 6-3. It is likely that hetero trimeric G protein/PKC signaling and Src kinase-dependent signaling influence whether or not activated ER K1/2 enters the nucleus or remains in the cytoplasm through a combination of the mechanis ms just listed. From preliminary data generated by our group, it appears that not a ll ERK1/2 and RSK co-localized within the cell in the absence of ligand treatment (Figure 6-4). Therefore, it is highly likely that differences in ERK1/2 subcellular distribution affect whether or not ERK1/2 is activated by either Src kinase-dependent signal ing or heterotrimeric G protein/PKC -dependent signaling. More work will need to be done in order to determine exactly how ERK1/2 is targeted to the nucleus and cytoplasm. Since MAP kinase signaling has been im plicated in so many processes, many groups have already begun to recognize the importance of identifying the mechanisms underlying ERK1/2 signaling proce sses in order to selectivel y inhibit cert ain signaling events without disrupting ERK1/2 signaling as a whole. For example, selective pharmacological inhibitors have already been generated which block ERK1/2 binding to either the DEF or D domains within ERK1 /2 substrates (27). These compounds, along with other targeting strategi es, will be important for dis cerning ERK1/2 function as it relates to angiotensin II si gnaling, as well as signaling through other receptors.

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122 What is clear from the studies contained in Chapters 2-4 is that Src kinase signaling and heterotrimeric G protein signaling are not separate events leading to different, Figure 6-4. Preliminary data supporting th e hypothesis that ERK1/2 subcellular distribution effects the mechanis m of upstream activation. The immunofluorescent image is of an unstimulated WT/AT1 cell. ERK2 is shown in red, RSK2 in green. DAPI stai ning (blue) shows the position of the nucleus. RSK2 fluorescence does not overlap entirely with ERK2 fluorescence, indicating that some ERK1/2 may not be positio ned to interact with RSK in the cytoplasm. distinct cellular outcomes. Both Src kinase-dependent ERK1/2 activation and heterotrimeric G protein/PKC -dependent ERK1/2 activation regulate a portion of Ang II-induced cell proliferati on, albeit through different mechanisms. These two mechanisms seem to converge at the level of c-fos and mediate a portion of c-fos protein expression. Thus, the angioten sin II signaling paradigm is not as simplistic as once thought, with heterotrimeric G protein signali ng mediating all signaling associated with

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123 the pressor response and tyrosine kinase si gnaling mediating cell pr oliferation. Clearly, these two types of signaling cascades can act in concert with one another in order to achieve a common outcome. This is illustrated in other examples as well. For instance, calcium and heterotrimeric G protein/PK C-dependent activation of Pyk2 has been demonstrated to mediate Src activa tion by GPCRs (26, 108). In addition, G q/PLC signaling through GPCRs affects th e activity of tyrosine kina ses, including Src, focal adhesion kinase, PYK2 and epidermal grow th factor receptor (30, 32, 67, 144, 152). Third, intracellular calcium release—a pro cess necessary for muscle contraction—has been shown to be dependent upon both Ja k2 tyrosine kinase (150) as well as heterotrimeric G protein si gnaling (118). Whether through direct activation of one another or coordinate (but separate) signaling events, he terotrimeric G protein and tyrosine kinase signaling often work together. As such, a paradigm shift is beginning to emerge in the field of angiot ensin II signaling, causing res earchers to rethink the older model of angiotensin signaling. A question specifically regarding AT1 receptor-induced cell proliferation does arise as it relates to the rethinking of this para digm: why does the cell u tilize two independent pathways to achieve the same effect. These data presented in Chapters 2-4 suggest that heterotrimeric G protein/PKC signaling and Src kinase signali ng have an additive effect on Ang II-induced ERK1/2 activation and cell pr oliferation. Therefore, one explanation is that these pathways may be somewhat redundant with one anot her in that ERK1/2 activation and cell proliferati on can persist (albeit at reduc ed amounts) when one pathway is disrupted. Perhaps this redundancy exists as an evolutionary conserved mechanism to protect the cell’s ability to divide and proliferate. If a spontaneous mutation suddenly

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124 arises in say the gene encoding PKC for example, the cell ultimately has another mechanism capable of initiati ng cell proliferation in a PKC -independent manner. However, angiotensin II-induced cell prolif eration has to date only been implicated in aberrant cell proliferation during disease states, including both cardiovascular disease and cancer (24, 31, 49, 56, 60, 72, 89, 104, 112, 120, 137). Therefore, another explanation for why the cell may use two path ways to achieve the same effect may be due to the role of angiotensi n II in the progression of dis ease. Many studies have shown that the magnitude and duration of ERK1/2 ac tivation determines th e outcome of ERK1/2 activation (101, 102, 120). Perhaps a more sustained, robust and simultaneous Ang IIinduced activation of ERK1/2 by two independe nt signaling pathways results in abnormal cell proliferation, whereas activation of ERK1 /2 at comparatively reduced levels by one pathway may help maintain normal levels of cell proliferation in cells expressing the AT1 receptor. Work by Lenormand and colleagues suggests that the magnitude and temporal pattern of ERK1/2 activation is carefully controlled by interac tions with different scaffolding proteins, which effectively insu late proteins from pathways which could otherwise influence their activity (101). Pe rhaps protein scaffolding determines whether ERK1/2 is activated by one pathway or two. As such, the simultaneous activation of ERK1/2 by two-independent signaling cas cades may be key to causing the Ang IIinduced cell proliferation commonly a ssociated with many disease states. It should be noted that the mechanisms of Ang II-induced ERK1/2 activation described in the preceeding chap ters describe ERK1/2 activati on as it relates to cellular proliferation. Angiotensin II also binds to receptors in cells which do not proliferate, including neurons and cardiac myocytes. Theref ore, these cellular pr oliferation pathways

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125 may only be applicable to cells which are cap able of proliferating, including vascular smooth muscle cells and fibroblasts. These cells likely possess the right combination of proteins in order for angiot ensin II to elicit cellular pr oliferation duri ng pathological conditions (i.e. cardiovascular diseases and cancer). ERK1/2 is also phosphorylated in other non-proliferative AT1 receptor-expressing cells, but may lead to different effects. For example, Ang II-induced ERK1/2 activation in cardiac myocytes may primarily trigger hypertrophy of these ce lls instead of cellular prol iferation while in neurons, ERK1/2 may modulate ion channel activity. The role of SHP-2 in angiot ensin II, Jak2/STAT signaling Phosphatases are often thought of as “the off switch” for many different signaling events through dephosphorylation at tyrosine re sidues. This is sometimes the case during Jak2 signaling. For example, MAP kinase phos phatase 1 (MKP-1) has been shown to be activated in a Jak2-dependent manner, and effectively turns off Ang II-induced ERK2 activation through dephos phorylation of ERK2 (111). Howe ver, it is demonstrated in Chapter 5 that SHP-2, a classic example of a phosphatase, mediates angiotensin II, Jak2/STAT signaling by facilitati ng Jak2 co-association with the AT1 receptor. Thus, in the case of angiotensin II, SHP-2 serves ini tially as an adaptor molecule necessary to positively mediate Jak2/STAT signaling. As su ch, SHP-2 is not acting to “turn off” Jak2/STAT signaling through dephosphorylati on of specific tyrosine residues. Previous work has generated discrepancie s as far as the role of SHP-2 in Jak2dependent signaling, which have often been attrib uted to the cell type and ligand utilized. In COS7 cells expressing the GH receptor, SHP-2 appears to positively regulate Jak2 signaling (61). Likewise, in Chapter 5, SHP2 appears to positively mediate Jak2/STAT signaling in response to angiot ensin II. However, SHP-2 a ppears to negatively regulate

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126 Jak2 signaling in fibroblasts treated with interferons ( 160). Perhaps SHP-2 signaling positively regulates Jak2 signaling in the c ontext of growth promoting signals, but negatively regulates Jak2/STAT signaling when ac tivated by other factor s. In the context of GPCRs like the AT1 receptor, it is clear that SHP-2 helps recuit Jak2 to the receptor and facilitate downstream signa ling. In the case of inte rferons, SHP-2 dephosphorylates phospho-tyrosine residues in order to shut o ff Jak2 signaling. It is unclear how SHP-2 positively regulates Jak2/STAT signaling in the case of the GH receptor, since this receptor already contains intrinsically bound Jak2 proteins which become phosphorylated upon receptor dimerization. Perhaps SHP-2 is play ing a role as an adaptor protein here as well, helping to facilitate the formation of the Jak2/STAT signaling complex at the receptor. More work will need to determine exactly why SHP-2 serves as both a positive and negative regulator of Jak2/STAT signaling. Angiotensin II signaling and disease As demonstrated in the dissertation, angi otensin II signaling is complex. This is illustrated by the fact that multiple signaling cascades can influence the same processes, such as angiotensin II-induced cell growth a nd proliferation. Additi onally, some proteins can have more than one function inside the cell. For example, SHP-2 is a phosphatase which can also serve to positively regula te Jak2/STAT signaling in response to angiotensin II. Current AT1 receptor antagonists, such as losartan or candesartan, are excellent AT1 receptor blockers and effective blood pressure reducers when administered in vivo (56, 104, 136, 146). Simply bl ocking the receptor may not always be the most effective means to achieve a desired cellular outcome, though, since a number of downstream signaling events will be effected in addition to the activity of a few desired proteins. For example, administering an AT1 receptor antagonist in vivo could be

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127 beneficial for inhibiting aber rant cell proliferation, but could also cause an undesired decrease in blood pressure. Understanding the complexity of angioten sin II signaling may aid in designing a more tailored approach for treating Ang II-associated diseases. From these studies, it has been observed that RSK inhibition via SL0101 is an effective means for lowering Ang IIinduced cell proliferation. In combination with a PKC inhibitor, one could theoretically completely attenuate Ang II-induced cell prolif eration as demonstrated in the cell culture systems utilized in this work. Thus, perhaps dual PKC inhibition and RSK inhibition, achieved via administering two compounds, c ould effectively reduce Ang II-induced cell growth and proliferation during disease st ates. Theoretically, these compounds would have little too no effect on vascular tone since this process is thought to occur via different signaling mechanisms (118). As such, this dissertation provides valuable insight into the underlying signaling associated with AT1 receptor activation and ultimately into the treatment of Ang II-associated diseases.

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143 BIOGRAPHICAL SKETCH Mr. Godeny was born on October 10, 1979, in Flemington, New Jersey. His love for the life sciences was sparked early in his childhood as he enj oyed collecting rocks, reading about animals and lear ning about space. In the 7th Grade, Mr Godeny decided that he wanted to become a cardiologist while writing an essay about the heart. In June of 1998, Mr. Godeny graduated with honors fr om Freedom High School in Bethelehem, Pennsylvania. He later enrolled as a freshm an at The Pennsylvania State University in University Park, Pennsylvania, where he started as a premedicine major. In the spring semester of his freshmen year, Mr. Godeny wo rked as a work-study in the laboratory of Dr. Regina Vasilatos-Younken in the Department of Poultry Science. There, he gained his first exposure to scientific researc h. In 2000, Mr. Godeny was accepted into the Ronald E. McNair Scholars program at Penn State. As part of the requirements, Mr. Godeny spent two semesters and summers doing re search in the laboratory of Dr. Richard Ordway studying the mechanisms of synaptic transmission in Drosophila melanogaster Mr. Godeny graduated from Penn State in Ma y of 2002 with a bachelor’s degree in biology. In August of 2002, he enrolled as a gra duate student at the University of Florida in Gainesville, Florida. There, Mr. G odeny worked as a graduate student in the laboratory of Dr. Peter Sayeski studying angi otensin II signaling. Mr. Godeny will attain a Ph.D. in biomedical science in August of 2006, and plans to work as a Postdoctoral Research Fellow at St. Jude Children’s Research Hospital in Memphis, Tennessee.


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Title: JAK2 and SRC Family Tyrosine Kinase Signaling via the Angiotensin II Type 1 Receptor
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
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Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
        Page v
    Table of Contents
        Page vi
        Page vii
        Page viii
        Page ix
    List of Tables
        Page x
    List of Figures
        Page xi
        Page xii
        Page xiii
    Abstract
        Page xiv
        Page xv
    Introduction
        Page 1
        Page 2
        Page 3
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    C-SRC/YES/FYN tyrosine kinases mediate a portion of angiotensin II-induced ERK ½ activation and cellular proliferation
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    SRC kinase-independent ERK ½ activation and cell proliferation is mediated by heterotrimeric G proteins and PKC-dependent signaling
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    ERK ½ regulates angiotensin II-dependent cell proliferation via the cytoplasmic activation of RSK2 and nuclear activation of ELK1
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    The N-terminal SH2 domain of the tyrosine phosphatase, SHP-2, is essential for JAK2-dependent signaling via the angiotensin II type 1 receptor
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    Conclusions and implications
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    References
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    Biographical sketch
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Full Text












JAK2 AND SRC FAMILY TYROSINE KINASE SIGNALING VIA THE
ANGIOTENSIN II TYPE 1 RECEPTOR














By

MICHAEL D. GODENY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006



























Copyright 2006

by

Michael D. Godeny
































This dissertation is dedicated to my family, for their constant love, support and guidance
















ACKNOWLEDGMENTS

I would like to first thank my mentor, Dr. Peter Sayeski. His guidance, patience

and support have enabled me to successfully navigate my way through graduate school.

Peter's passion for research coupled to his enthusiasm for mentoring always kept me

excited about scientific research. Furthermore, Peter has gone above and beyond the role

of a graduate studies mentor by always being readily available to answer any questions

about my research, my career path and life in general. In short, Peter has been the perfect

role model, and I consider myself fortunate to have worked with such an outstanding

individual.

I would also like to thank my committee members for their insight and guidance:

Dr. Hideko Kasahara, Dr. Colin Sumners, and Dr. Lei Xiao. These individuals have

played an invaluable role in my success in graduate school.

Additionally, I would like acknowledge a few individuals who have provided

technical assistance and reagents. I would first like to thank Tim Vaught (MBI

Microscopy Facility) and Michael Poulos (Swanson Lab) for their technical assistance, as

I would not have been able to obtain any microscopy images without their help. In

addition, I would like to thanks Dr. Phillip Soriano (University of Washington), Dr.

Kenneth Bernstein (Emory University), Dr. Brad Berk (University of Rochester), Dr.

Phillip J. Stork (Oregon Health Science Center) and Dr. Jessica Schwartz (University of









Michigan) for donating various plasmids and cell lines which have been essential for the

completion of this work.

Finally, I would like to thank all of the members of the Sayeski lab, both past and

present, with whom I have been fortunate enough to work: Dr. Ma Xianyue, Dr. Tiffany

Wallace, Dr. Eric Sandberg, Issam McDoom, Jacqueline Sayyah, Dannielle

VonDerLinden, Melissa Johns, Bobby Blair and Andrew Magis. Each one of them has

made the laboratory environment a joy to be in. I thank them for sharing their insight,

ideas and reagents. I will miss all of them!

Finally, I would like to thank my family and friends for being there when times

were rough, and for being supportive and keeping me sane during those times. In

addition, I am indebted to Rick Swenson and Dana Moser, organizers of the Gainesville

Arthritis Foundation's Marathon Training Team. These individuals provided me with a

new life-long hobby and helped to keep my fitness and stress in check while I was a

graduate student.















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .. ............ ................................................... ............... x...... .... ..x

LIST OF FIGURES ........................................ .............. xi

ABSTRACT ................................................... ................. xiv

1 IN TR O D U C T IO N ........ .. ......................................... ..........................................1.

O v e rv ie w ....................................................................................................................... 1
A n g ioten sin II ....................................................... ............................................. . 2
H history of the R enin A ngiotensin System ...........................2.... ........................... 2
Physiological Effects of Angiotensin II Associated With Binding of the AT1
R ecep to r ..................................................... ........................................... . 2
A ngioten sin II Signaling ......................................... ....................... ...............4...
The JAK Fam ily of Tyrosine K inases ....................... ..........................................6...
Structure .................................................................................. . .. ...............6
Jak2/STA T Signaling .............. ................. ...............................................7....
The Src Fam ily of Tyrosine K inases ....................................................... ...............8...
S tru ctu re ....................................................... ............................................. . .8
c-Src/Y es/Fyn K nockout M ice ......................................................... ............... 8
A T1 R eceptor-induced Signaling .................................................... ................ 10
ERK 1 and ERK 2 M A P K inases ....................... .............................................1...... 1
The M A P K inase Superfam ily ....................... ...............................................11
E R K I/2 Signaling ... ................................................................... .. .......... ... 12
R ib osom al S6 kinase ... ... ........................................... ....................... ............... 12
The R SK Fam ily of Proteins .......................................................... ............... 12
Structure and Function of RSK Proteins ........................................ ................ 13
The A ngiotensin II Signaling Paradigm ................................................ ................ 15
Sum m ary and R ationale ...................................................................... ............... 16

2 c-SRC/YES/FYN TYROSINE KINASES MEDIATE A PORTION OF
ANGIOTENSIN II-INDUCED ERK1/2 ACTIVATION AND CELLULAR
P R O L IF E R A T IO N ..................................................................................................... 18

In tro d u ctio n ............................................................................................................... .. 1 8
M materials an d M eth o d s ............................................................................. ............... 2 1
Creation of WT/AT1 and SYF/AT1 Stable Cell Lines....................................21









C ell C culture and R agents ................................................................. ............... 2 1
Immunoprecipitation and Western Blotting .............. ....................................22
A n tib o d ie s............................................................................................................ 2 3
Im m unofluorescence ....................................... ..... ................ 23
Measurement of Cellular ATP Levels............................................................24
Measurement of Formazan Production ..........................................................24
C e ll C o u n t............................................................................................................ 2 4
D ensitom etric A naly sis ........................................ ....................... ................ 25
Statystical A analysis ............. ................. .............................................. 25
R e su lts ................................................. .. ............... ....... ................................. ........ 2 5
Characterization of WT/AT1 and SYF/AT1 Cells .............................................25
Angiotensin II-induced ERK1/2 Activation Is Reduced By About 50% in Src
K inase D efficient C ells ................................................................ ................ 26
Angiotensin II-induced ERK1/2 Nuclear Translocation Is Not Dependent
U p on Src K in ases .............................................. .. ................. ..... ......... .....2 9
Angiotensin II Induced Cell Proliferation Is Reduced in Src Kinase Deficient
C ells ... . ...... ..................................................................................... . 30
D isc u ssio n ............................................................................................................... ... 3 4

3 SRC KINASE-INDEPENDENT ERK1/2 ACTIVATION AND CELL
PROLIFERATION IS MEDIATED BY HETEROTRIMERIC G PROTEINS
AND PKC(-DEPENDENT SIGNALING ....................................... ..................... 36

In tro d u ctio n ................................................................................................................. 3 6
M materials and M ethods .. ..................................................................... ................ 38
C e ll C u ltu re ......................................................................................................... 3 8
P harm ecological Inhibitors............................................................. ................ 39
siRNA Treatment of WT/AT1 Cells...............................................................39
Immunoprecipitation, Western Blotting and Densitometric Analysis .............40
Immunofluorescence and Quantification of Fluorescence................................40
C e ll C o u n t............................................................................................................ 4 0
Statistical A analysis .............. ...... ............ ................................................ 40
R e su lts ............................ ......... ... ....... ..... ............... ......... ............................... 4 1
Src Kinase Independent ERK1/2 Activation Does Not Require EGF
Receptor, PDGF Receptor or PI3K Activity ...................................................41
Src Kinase Independent ERK1/2 Activation Is Dependent on MEK1/2, But
N ot R afl ...................................... ...... .. ....... ............................ 43
Heterotrimeric G Proteins Mediate A Portion of ERK1/2 Activation In A Src
K inase-independent M anner ....................................................... ................ 45
Protein Kinase C C Mediates ERK1/2 Activation In A Src Kinase-
independent M anner.................................................................... ................ 48
PKC( Mediates MEK1/2 Activation Independent of Src Kinases...................52
ERK1/2 Nuclear Translocation Is Dependent Upon PKC( In Response to
A ngioten sin II ....................................................................... . ... ............... 53
Cell Proliferation Is Attenuated Through Inhibition of PKC( Signaling In
R response to A ngiotensin II......................................................... ............... 55
D isc u ssio n ............................................................................................................... ... 5 7









4 ERK1/2 REGULATES ANGIOTENSIN II-DEPENDENT CELL
PROLIFERATION VIA THE CYTOPLASMIC ACTIVATION OF RSK2 AND
NUCLEAR ACTIVATION OF ELK1 ..................................................................60

In tro d u ctio n ................................................................................................................ 6 0
M materials and M ethods ................................................... ..... ............... ................ 62
Antibodies and Pharmacological Inhibitors ...................................................62
C ell lines and C ell C culture ................................................................ ............... 63
Cell Lysate Preparation, Immunoprecipitation and Western Blotting .............63
D ensitom etric A naly sis ........................................ ....................... ................ 63
Im m unofluorescence ................ .. ...... ..... ........ ......... ................ 63
Quantification of nuclear and cytoplasmic fluorescence ..........................64
c-fos transcriptional activity .................................................... ................ 64
Cell M migration A ssay..................................................................................... 65
C e ll C o u n t............................................................................................................ 6 5
Statistical A analysis .............. ...... ............. .............................................. 66
R e su lts ........................................ ........ ........ ........................ ..... ................... ........ 6 6
RSK Phosphorylation and ERK1/2-RSK Co-association Are Dependent
Upon Src Kinases in Response to Angiotensin II..........................................66
RSK Nuclear Translocation Is Src Kinase Dependent, While ERK1/2 Nuclear
Translocation Is PKC( Dependent in Response to Angiotensin II............... 69
SRF and TCF Binding Within the c-fos Promoter Are Mediated in A RSK
And A ERKl/2-dependent Manner, Respectively ...........................................71
c-fos Protein Expression Is Dependent Upon Src Kinase Signaling And PKC(
Signaling ............. ........... .............. .................... ............ 74
c-fos Phosphorylation Is Dependent Upon Src Kinase-RSK Signaling ........... 75
Angiotensin II-induced Cell Proliferation Requires RSK And PKC( Activity...77
Angiotensin II-induced ERK1/2 Activation Is Mediated By Both Src Kinases
and PKC in Vascular Smooth Muscle Cells................................................ 81
Angiotensin II-induced Cell Migration Is Attenuated in VSMCs Treated With
S L 0 1 0 1 ........................................................................................................ . 8 2
D isc u ssio n ............................................................................................................... ... 8 3

5 THE N-TERMINAL SH2 DOMAIN OF THE TYROSINE PHOSPHATASE,
SHP-2, IS ESSENTIAL FOR JAK2-DEPENDENT SIGNALING VIA THE
ANGIOTENSIN II TYPE 1 RECEPTOR ............... .............. ..................... 89

In tro d u ctio n ............................................................................................................... .. 8 9
M materials and M ethods .. ..................................................................... ................ 9 1
C ell C culture .............. ................................................ ....................... . 9 1
Im m unoprecipitation .............. ............. .............................................. 91
Western Blotting...................................................................... 91
G ST P ull D ow n A ssay s ........................................ ....................... ................ 92
L uciferase A ssay .............................. ............................................ 92
M olecular M odel of Jak2....................................... ...................... ................ 92
Statistical A analysis .............. ...... ............. .............................................. 93
R e su lts....................................................................................................... ....... .. 9 3









SH P-2A46-110 and SH P-2 W T C ells.................................................. ................ 93
Jak2 Phosphorylation Is Not Influenced by the N-terminal SH2 Domain of
SH P -2 ................ . .. ........................... ... ... ... ................... 93
STAT1 and STAT3 Phosphorylation and STAT-mediated Gene Transcription
Require the N-terminal SH2 Domain of SHP-2 ........................................95
Jak2-AT1 Receptor Co-association Is Mediated by SHP-2..............................97
Transfecting Wild Type SHP-2 back into SHP-2A46-110 Cells Restores STAT1
and STAT3 Phosphorylation and STAT-mediated Gene Transcription........ 100
Jak2 Tyrosine 201 Mediates Jak2-SHP2 Interactions.................................. 101
Jak2 Tyrosine 201 Mediates AT1 Receptor-Jak2 Co-association, STAT1 and
STAT3 Activation and STAT-mediated Gene Transcription..................... 106
D isc u ssio n ............................................................................................................. .. 1 0 8

6 CONCLUSIONS AND IMPLICATIONS ................. ......................................113

Su m m ary of R esu lts ......1.......... .... .......... .. ...... .... .................... ........ .............. 113
Src Kinases and Angiotensin II-induced Cell Proliferation ................................114
The role of SHP-2 in angiotensin II, Jak2/STAT signaling.................................125
Angiotensin II signaling and disease ....... ... ...... ..................... 126

LIST O F R EFEREN CE S .. .................................................................... ............... 128

BIOGRAPH ICAL SKETCH ................. ............................................................... 143
















LIST OF TABLES


Table page

1-1 K n ow n R SK sub states ............................................................................................. 15

1-2 Pharmacological inhibition of Src kinase-independent ERK1/2 inhibition.............41















LIST OF FIGURES


Figure page

1-1 Sum m ary of the renin-angiotensin system ........................................... ...............3...

1-2 D iagram of the rat A T ia receptor........................................................... ...............5...

1-3 Jak2 structural dom ain s ............................................ .......................... ...............7...

1-4 The Jak/STA T signaling paradigm ...................... ............................................9...

1-5 Structure/function of conserved domains within Src family tyrosine kinases ........ 10

1-6 Structure and signaling of ribosmal S6 kinase....................................................14

2-1 Characterization of WT/AT1 and SYF/AT1 cells................................................27

2-2 Quantification of ERK1/2 activation in response to Ang II in WT/AT1 and
SY F/A T I cells .......................................................................... .......... ............... 28

2-3 Nuclear translocation of active ERK2 is unaffected by the loss of c-
S rc/Y e s/F y n ............................................................................................................. 3 1

2-4 Ang II-induced cell proliferation is reduced in Ang II-stimulated SYF/AT1
c e lls ......................................................................................................... ....... .. 3 3

3-1 ERK1/2 activation in SYF/AT1 cells does not require transactivation of the
PDGFR or the EGFR or activation of PI3K ................ ....................................42

3-2 ERK1/2 activation in SYF/ATi cells requires MEK1/2 activation, but not Rafl
a c tiv a tio n ............................................................................................................... .. 4 4

3-3 Ang II-induced ERK1/2 activation is partially dependent upon heterotrimeric G
p ro te in s .................................................................................................................. ... 4 7

3-4 Ang II-induced ERK1/2 activation is partially dependent upon PKC.. ................. 49

3-5 PKC( mediates Ang II-induced ERK1/2 activation independent of c-
S rc/Y e s/F y n ............................................................................................................. 5 1

3-6 PKC(-specific siRNA attenuated Ang II-induced ERK1/2 activation in WT/AT1
c e lls ........................................................................................................................... 5 2









3-7 MEK phosphorylation is dependent upon PKC. ............. ............... 53

3-8 Nuclear translocation of active ERK2 is controlled by PKC-dependent
sig n a lin g ............................................................................................................. .. 5 6

3-9 Ang II-induced cell proliferation is completely attenuated by blocking c-
Src/Y es/Fyn and PK C-dependent signaling. ........................................ ............... 57

4-1 RSK2 phosphorylation and RSK2-ERK1/2 co-association are decreased in
S Y F /A T 1 cells. ............. ................................................ ................... .. 6 7

4-2 RSK2 and ERK1/2 nuclear phosphorylation in response to Ang II in WT/AT1
and SYF/AT1 cells ....................... ........... .........................70

4-3 Ang II-induced SRF and elkI nuclear phosphorylation in WT/AT1 and SYF/AT1
c ells ......................................................................................................... ....... .. 7 3

4-4 c-fos transcriptional activity in WT/AT1 and SYF/AT1 cells in response to Ang
II. ............................................................................................................ ....... .. 7 5

4-5 c-fos protein levels in response to Ang II in WT/AT1 and SYF/AT1 cells ..............76

4-6 Ang II-induced c-fos phosphorylation in WT/AT1 and SYF/AT1 cells.................. 78

4-7 Ang II-induced cell proliferation in response to RSK and PKC inhibition........... 80

4-8 Ang II-induced ERK1/2 activation is mediated by c-Src/Yes/Fyn and PKC(-
dependent signaling in V SM C .................................... ..................... ................ 82

4-9 Angiotensin II-induced cell migration is attenuated through selective RSK
in h ib itio n .............................................................................................................. .. 8 4

4-10 Mechanistic diagram illustrating how Src kinase and PKC-dependent ERK1/2
activation pathways dually regulate Ang II-induced cell proliferation.................85

5-1 Jak2 tyrosine phosphorylation in SHP-2 WT or SHP-2A46-110 fibroblast cells........94

5-2 STAT1/3 phosphorylation and STAT-induced luciferase activity in SHP-2 WT
or SHP-2A46-110 transfected fibroblast cells......................................... ................ 96

5-3 The recruitment of Jak2 to the AT1 receptor is dependent upon SHP-2...............99

5-4 AT1/Jak2 co-association, STAT1 phosphorylation and STAT-induced luciferase
activity are restored in SHP-2A46-110 fibroblasts transfected with wild type SHP-
2 .............................................................................................................................. 1 0 2

5-5 SHP-2/Jak2 co-association occurs mainly through interaction of Jak2 amino
acids 1-294 and the N terminal SH2 domain of SHP-2. .............. ...................103









5-6 Jak2 tyrosine 201 is critical for Jak2/SHP-2 interaction.................................. 105

5-7 Mutation of Jak2 tyrosine 201 reduces Jak2-dependent signaling in response to
an g io ten sin II.......................................................................................................... 10 7

5-8 Proposed mechanism for Jak2/SHP-2 interactions upon stimulation of the AT1
re c e p to r .............................................................................................................. ... 1 1 2

6-1 Possible CRM-1-dependent mechanism influencing ERK1/2 localization. .......118

6-2 Possible mechanism whereby phosphorylation of tyrosine 314 affects ERK1/2
subcellular localization ...................................... ......................... ............... 119

6-3 Proposed mechanism whereby substrate recognition influences ERK1/2
subcellular localization ..................................................................................... 120

6-4 Preliminary data supporting the hypothesis that ERK1/2 subcellular distribution
effects the mechanism of upstream activation.. ................................ ................ 122















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

JAK2 AND SRC FAMILY TYROSINE KINASE SIGNALING VIA THE
ANGIOTENSIN II TYPE 1 RECEPTOR

By

Michael D. Godeny

December 2006

Chair: Peter P. Sayeski
Major Department: Medical Sciences- Physiology and Pharmacology

The AT1 receptor is a prototypical G protein-coupled receptor activated through

high affinity binding of the hormone, angiotensin II (Ang II). More recent work has

demonstrated that the AT1 receptor can activate tyrosine kinases independent of

heterotrimeric G proteins. This dissertation focuses on tyrosine kinase-mediated

signaling events downstream of the angiotensin II type 1 (ATi) receptor. Specifically,

the involvement of Src family tyrosine kinases in angiotensin II-induced ERK1/2

activation and cell proliferation is explored. In addition, angiotensin II, Jak2 signaling is

explored in detail with respect to Jak2's interaction with the phosphatase, SHP-2. As

such, this work provides valuable insight into angiotensin II signaling.

Src family tyrosine kinases mediate as much as 50% of angiotensin II-induced

ERK1/2 activation and cell proliferation. The remaining 50% is mediated by

heterotrimeric G protein and PKC signaling. In addition, these two signaling cascades

activate ERK1/2 and initiate cell proliferation independent of one another. Interestingly,









the cellular consequence associated with ERK1/2 activation by each of these two distinct

pathways is different. When ERK1/2 is activated by heterotrimeric G protein and PKC-

dependent signaling, it translocates into the nucleus and initiates cellular proliferation

through the activation of the transcription factor, elkl. When ERK1/2 is activated by Src

kinase-dependent signaling, it remains in the cytoplasm and phophorylates ribosomal S6

kinase (RSK). Ultimately, RSK translocates into the nucleus and modulates cell

proliferation via the activation of the serum response factor (SRF), another known

transcription factor. Thus, the cell mediates angiotensin II-induced cell proliferation

through the activation of ERK1/2 via two independent signaling pathways.

Jak2 is another tyrosine kinase phosphorylated by angiotensin II. Here, it is

demonstrated that angiotensin II, Jak2-dependent signaling requires SHP-2. SHP-2 acts

as an adaptor molecule (at the site of the N terminal SH2 domain), serving to recruit Jak2

to the AT1 receptor via interactions with the Jak2 tyrosine 201 residue. Jak2 then recruits

STAT1 and STAT3 proteins, which dimerize and translocate into the nucleus. STAT

nuclear translocation initiates the transcription of STAT responsive genes. As such,

SHP-2 positively regulates Jak2 signaling in response to Ang II.

Collectively, this work helps to redefine the angiotensin II signaling paradigm, and

may aid in the future treatment Ang II-associated diseases.














CHAPTER 1
INTRODUCTION

Overview

Angiotensin II (Ang II) is responsible for a wide array of biological effects which

are primarily mediated by the angiotensin II type 1 (ATi) receptor. The intracellular

signaling pathways associated with AT1 receptor activation relay an Ang II-induced

signal from the cell surface to the appropriate intracellular proteins, resulting in a desired

cellular outcome. Since the ATi receptor is a prototypical G protein-coupled receptor

(GPCR), many of these signaling events are dependent upon heterotrimeric G protein-

mediated signaling. However, in the 1990's a paradigm shift emerged in the field of

angiotensin II signaling when it was discovered that the ATi receptor could also activate

tyrosine kinases and induce signaling independent of heterotrimeric G proteins. Since

then, ongoing work has focused upon the characterization of these tyrosine kinase-

mediated signaling events. This dissertation will emphasize two specific tyrosine kinase

families activated by the ATi receptor: the Janus kinases (JAK) and Src kinases. The

following chapter will serve as an introduction to angiotensin II signaling as well as

important signaling molecules involved in ATi receptor-induced processes, including

Janus and Src kinases. The remaining chapters will examine two specific JAK and Src

kinase-dependent signaling events. First, the role of Src family tyrosine kinases will be

explored in Ang II-induced cell proliferation, and a mechanism will be proposed for this

process. Second, the activity of an important Janus kinase family member, Jak2, will be

explored with respect to its interactions with a well-known phosphatase, SHP-2. As such,









the following chapters provide information about the signaling mechanisms of two

important tyrosine kinase families activated by angiotensin II.

Angiotensin II

History of the Renin Angiotensin System

Angiotensin II was initially identified as a product resulting from the direct

cleavage of a plasma substrate (later named angiotensinogen) by the kidney-produced

enzyme renin (11, 94). The groups of Braun-Menendez and Page and Helmer

independently made this discovery, and named the peptide hypertension and

"angiotonin" respectively. Both groups settled on the name "angiotensin" and

demonstrated that this peptide was a remarkable inducer of vasoconstriction. Leonard T.

Skeggs and colleagues later purified the immediate precursor of Ang II, the 10 amino

acid peptide angiotensin I, from hog renin and horse plasma (127). In one study, Skeggs

accidentally purified angiotensin I in the presence of 0.15M NaC1, and noticed that an 8

amino acid variant of this protein was formed. This octapeptide turned out to be

angiotensin II, which was subsequently shown by Skeggs and colleagues to be produced

from the cleavage of angiotensin I by angiotensin-converting enzyme (ACE) (126).

Finally, the lung was identified as the tissue-source for ACE (88, 107), and the

biochemical pathway known today as the "renin-angiotensin" system was established

(Figure 1-1). To this day, angiotensin II remains the primary effector molecule of the

renin-angiotensin system.

Physiological Effects of Angiotensin II Associated With Binding of the ATi Receptor

The angiotensin II type 1 (ATi) receptor is regarded as the receptor which mediates

the majority of the physiological responses associated with Ang II. It is expressed in a

number of tissues, including the vasculature, heart, kidney, lung, adrenal gland, intestine








and brain. Due to the presence of the AT1 receptor in a number of distinct tissue beds,

Ang II is implicated in an array of physiological responses. In the kidney, for example,

Ang II increases glomerular filtration rate by stimulating constriction of the efferent

arteriole (130). AT1 receptor stimulation in the adrenal cortex initiates aldosterone

synthesis, resulting in the reabsorption of sodium in the distal convoluted tubule of the

kidney (92). Ang II also induces sodium reabsorption in the intestine (68), while in the

brain Ang II triggers a thirst response by directly stimulating regions of the

NH2 COOH
Angiotensinogen DF-H-L
(453 AA) D-R-V-Y-1-H-P-F-H-L V-1-H-------------

4 Renin
Angiotensin I
(10 AA) D-R-V-Y-I-H-P-F H-L

A ACE &'C
*Angiotensin II I AT1R
(8 AA) D R-V-Y-I-H-P-F ATR2
SAT2R
iten Aminopeptidase
Angiotensin III
(7 AA) R-V-Y-I-H-P-F

T ^ Various Peptidases
Inactive
Metabolites
Figure 1-1. Summary of the renin-angiotensin system. Angiotensinogen is cleaved by
renin to yield angiotensin I. Angiotensin I is then further cleaved by
angiotensin-converting enzyme (ACE) to produce Ang II. Ang II binds to
either the ATi or AT2 receptors, or is degraded to angiotensin III and other
inactive metabolites by various peptidases.
hypothalamus (99). Finally, Ang II acts as a potent vasoconstrictor within the vasculature

(48). These Ang II-induced effects within different tissues collectively help maintain

mammalian blood pressure and fluid electrolyte amounts at homeostatic levels. In









addition to its role as a regulator of blood pressure and fluid osmolality, angiotensin II

also acts as a potent growth factor. These Ang II-induced cellular growth and

proliferative responses are associated with disease states. For example, Ang II has been

linked to cardiac hypertrophy. Cardiac hypertrophy is classified as a thickening of the

muscles in the wall of the left ventricle to compensate for increases in preload volume,

and Ang II has been directly implicated in this condition (25). In addition, Ang II

contributes to aberrant vascular smooth muscle cell proliferation during neointimal

formation as well as following balloon-injury from angioplasty (55, 93). Finally, Ang II

has been linked to angiogenesis during cancer, a process by which new blood vessels

form and grow in order to feed rapidly-proliferating tumor cells (24). As such, Ang II is

a major contributor to maladaptive growth and proliferative responses associated with

cardiovascular diseases and cancer.

Angiotensin II Signaling

The diversity of systemic effects attributed to angiotensin II is in part due to the

multitude of intracellular signaling pathways activated by the AT1 receptor (Figure 1-2).

For example, the AT1 receptor couples to and activates heterotrimeric G proteins, and

many downstream signaling events are dependent upon these events. Upon ATi receptor

activation, the Ga subunit binds GTP and dissociates from the G3y subunits, allowing

each subunit to interact with other signaling molecules. Specifically, angiotensin II can

couple to Gaq, which stimulates Phospholipase C beta (PLCO) to convert

Phosphatidylinositol 4,5-bisphosphate (PIP2) to Inositol 1,4,5 Triphosphate (IP3) and

Diacylglycerol (DAG) (113, 151). DAG activates some isoforms of Protein Kinase C

(PKC), a serine/threonine kinase that can phosphorylate a number of substrates, while IP3










1 --7





Bilipid membrane





219W 312y

Amino Acid Residue Function

1-309 Src kinase Activation
219-225 Heterotrimeric G protein
activation
309-359 IP3 release
312-314 Heterotrimeric G protein
activation
319-322 Jak2 co-association

Figure 1-2. Diagram of the rat ATia receptor. The seven transmembrane spanning
domains are drawn from left to right as indicated. The positions and functions of amino
acid residues important for angiotensin II signaling are also indicated.

binds IP3 receptors on the endoplasmic reticulum and causes a calcium efflux into the

cytoplasm. In addition, the AT1 receptor can couple to Gpy, and, in combination with

Gal2, activate phospholipase D in vascular smooth muscle cells (147). A final example

of AT1 receptor, heterotrimeric G protein-dependent signaling occurs when Gas

stimulates adenylate cyclase, which converts ATP to cyclic adenosine monophosphate

(cAMP) (135). cAMP then stimulates PKA, an AGC kinase capable of phosphorylating

a variety of cellular substrates. Thus, the ATi receptor can activate numerous secondary

messengers via heterotrimeric G protein-dependent signaling. However, there appears to









be disparity in heterotrimeric G protein-mediated signaling between cell types, which can

be explained by cell-specific differences in the expression of a and Py subtypes (118).

In addition to being a prototypical GPCR, the AT1 receptor can also activate a

variety of non-receptor associated tyrosine kinases. These include c-Src, Yes, Fyn, Pyk2,

Jak2, and FAK (118). The mechanisms of tyrosine kinase activation by the AT1 receptor

are poorly defined in many cases; however, some of the specific amino acid residues on

the receptor necessary for tyrosine kinase activation have been identified. Interestingly,

many of these residues are different from residues linked to heterotrimeric G protein

activation. For example, Jak2 tyrosine kinase has been shown to co-associate with the

AT1 receptor through a specific 319YIPP motif on the carboxyl terminal tail (3). In

contrast, heterotrimeric G protein activation has been linked to W219-A225 and Y312-

L314 motifs on the third intracellular loop and C terminal tail (113, 151). Thus, a

number of tyrosine kinases are activated downstream of the AT1 receptor, and often

signal independently of heterotrimeric G proteins.

The JAK Family of Tyrosine Kinases

Structure

The Janus kinase (JAK) family of tyrosine kinases is activated by a number of receptors,

including the AT1 receptor (79). Each member of the Janus family shares the distinct

structural feature of having a kinase domain directly adjacent to a pseudokinase domain,

and therefore cleverly received their namesake after "Janus," the Roman god of two

opposing faces. Members of the JAK family include Jakl, Jak2, Jak3, and Tyk2. Each

of these proteins is approximately 130 kDa in mass, and are structurally similar since

they contain seven conserved JAK homology domains. The structure of Jak2 is

illustrated as an example of JAK structure (Figure 1-3).












Pseudokinase
FERM SH2-like Domain Kinase Domain
A t -- -- r\/\ f ---- "


NH2 JH7 JH6 JH5 JH4 JH3 JH2 JH1 COOH



Y201 V617F Y1007
Y1008

Domain Amino Acid

JH7 38-122
JH6 144-284
JH5 288-309
JH4 322-440
JH3 451-538
JH2 543-827
JH1 836-1123



Figure 1-3. Jak2 structural domains. Shown are the positions of the seven-conserved Jak
homology (JH) domains as well as the amino acid sequence for each domain
within Jak2. Amino acid residues and mutations known to affect Jak2 activity
are also indicated. Reproduced with permission from Current Medicinal
Chemistry, in press, Copyright 2006 American Chemical Society.

Jak2/STAT Signaling

Of all the JAK family members, Jak2 has been perhaps implicated the most in

angiotensin II signaling. Even though Jak2 lacks canonical SH2 and SH3 domains, it is

still able to associate with the AT1 receptor and induce gene transcription. Jak2-

dependent gene transcription occurs through the phosphorylation of STAT (signal

transducers and activators of transcription) proteins (8, 9, 79). When phosphorylated,

STAT proteins form hetero/homodimers and migrate into the nucleus, where they bind

STAT recognition sequences within gene promoters. These events trigger STAT-

mediated transcription in a variety of early response genes. The Jak2/STAT signaling

paradigm was originally identified in the context of cytokine receptors, but more recent









work has established a mechanism by which GPCRs like AT1 signal through Jak2 (112).

The specific events associated with Jak2/STAT signaling are described in detail in Figure

1-4. As such, Jak2 serves as a conduit between angiotensin II binding at the surface of

the cell and gene transcription within the nucleus.

The Src Family of Tyrosine Kinases

Structure

The Src family oftyrosine kinases was the first tyrosine kinase family identified. This

family of tyrosine kinases is comprised of fourteen different family members, including

c-Src, Yes, Fyn, Yrk, Fgr, Lyn, Hck, Lck and Blk. The fourteen different Src kinases are

derived from nine separate genes, with alternative splicing accounting for a portion of

these gene products. All Src kinase family gene products are similar in size (55 62

kDa), and share common structural features, including an SH2 domain, an SH3 domain

and a tyrosine kinase domain (Figure 1-5). Three of the Src kinases-c-Src, Yes, Fyn-

are ubiquitously expressed, while the expression of other family members is restricted to

hematapoietic cells. Due to a common structure and similar expression patterns, there is

functional redundancy amongst certain family members, namely c-Src, Yes and Fyn, as

demonstrated through the generation of specific knockout mice (62, 131).

c-Src/Yes/Fyn Knockout Mice

Mutations in either the c-src orfyn genes were shown previously to lead to

restricted nonoverlapping phenotypes only in a subset of cells in which these kinases are

expressed, while a mutation in the yes gene does not lead to an overt phenotype (131).

Except for brain, the level or distribution of related Src family kinases is not altered in

major tissues, demonstrating that there may be functional redundancy among these Src

















Receptor


Figure 1-4. The Jak/STAT signaling paradigm. Ligand binding to the receptor initiates
receptor activation. For cytokine receptors (A), receptor dimerization triggers
Jak2 autophosphorylation, resulting in the Jak2-dependent phosphorylation of
STATs. In the case of the AT1 receptor (B), Jak2 becomes activated in the
cytoplasm and is recruited to the receptor, where it phosphorylates the STATs.
STAT molecules form homo/hetero dimers upon phosphorylation (C), and
translocate into the nucleus. There, STATs bind STAT-responsive elements
within gene promoters (D) and initiate transcription. Reproduced with
permission from Current Medicinal Chemistry, in press, Copyright 2006
American Chemical Society.

family members. Generation of c-src, yes, or fyn double mutants sought to provide more

evidence for this redundancy of function. The src/fyn or src/yes double mutants die

perinatally, while a substantial proportion of fyn/yes double mutants are viable but

undergo degenerative renal changes leading to diffuse segmental glomerulosclerosis

(131). Finally, c-Src/Yes/Fyn triple knockout mice die during development (62). Taken










Catalytic Domain


Unique N Terminus







NH2 M --SH3


Activation Loop


Region
N terminal myristoylation sequence (M)
SH3 domain
SH2 domain

Catalytic domain

Activation loop

C terminal tail


Function
Anchors protein to membrane
Binds proline rich sequences
Binds phosphorylated tyrosine
residues
Enzymatic activity; divided into two
lobes
Phosphorylation regulated protein
activity
When phosphorylated, binds SH2
domain


Figure 1-5. Structure/function of conserved domains within Src family tyrosine kinases.
Shown above are the relative positions of conserved domains and regions
characteristic of the Src family members. Conserved phosphorylation sites
are also indicated. The putative function of each domain/region is listed in the
corresponding table.

together, these data are consistent with the hypothesis that c-Src, Yes, and Fyn tyrosine

kinases are able to compensate for the loss of one or more related Src kinases.

AT1 Receptor-induced Signaling

Like other tyrosine kinases, Src kinases are activated by a wide variety of receptors,

including the AT1 receptor. Work in vascular smooth muscle cells and cardiac myocytes

has shown that Src kinases are phosphorylated in response to Ang II treatment (54, 109).









Ang II-induced activation of Src kinases precedes the activation of important downstream

signaling events, including the mitogen-activated protein (MAP) kinase (53, 109, 115,

140) and PLCyl/IP3/Ca2+ (80) signaling pathways. Work by the Sadoshima laboratory

has shown that amino acid residues 1-309 are critical for Src kinase activation and

downstream signaling, but not for other Src-independent events such as IP3 release (122).

Thus, Src kinases must bind the C-terminal tail of the AT1 receptor in order to become

activated. However, the molecular events describing how AT1 receptor activation leads

to the activation of Src kinases are not well understood.

ERK1 and ERK2 MAP Kinases

The MAP Kinase Superfamily

MAP kinases are evolutionary conserved enzymes that phosphorylate their

substrates on serine/threonine residues. MAP kinase family members are grouped into

three sub-families based on their activation sequences: the c-Jun NH2-terminal kinases

(JNKs), the p38 MAP kinases, and the extracellular signal-regulated kinases (ERKs). All

of these MAP kinases mediate an intracellular effect in response to extracellular stimuli,

although different types of stimuli preferentially activate certain MAP kinases (98). For

example, JNKs and p38 MAP kinases are often activated in response to extracellular

stress, including UV irradiation, heat shock, osmotic stress and inflammatory kinase

stimulation. ERKs are often activated by growth factors and hormones, and mediate

different cellular responses, including cell growth, differentiation, proliferation and

growth arrest. In addition, the cellular outcome associated with MAP kinase activation

will also depend upon the duration and magnitude of activation. For example, transient

or sustained patterns of ERK1/2 activation have been shown to differentially affect

developmental and adult mammalian cell processes (1, 13, 21, 64, 76, 138).









ERK1/2 Signaling

A general three-tiered signaling cascade describes the activation of all MAP

kinases. This starts with the activation of a MAP kinase kinase kinase by a receptor,

which phosphorylates a MAP kinase kinase, which in turn phosphorylates a MAP kinase

in order to achieve the desired cellular effect. In the case of ERK1/2, the MAP kinase

kinase kinase activated is Ras, which phosphorylates Raf (MAP kinase kinase). Raf then

phosphorylates MEK1/2, which is the immediate upstream activator of ERK1/2. MEK-

induced ERK1/2 phosphorylation occurs via dual threonine and tyrosine phosphorylation

at a conserved TEY motif within the activation loop. Activated ERK1/2 then

phosphorylates substrates at proline-directed serine and threonine residues. The

substrates acted upon by ERK1/2 are often dependent upon the scaffolding proteins

bringing the MAP kinase signaling complex together, the ligand-receptor interaction

responsible for ERK1/2 activation, the cell type and the presence of substrate within the

cell (19, 20). Finally, ERK1/2 signaling is terminated via dephosphorylation by

phosphatases. For example, MAP kinase phosphatase 1 dephosphorylates ERK1/2 in a

Jak2-dependent manner, effectively shutting off ERK1/2 signaling (111). As such,

ERK1/2 signaling occurs in a coordinate manner and is controlled by many proteins. The

regulation of ERK1/2 signaling is required in order to control such processes as cell

growth, proliferation, differentiation, and growth arrest (19, 105).

Ribosomal S6 kinase

The RSK Family of Proteins

Ribosomal S6 kinase (RSK) is a member of the AGC family of serine/threonine

kinases, which also includes protein kinases A, G and C. RSK is the 90 kDa protein

within this family, and was originally discovered in Xenopus laevis oocytes by Erikson









and Maller in 1985 (35). To date, three isoforms of RSK have been identified (RSK1-3),

and each of these proteins are products of separate genes (37). RSK1-3 are

approximately 90% identical, and exhibit functional redundancy in tissues where each is

expressed, including the brain. Furthermore, RSK homologues are present in a variety of

organisms, including human, mouse, rat, chicken, Drosophila and C. elegans. As such,

RSK proteins appear to be physiologically important, which is directly demonstrated by

the fact that RSK defects have been linked to human Coffin-Lowry syndrome, a disease

characterized by mental retardation, facial and digital dysmorphologies and progressive

skeletal malformations (143).

RSK was originally identified for its ability to phosphorylate the 40S ribosomal

subunit protein, S6 (36). The phosphorylation of S6 promotes the translation of selected

mRNAs important for cell growth. In 1990, however, a 70 kDa S6 kinase (p70s6K) of

60% sequence homology to RSK was identified, and this protein was found to be the

primary protein responsible for S6 phosphorylation (6, 7, 17, 63). It is now believed that

RSK phosphorylates S6 only under special circumstances, leaving the main function of

RSK unknown (37, 38). What is agreed upon is that RSK is a substrate for ERK1/2,

although the function of ERKl/2-induced RSK activation is still unknown in most cases,

including during AT1 receptor activation.

Structure and Function of RSK Proteins

The structure of RSK is undoubtedly tied to its function. RSK proteins are unique

since they contain both an N terminal and a C terminal kinase domain, connected by a

linker region (Figure 1-6). The N terminal kinase domain is responsible for the activation

of other kinases, and recognizes the following consensus motifs: Arg/Lys-X-Arg-X-X-









Ser/Thr or Arg-Arg-X-Ser/Thr (66). The C terminal kinase domain is most closely

associated with the calcium/calmodulin-dependent group of kinases, and phosphorylation

of this domain precedes the activation of the N terminal kinase domain. In addition,

phosphorylation of amino acid residues within the linker region has also been shown to

play a role in the activation of RSK proteins (37). As such, RSK activation is regulated

by ERK1/2 and phosphorylates a variety of substrates containing specific consensus

motifs, including transcription factors, cytoplasmic proteins, steroid receptors and

ribosomal proteins (Table 1-1).



B

C p P P P ERK1/2 A


NH2 NTK CTK EB COOH






p Region Amino Acid
N terminal kinase (NTK) 68-323
D Substrate Linker 323-422
C terminal kinase (CTK) 422-675
ERK binding (EB) 727-728,
730-731


Figure 1-6. Structure and signaling of ribosmal S6 kinase. Shown above are the positions
of the N terminal kinase (NTK) domain, the C terminal kinase (CTK) domain,
and the ERK-binding (EB) domain. Upon ligand stimulation, ERK1/2 bind to
the EB domain (A), which initiates phosphorylation of residues within the
CTK and linker region (B). In addition, the CTK phosphorylates the linker
region (C). These events are necessary for the activation of the NTK (C),
which phosphorylates RSK substrates (D) at specific consensus sequences.
Specific amino acid sequences for RSK2 domains and the linker region are
indicated.












Table 1-1. Known RSK substrates.

RSK Substrate Category Reference
Glycogen synthase kinase 3 Cytoplasmic protein (34, 139)
Sos Cytoplasmic protein (29)
L1 cell adhesion molecule Cell Adhesion (155)
Polyribosomes Translation (5)
IKBa/NFKp3 Transcription factor (39, 119)
Estrogen receptor Steroid receptor (59)

CREB Transcription factor (23, 40, 156)
c-fos Transcription factor (14, 15)
CBP Transcription co-activator (87)


The Angiotensin II Signaling Paradigm

A number of proteins, including those just listed, are activated in response to

angiotensin II. It was originally thought that the ability of angiotensin II to produce

either a pressor response or a growth and proliferative response was dependent upon

whether the signaling downstream of the AT1 receptor was mediated by tyrosine kinases

or heterotrimeric G proteins. For example, increases in Ang II-induced vasoconstriction

have been shown to be dependent upon Ang II-induced calcium release, which occurs in

part through heterotrimeric G protein, PLC3 signaling (113, 151). On the other hand,

many growth and proliferative responses have been linked to Ang II-induced tyrosine

kinase activation, primarily during cardiovascular and cancer disease states (24, 47, 65,

82, 100, 134, 140, 141). As such, heterotrimeric G protein and tyrosine kinase-mediated

signaling have often been viewed as independent signaling events, determining the

cellular outcome associated with AT1 receptor activation in a given tissue.

More recent work, however, has shown that heterotrimeric G protein and tyrosine

kinase signaling events are often interwoven with respect to the AT1 receptor. For

example, work from our group has shown that Jak2 tyrosine kinase regulates intracellular









calcium release (150), a process which is also highly dependent upon heterotrimeric G

proteins (51). Furthermore, Ang II-induced IP3 production has been shown to be

dependent on both heterotrimeric G proteins (through PLC 31) as well as Src tyrosine

kinases (via PLCy1) (80, 113, 151). Finally, this dissertation will focus extensively on

the mechanisms of Ang II-induced cell proliferation, which (as described later) is both

heterotrimeric G protein-dependent and tyrosine kinase-dependent. Thus, the portrayal of

AT1 receptor signaling as occurring through two types of linear cascades with no

interaction seems oversimplified, since some signaling events are facilitated by

interaction between both heterotrimeric G protein and tyrosine kinase signaling. Clearly,

more work needs to be done in order to understand the complexity behind angiotensin II

signaling.

Summary and Rationale

Angiotensin II signaling occurs in part through heterotrimeric G protein as well as

tyrosine kinase-mediated signaling events. Of the kinase-induced signaling pathways,

many are reliant upon members of the Janus and Src families of tyrosine kinases. Since

Ang II initiates a multitude of tissue-specific effects, it is important to understand how

these tyrosine kinases regulate the signaling associated with such processes. In addition,

it is also important to understand how JAK and Src kinase activity is regulated since

these proteins essentially serve as the "on" switch for many AT1 receptor-driven

signaling processes.

Angiotensin II has already been linked to both cardiovascular disease and cancer,

and the ATi receptor is currently a target for the treatment of these disease states (24, 31,

49, 60, 137). Not surprisingly, Jak2 and Src kinases have also been implicated in many

of these same disease states, and are also promising candidates for therapeutic






17


intervention (47, 65, 82, 100, 134, 140, 141). The goal of this study is to provide a better

understanding of Src family tyrosine kinase and Jak2 tyrosine kinase function with

respect to the AT1 receptor. Specifically, the role of c-Src/Yes/Fyn in angiotensin II-

induced cell proliferation will be examined. In addition, the role of SHP-2 in Jak2

signaling will also be explored. Since Src and Janus kinases play such a pivotal role in

angiotensin II signaling, it is the hope that the knowledge generated from this work will

aid in the treatment of Ang II-associated diseases.














CHAPTER 2
C-SRC/YES/FYN TYROSINE KINASES MEDIATE A PORTION OF ANGIOTENSIN
II-INDUCED ERK1/2 ACTIVATION AND CELLULAR PROLIFERATION

Introduction

Src family tyrosine kinases mediate an array of signaling processes in response to

high affinity binding of angiotensin II to the AT1 receptor (118). Included in this list is

the activation of a pro-mitotic MAP kinase signaling cascade, resulting in the

phosphorylation of intracellular ERK1/2. Berk et al have demonstrated that Ang II-

induced ERK1/2 activation is critically mediated by c-Src in vascular smooth muscle

cells (53). The authors claim that Ang II-induced ERK1/2 activation was blocked by

each of the following: pharmacological inhibition using broad tyrosine kinase inhibitors

(CP-188,556 or Genistein), c-Src knockout VSMCs or retroviral transduction of

dominant-negative c-Src into rat VSMCs. Thus, they conclude that ERK1/2 activation is

completely dependent upon c-Src.

While it is clear from these studies that c-Src is indeed implicated in Ang II-

induced ERK1/2 activation, an essential role for c-Src in these signaling events may be

over-interpreted. This is demonstrated by the fact that complete inhibition of ERK1/2

activation was only achieved when cells were pretreated with 100 PM of pharmacological

inhibitor, while only partial inhibition of ERK1/2 activation occurred in c-Src -/- mouse

VSMCs or in rat VSMCs transduced with dominant-negative c-Src. These findings also

raise speculation as to whether the complete inhibition of Ang II-induced ERK1/2

activation was achieved through the simultaneous, non-specific inhibition of other









signaling proteins via the pharmacological inhibitors utilized in these experiments. In

addition, it is not clear if the remaining ERK1/2 activation present in the c-Src dominant-

negative transduced VSMCs or c-Src -/- VSMCs was due to functional redundancy by

other Src kinases expressed within these cells, incomplete inhibition of c-Src function or

activation of ERK1/2 by c-Src-independent signaling. Therefore, the dependency of Ang

II-induced ERK1/2 activation on Src family tyrosine kinases is still in question.

Other work by Schiffrin and colleagues has demonstrated a critical role for c-Src in

growth signaling by angiotensin II in VSMCs from arteries of hypertensive patients

(140). This growth response occurred through the c-Src-dependent activation of ERK1/2

in response to Ang II treatment, and thus implicated c-Src in growth and proliferative

effects associated with Ang II. Work done by our group identified a mechanism whereby

c-Src activates ERK1/2 via coupling to the Shc/Grb2/Sos signaling cascade, leading to

the activation of RAS in response to Ang II (115). However, it should be noted that in

each of these reports, ERK1/2 activation was not completely achieved though c-Src

inhibition, again raising the question that Ang II-induced ERK1/2 activation is also

occurring through other Src kinases or through Src kinase-independent signaling.

Additional work has shown that other Src family tyrosine kinases are also

implicated in Ang II-induced ERK1/2 activation. Sadoshima and Izumo demonstrated

that in cardiac myocytes, Ang II activates Fyn and stimulates the association of Shc with

Fyn and the subsequent formation of a Shc-Grb-Sos complex (109). These authors

further demonstrated that Fyn activates Ras through the formation of this complex, thus

leading to ERK1/2 activation. As such, Fyn can also activate ERK1/2 in response to

angiotensin II. In addition, the Src kinase Yes cannot be ruled out as a mediator of Ang









II-induced ERK1/2 activation due to a similar structure, expression pattern, and already

proven ability to compensate for a loss of related Src kinase function (131). Thus, it is

apparent that c-Src is not the only Src family tyrosine kinase mediating ERK1/2

activation in response to Ang II, as suggested from previous studies.

Clearly, more work needs to be done in order to determine the contribution that all

Src family tyrosine kinases have on Ang II-induced ERK1/2 activation since previous

reports have generated conflicting results. This is likely due to the fact that ERK1/2

activation was not examined in a completely Src kinase-deficient background. Here, c-

Src/Yes/Fyn-deficient mouse embryonic fibroblast (VIMEF) cells stably transfected with

the AT1 receptor were utilized. These cells will be described in detail later in this

chapter, and provide a powerful system for identifying the dependency of Ang II-induced

ERK1/2 activation on Src family tyrosine kinases because they completely lack

functional c-Src/Yes/Fyn (62). Therefore, these cells lack the ubiquitously expressed Src

kinases present in mammalian cells, and also lack the remainder of Src family kinases

since the expression of these proteins is restricted to cells of hematopoietic lineage. As

such, the possibility of compensation for the loss of one Src kinase by another is

eliminated by the use of the c-Src/Yes/Fyn-deficient cells.

From these studies, it was found that Src kinases mediate approximately 50% of

angiotensin II-dependent ERK1/2 activation. It appears that other signaling processes

mediate the remainder of ERK1/2 activation independent of Src kinases. Interestingly, a

loss of c-Src/Yes/Fyn did not affect the ability of ERK1/2 to translocate into the nucleus,

but did cause a reduction in Ang II-induced cell proliferation. As such, c-Src/Yes/Fyn-

activated ERK1/2 mediates cell proliferation independent of nuclear translocation. These









findings therefore provide valuable insight into signaling via the AT1 receptor and the

intracellular events associated with Ang II-induced cell proliferation.

Materials and Methods

Creation of WT/ATi and SYF/ATi Stable Cell Lines

Immortalized SYF and WT MEF cells were a gift from Dr. Philippe Soriano, and

were previously isolated from c-Src/Yes/Fyn triple knockout and WT mice respectively

at E9.5 (62). Both cell lines lack endogenous AT1 receptor expression, and were

therefore stably transfected by our group with 20 atg of an HA-tagged AT1 receptor wild

type cDNA plasmid as previously described (111). The AT1 receptor used for

transfection has an HA-tag present after the methionine initiation sequence. Two days

after transfection, the cells were switched to medium supplemented with 500 [tg/ml

Zeocin (Invitrogen) to select for stable transfectants. Surviving colonies were ring

cloned, and AT1 receptor binding assays were performed using [125I-Sarl,Ile8]

angiotensin II (PerkinElmer Life Sciences) as described previously (3). Nonspecific

binding was defined as binding in the presence of 1.0 atM unlabelled angiotensin II.

Scatchard analysis was used to identify respective WT/AT1 and SYF/AT1 clones in which

the binding parameters were similar.

Cell Culture and Reagents

WT/AT1 and SYF/AT1 cells were cultured in Dulbecco's Modified Eagle's

Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum

(Hyclone), 1 mM sodium pyruvate, 10 units/mL penicillin, 10 atg/mL streptomyocin, 2

mM L-glutamine, 10 mM HEPES and 100 atg/mL Zeocin. WT/ATi and SYF/ATi cells

were growth arrested in serum-free DMEM for 48 hours prior to experiments. PP2 was

obtained from Calbiochem and used at concentrations found to have maximum inhibitory









effect (122). Cells were pretreated with inhibitor for the indicated time and stimulated

with 100 nM Ang II as described. All cell culture reagents were obtained from

Invitrogen. All other reagents were obtained from SIGMA or Fisher.

Immunoprecipitation and Western Blotting

Immunoprecipitation and Western blot analysis were performed in order to assess

protein expression and phosphorylation in the indicated cells. To prepare whole cell

protein lysates, cells were washed in two volumes of ice-cold PBS containing 1 mM

Na3VO4 and lysed in 0.8 ml ice-cold RIPA buffer (20 mM Tris [pH 7.5], 10% glycerol,

1% Triton X-100, 1% deocycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM

Na4P207, 4 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4 and

10 tg/mL aprotinin). The samples were then sonicated at a medium power setting and

incubated on ice for 30 min. Samples were centrifuged at 13,200 rpm for 5 min at 4C.

Supernatants were normalized for protein content using the Bio-Rad Dc assay.

Normalized lysates were then either directly resuspended in SDS sample buffer and

separated by SDS-PAGE for Western blot analysis or immunoprecipitated.

Immunoprecipitations were performed for 2 hrs at 4C using 2 [tg of the indicated

antibody and 20 tL of Protein A/G Plus agarose beads (Santa Cruz Biotechnology).

Following immunoprecipitation, samples were centrifuged for 2 min at 7,000 x g.

Protein bound A/G beads were then were then washed in wash buffer (25 mM Tris [pH

7.5], 150 mM NaCl and 0.1% Triton X-100). Samples were washed a total of three

times, and resuspended in SDS sample buffer. Protein-A/G bead complexes were boiled

for 10 min in order to separate bound proteins. Samples were separated by SDS-PAGE,

and transferred onto nitrocellulose membranes.









Whole cell lysates or immunoprecipitates were Western blotted with the indicated

antibody for each experiment. Antibodies were used at a final concentration of 1:1000 in

5% milk/TBST plus sodium azide. Membranes were subsequently stripped for 18 min,

and then reprobed with the indicated antibody to confirm equal protein loading of all

samples. Proteins were detected using enhanced chemiluminescence following the

manufacturer's instructions (Amersham).

Antibodies

The cocktail of anti-ERK1/2(P) antibodies were from Promega and Santa Cruz

Biotechnology. Note that these antibodies employed in the cocktail recognize the same

phospho-tyrosine residues, and were used in order to increase signal:noise ratio. The

anti-ERK1/2, the anti-MEK1/2, the anti-MEK1/2(P) and the anti-PKC( antibodies were

from Santa Cruz Biotechnology. The anti-phosphotyrosine antibody (PY20) was from

BD Transduction Laboratories. The immunoprecipitating anti-PLCy antibody was from

Santa Cruz Biotechnology. The immunoprecipitating anti-GFP antibody was from Cell

Signaling Technology.

Immunofluorescence

GFP-ERK2 plasmid was kindly provided by Philip J.S. Stork (50). WT/AT1 and

SYF/AT1 cells were plated onto four-chambered slides (Lab-Tek) and grown to 50%

confluency. The cells were washed one time with PBS (pH 7.4) to remove dead cells and

debris. Cells were transfected for 5 hrs with 10 [tg of GFP-ERK2 plasmid using

Lipofectin (Invitrogen) and following the manufacturer's instructions. The medium was

replaced with serum-containing medium and incubated at 37C for two days. Cells were

washed twice in PBS and starved for 48 hr in serum-free medium. Following starvation,

all cells were ligand-treated with 100 nM Ang II for 0, 5 or 10 min. Cells were rinsed









once in PBS and fixed in 4% paraformaldehyde for 10 min. Slide chambers were

removed and the slides were dipped twice into chilled PBS. Excess PBS was drained

from each slide and a coverglass was mounted to each slide using Vectashield + DAPI

mounting medium (Vector Labs). The edges of the slide were sealed with nail polish

(Maybelline LLC). Slides were viewed on a Zeiss Axioplan II Fluorescence microscope.

Measurement of Cellular ATP Levels

WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to

80% confluency. Cells were serum-starved for 48 hr and then treated with 100 nM Ang

II. Cellular ATP levels were assessed using the ViaLight HS proliferation/cytotoxicity

kit (Cambrex) following the manufacturer's protocol. A luminometer (Monolight model

2030) was used to measure bioluminescence.

Measurement of Formazan Production

WT/ATi and SYF/ATi cells were plated onto 96 well plates and grown to 80%

confluency. Cells were then starved for 48 hr in serum-free medium and treated with

100 nM Ang II as indicated. Formazan production was measured using the CellTiter 96

Aqueous One Solution Reagent (Promega) following the manufacturer's instructions.

Production of formazan was proportional to increased absorbance at 490 nm as measured

by spectrophotometry.

Cell Count

WT/ATi and SYF/ATi cells were plated onto 100 mm culture dishes and grown to

80% confluency. The cells were then serum-starved and treated with 100 nM Ang II as

indicated. Both cell types were then counted using a hemacytometer as described (122).









Densitometric Analysis

Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk

Scientific). The average pixel value minus background was obtained for each cell type

and normalized to the average pixel value for the respective non-Ang II-treated cells.

Statystical Analysis

Data were analyzed by two-way ANOVA. All data passed a Normality Test as

well as Equal Variance Test. Pairwise comparisons were made following the Holm-

Sidak method. All data are expressed as mean +/- SEM of replicates from three

independent experiments. = p<0.05, ** = p<0.01.

Results

Characterization of WT/ATi and SYF/ATi Cells

Previous reports have indicated that c-Src is a critical mediator of intracellular

ERK1/2 activation (53, 115, 140). The role of Src kinases in intracellular ERK1/2

activation was first explored, specifically in response to angiotensin II using c-

Src/Yes/Fyn-deficient (SYF) and wild type (WT) MEF cells. These SYF fibroblasts

were previously isolated at E9.5 from a developing c-Src/Yes/Fyn-deficient mouse

embryo and have been shown to be completely devoid of these proteins (62). MEF cells

containing functional c-Src/Yes/Fyn were also isolated from WT littermates and served

as controls (WT cells).

Both the SYF and WT cells do not endogenously express the AT1 receptor (data

not shown). Therefore, the AT1 receptor was stably transfected into both cell types to

constitute angiotensin II signaling. These ATi receptor stable cell lines have been named

SYF/ATi and WT/ATi respectively. Saturation binding studies were then performed in

order to identify respective SYF/ATi and WT/AT1 clones in which the binding









parameters were similar. SYF/AT1 (clone #16) and WT/AT1 (clone #2) both had a KD of

0.4 nM and a Bmax of 140-150 fmol/mg protein (Figure 2-1A). To demonstrate that these

two cell lines were similar in all aspects other than the levels of c-Src/Yes/Fyn, the

expression levels of Jak2 and STAT3, two non-Src kinase dependent genes, were

examined. It was found that these two genes were expressed at similar levels in the two

cell types (Figures 2-1B and 2-1C). Next, the ability of Ang II to activate PLCyI was

examined in WT/AT1 and SYF/AT1 cells. It was found that both cell types were capable

of increasing PLCyI tyrosine phosphorylation levels to roughly equal levels, an

indication that the ATi receptor can signal similarly in both cell types when examining

signaling events independent of c-Src/Yes/Fyn (Figure 2-1D). Collectively, these data

suggest that the WT/ATi and SYF/ATi cells are similar in all aspects except for c-

Src/Yes/Fyn expression and signaling pathways that are dependent on these three

proteins.

Angiotensin II-induced ERK1/2 Activation Is Reduced By About 50% in Src Kinase
Deficient Cells

Ang II-induced ERK1/2 activation was next assessed in WT/ATi and SYF/AT1

cells. Cells were stimulated with 100 nM Ang II for 0, 5 and 10 min, and ERK1/2

activity assessed via Western blot. Ang II-dependent ERK1/2 activation was decreased

in SYF/ATi cells when compared to WT/ATi cells after 5 and 10 min of Ang II treatment

(Figure 2-2A). Furthermore, Ang II-induced ERK1/2 activation was reduced in WT/ATi

cells pretreated with the Src family kinase inhibitor, PP2, to levels comparative to Ang II-

stimulated SYF/ATi cells (Figure 2-2B). Finally, ERK1/2 activation in the SYF/AT1

cells was partially restored by transiently-transfecting these cells with c-Src (Figure 2-











2C). Taken together, these results demonstrate that Src kinases mediate a portion of Ang

II-induced ERK1/2 activation.

It was next determined whether reductions seen in SYF/ATi cell ERK1/2 activation


were indeed due to the loss of c-Src/Yes/Fyn and not due to clonal artifact. Previous


A. B.

140 WT/AT, SYF/AT,
E .............. Ang II (m in)
120 A 0 5 10 0 5 10
1Jak2
100 111 kDa- -
S 80 WT/AT, IB aJak2 pAbs
60 o" SYF/AT,
0 / C.
o 40
20i WT/AT, SYF/AT,
20 Angll (mln) 0 5 10 0 5 10
0 TT3
79 IL. -
00 02 04 06 0 8 1 0 1 2 IB aSTAT3 pAbs
Free 1251-Sarlle (nM) D.

IP aPLCy1-pAbs
WT/AT1 SYF/AT1
Ang II (min) 0 5 10 0 5 10

111 kDa PLCy71(P)
IB aPhospho-Tyr mAbs

111 kDaPLC
IB aPLCy1-pAbs


Figure 2-1. Characterization of WT/ATi and SYF/ATi cells. A: Saturation binding curve
for WT/ATi and SYF/ATi cells. B-D: WT/ATi and SYF/ATi cells were
stimulated with 100 nM Ang II for 0, 5, and 10 min, and whole cell lysates
were prepared. B & C: Cell lysates were Western blotted with the indicated
antibodies. D: Cell lysates were immunoprecipitated (IP) and then Western
blotted with the indicated antibodies (top panel). Western blots were then
stripped and reprobed with the indicated antibody to demonstrate equal
protein loading. This figure is used with permission from (42).

work in MEF cells showed that PDGF activates ERK1/2 in a c-Src/Yes/Fyn-independent


manner (62). Therefore, both WT/ATi and SYF/ATi cells were stimulated with 30 ng/ml


PDGF for 0, 5, and 10 min. All cells were then lysed, and total protein extract was


immunoblotted with anti-active ERK1/2 pAbs. Contrary to Ang II treatment, stimulation


with PDGF resulted in a similar activation of ERK1/2 in both the WT/ATi and SYF/ATi











A. D.
WT/AT1 SYF/AT1 WT/AT1 SYF/AT1
Ang II (min) 0 5 10 0 5 10 PDGF(min) 0 5 10 0 5 10
4- ERK1(P) 36 Pa- EKI.-ER P
36 kDa -ERK2(P) : --EPK2iP)
IB: ERK1/2(P)-pAbs IERK1/2(P)-pAbs
36 kDa ---ERK1 36 _-EPK
IB: aERK1/2-Abs IB: aERK1/2-Abs
B. E.

WT/AT1 SYF/AT1
Ang II (min) 0 5 10 0 5 10 0 5 10
PP2 + + + E 6 1 W-
5 4 *Y FT/A T
36 kDa- -- ERK2(P) SYF/AT
IB: aERK1/2(P)-pAbs 30 2
36kDa- -*- 4-ERK2 0 I I
0 5 10
IB: aERK1/2-Abs Ang 1 (min)l

C.
IP: aGFP-pAb
WT/AT1 SYF/AT1
Ang II (min) 0 5 10 0 5 10
c-Src + + +
GFP-ERK2 + + + + + +

61 kDa i 4--GFP-ERK2(P)
IB: xERK1/2(P)-pAbs

61 kDa -4GFP-ERK2
IB: aGFP-pAb


Figure 2-2. Quantification of ERK1/2 activation in response to Ang II in WT/AT1 and
SYF/AT1 cells. A: WT/AT1 and SYF/AT1 cells were treated with 100 nM
Ang II, and ERK1/2 activation assessed via Western blot analysis B: WT/ATi
and SYF/ATi cells were pretreated with 30 [M PP2 or DMSO for 60 min, and
stimulated with 100 nM Ang II. Active ERK1/2 levels were assessed via
Western blot analysis using the indicated antibodies. C: SYF/ATi cells were
co-transfected with a plasmid encoding GFP-ERK2 and a c-Src-encoding
plasmid or empty vector control as indicated. Cells were stimulated with 100
nM Ang II, and whole cell lysates immunoprecipitated with GFP antibody.
ERK1/2 activation was assessed via Western blot. D: Cells were pretreated
with 30 ng/mL PDGF, and ERK1/2 activation assessed via Western blot. E:
Quantification of active ERK2 amounts from A. Fold changes in active
ERK2 in response to Ang II treatment were calculated by dividing average
ERK2 pixel density in Ang II-treated cells by average pixel density in non-
treated controls. This figure is used with permission from (42).

cells (Figure 2-2D). Thus, while SYF/ATi cells are capable of activating ERK1/2 to the

same degree as WT/AT1 cells in response to PDGF, the decreased ERK1/2 activation









seen in SYF/AT1 cells in response to Ang II is in fact due to the specific absence of c-

Src/Yes/Fyn-dependent signaling.

Quantification of band densities from 2-2A revealed that decreased ERK1/2

phosphorylation in the SYF/AT1 cells was statistically significant (Figure 2-2E).

Furthermore, maximum Ang II-induced ERK1/2 activation in the SYF/ATi cells was

observed after 5 min of Ang II treatment, whereas maximum ERK1/2 activation occurred

10 min post-Ang II treatment in the WT/ATi cells. Maximum ERK1/2 phosphorylation

levels were reduced by about 50% in the SYF/ATi cells when compared to peak levels of

ERK1/2 phosphorylation in the WT/ATi cells. As such, c-Src/Yes/Fyn tyrosine kinases

mediate at most 50% of Ang II-induced ERK1/2 activation since roughly half of Ang II-

activated ERK1/2 activation persists in their absence.

Angiotensin II-induced ERK1/2 Nuclear Translocation Is Not Dependent Upon Src
Kinases

Clearly, Src/Yes/Fyn-dependent signaling is responsible for a portion of Ang II-

induced ERK1/2 activation. The effect of a loss of c-Src/Yes/Fyn signaling on Ang II-

induced ERK1/2 nuclear translocation was next assessed. Previous reports have shown

that ERK1/2 translocates into the nucleus and initiates gene transcription of early

response genes via the phosphorylation of specific transcription factor targets (98). Other

work has shown that ERK1/2 nuclear translocation is dependent upon heterotrimeric G

protein signaling in response to Ang II (122). Therefore, it was next examined whether

the elimination of c-Src/Yes/Fyn and therefore a loss of about 50% of Ang II-induced

ERK1/2 activation would also affect ERK1/2 nuclear translocation.

WT/AT1 and SYF/AT1 cells were transfected with a GFP-ERK2 plasmid in order

to track ERK2 movement in response to Ang II treatment. Cells were then stimulated









with 100 nM Ang II, fixed and DAPI stained to visualize the nucleus. In the absence of

Ang II, GFP-ERK2 was distributed fairly evenly between both the nucleus and cytoplasm

in WT/AT1 and SYF/AT1 cells (Figures 2-3A, 2-3B and 2-3G). DAPI counterstain of

these images and merging of the GFP and DAPI images confirmed these findings

(Figures 2-3D, 2-3E and 2-3J). In contrast, ERK1/2 accumulation was markedly

increased in the nucleus of both WT/AT1 and SYF/AT1 cells treated with Ang II (Figures

2-3C, 2-3 H and 2-31), and this finding was confirmed by DAPI counterstain (Figures 2-

3F, 2-3K and 2-3L). As such, it appeared that ERK1/2 nuclear translocation was present

in both Ang II-stimulated WT/ATi and SYF/ATi cells. However, there was no

statistically significant difference in nuclear fluorescence between Ang II-stimulated

SYF/ATi cells when compared to WT/ATi controls. As such, it appears that c-

Src/Yes/Fyn do not influence ERK1/2 nuclear translocation. Quantification of nuclear

fluorescence relative to cytoplasmic fluorescence revealed a significant increase in

ERK1/2 nuclear fluorescence in both WT/AT1 and SYF/ATi cells stimulated with Ang II,

indicative of increased nuclear translocation (Figures 2-3M and 2-3N).

Angiotensin II Induced Cell Proliferation Is Reduced in Src Kinase Deficient Cells

Ang II-induced ERK1/2 activation has been shown to initiate cell proliferation

(33, 89, 90, 133). It has primarily been thought that this occurs through the translocation

of ERK1/2 into the nucleus and the subsequent initiation of growth response gene

transcription (122). Here, it has been demonstrated that Ang II-induced ERK2 nuclear

translocation is unaffected by the loss of c-Src/Yes/Fyn-mediated ERK1/2 activation









WTIAT,

Ang I + +



**l _0







SYFIAT,
Ang II + +











Figure 2-3. Nuclear translocation of active ERK2 is unaffected by the loss of
c-Src/Yes/Fyn. A C, G I: WT/ATi or SYF/ATi cells were transfected with
a GFP-ERK2 plasmid and then stimulated with Ang II for 0 and 10 min.
Nuclear translocation of ERK2 was assessed by fluorescent microscopy. A -
C: GFP-ERK2 images in non-treated and Ang II-treated WT/ATi cells. G I:
Merging of images A C respectively with DAPI stained images. G I: GFP-
ERK2 images in non-treated and Ang II-treated SYF/ATi cells. J L: Merging
of images G I respectively with DAPI stained images. M: Nuclear
fluorescence from A C was quantified and normalized to cytoplasmic
fluorescence. N: Nuclear fluorescence from G I was quantified and
normalized to cytoplasmic fluorescence. All images are representative of the
entire field and were taken at 40X magnification. Bar represents 15 microns.
Shown is one of two independent results. This figure is used with permission
from (42).
from (42).









(Figure 2-3). It was next determined whether eliminating c-Src/Yes/Fyn effects Ang II-

induced cell proliferation, independent of the ability of ERK1/2 to translocate into the

nucleus.

Ang II-induced cell proliferation was assessed via three different methodologies.

First, cellular ATP levels were measured since ATP amounts have previously been

reported to be excellent indicators of cell number (22). WT/AT1 and SYF/AT1 cells were

treated with 100 nM Ang II and intracellular ATP levels were measured. After 4 hours of

Ang II treatment, ATP levels had already increased over 3 fold in WT/AT1 cells (Figure

2-4A). ATP levels in SYF/AT1 cells increased slightly by about 1.75 fold, but were

markedly reduced after Ang II treatment compared to WT/ATi controls. These data

suggest that cell number was decreased in Ang II-stimulated SYF/ATi cells when

compared to WT/AT1 controls and therefore that Ang II-induced cell proliferation was

significantly reduced by the elimination of c-Src/Yes/Fyn.

Next, Ang II-induced cell proliferation was assessed through the measurement of

formazan levels, which has previously been reported to also be an excellent indicator of

increased cell number (12). WT/ATi and SYF/ATi cells were treated with 100 nM Ang

II and then formazan production was assessed. Ang II-induced formazan production was

significantly increased in WT/AT1 cells after 5 hours of Ang II treatment (Figure 2-4B).

However, formazan production barely increased in SYF/ATi cells treated for 5 hours with

Ang II. SYF/AT1 cell formazan production was significantly decreased relative to

WT/ATi controls.

Lastly, Ang II-induced cell proliferation was analyzed via direct cell count.

WT/ATi and SYF/ATi cells were treated with 100 nM Ang II for 0 and 24 hr. Cells were














_ 5
E 4.5
4
D 3.5
u 3
2.5
- 2
1.5
a- 0.5
I--
< 0
Angl -


II


'7 *
II


+ +


- 4 I
0 3.5
G) .. 3 **
.E 2.5 r i
z 2
0 1.5

L_ 0.5
0
Ang II + +


S2.5

E 2
o WT/AT,
o SYF/AT, E 1.5
0)
-5 1
E
v 0.5
Ang II


| I WT/AT,
--] SYF/AT,





- + +


5 WT/AT1
0 SYF/AT1


Figure 2-4. Ang II-induced cell proliferation is reduced in Ang II-stimulated SYF/AT1
cells. A: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 4
hr. Cellular ATP levels were then measured. Data are expressed as fold
change in ATP in Ang II stimulated cells relative to non-treated controls. B:
WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 5 hr.
Formazan production was then measured. Data are expressed as fold change
in formazan in Ang II stimulated cells relative to non-treated controls. C:
WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 and 24 hr. All
cells were then detached and counted using a hemacytometer. All data are
expressed as a fold change in Ang II-stimulated relative to non-stimulated
cells. All data are representative of three independent experiments. WT/ATi
control cells. Thus, these data also suggest that Ang II-induced cell
proliferation is markedly reduced in SYF/ATi cells relative to WT controls.
This figure is used with permission from (42).

detached and counted using a hemacytometer. WT/ATi cell number was increased by

over 3 fold when treated with Ang II for 24 hours relative to non-treated controls (Figure

2-4C). SYF/ATi cell number also increased, but this increase was significantly reduced

when compared to Ang II-treated WT/AT1 cells. As such, these data show that Ang II-









induced cell proliferation was markedly reduced in SYF/AT1 cells lacking c-

Src/Yes/Fyn-dependent signaling.

In summary, Ang II-induced cell proliferation is reduced when c-Src/Yes/Fyn are

eliminated from the cell. Eliminating the -50% of Ang II-induced ERK1/2 activation

dependent upon c-Src/Yes/Fyn therefore alters the ability of these cells to proliferate in

response to Ang II. Interestingly, the decrease in Ang II-induced cell proliferation

observed in SYF/AT1 cells occurs independent of the ability of ERK1/2 to translocate

into the nucleus. In addition, a portion of cell proliferation is dependent upon c-

Src/Yes/Fyn-independent signaling.

Discussion

Here, MEF cells completely devoid of c-Src/Yes/Fyn were utilized. In doing so, all

Src kinase function and the possibility that ERK1/2 activation can be mediated via any of

these very similar family members has been completely eliminated. It was found that

while c-Src/Yes/Fyn tyrosine kinases do play a role in the activation of ERK1/2 as

previously reported, ERK1/2 activation is not completely dependent on these proteins and

persists at reduced levels in their absence. c-Src/Yes/Fyn are capable of activating only

about 50% of intracellular ERK1/2. An explanation for these results is that the remaining

50% of intracellular ERK1/2 are activated by c-Src/Yes/Fyn-independent mechanisms.

As such, Ang II-induced ERK1/2 activation occurs through two independent signaling

cascades, and is not completely dependent upon Src kinases as previous work has shown

(53).

Interestingly, while the Src kinase dependent signaling pathways appears to

mediate as much as 50% of Ang II-induced ERK1/2 activation, these signaling events do

not influence Ang II-induced ERK1/2 nuclear translocation. It had previously been









thought that ERK1/2 must translocate into the nucleus in order to initiate events

necessary for the start of cell proliferation, including the transcription of early response

genes such as c-fos (14, 15, 109). Interestingly, the loss c-Src/Yes/Fyn had no effect on

the ability of ERK1/2 to translocate into the nucleus. ERK1/2 was able to enter the

nucleus in the absence of c-Src/Yes/Fyn; however, cell proliferation was still markedly

reduced.

Even more striking is the finding that c-Src/Yes/Fyn still influence cell

proliferation, independent of ERK1/2 nuclear translocation. An explanation for these

findings is that ERK1/2 activated via c-Src/Yes/Fyn-dependent signaling acts upon

cytoplasmic proteins to mediate proliferation. Previous work has already shown that

ERK1/2 can phosphorylate a number of cytoplasmic substrates, including members of the

RSK, MSK and MNK families of proteins (37). Furthermore, many of these proteins

have been shown to regulate the activity of transcription factors. For example, RSK has

been shown to modulate phosphorylation of both the serum response factor (SRF) as well

as CREB, both of which have been shown to be pro-mitotic (37). Therefore, ERK1/2

may also modulate transcription through the phosphorylation of cytoplasmic proteins,

which themselves activate transcription factors initiating pro-growth and proliferative

events. In addition, ERK1/2 activated via c-Src/Yes/Fyn-independent signaling may

translocate into the nucleus to directly mediate transcriptional events (122). These events

will be investigated and described in detail in the following chapters.














CHAPTER 3
SRC KINASE-INDEPENDENT ERK1/2 ACTIVATION AND CELL
PROLIFERATION IS MEDIATED BY HETEROTRIMERIC G PROTEINS AND
PKC(-DEPENDENT SIGNALING

Introduction

In the previous chapter, it was demonstrated that approximately 50% of Ang II-

induced ERK1/2 activation is mediated by Src family tyrosine kinases. In addition, these

proteins also mediate a portion of angiotensin II-induced cell proliferation. The

remaining 50% of intracellular ERK1/2 activation and portion of Ang II-induced cell

proliferation must therefore be occurring through Src kinase-independent signaling. In

this chapter, the events underlying Src kinase-independent ERK1/2 activation through the

AT1 receptor will be discussed.

Previous work has implicated numerous proteins other than Src kinases in Ang II-

induced ERK1/2 activation. For example, the AT1 receptor is capable of transactivating

other receptors with intrinsic tyrosine kinase activity, including the epidermal growth

factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), which in

turn activate ERK1/2 (32, 74, 85). In addition to receptor transactivation, numerous

cytoplasmic kinases have been shown to mediate ERK1/2 activation. Pharmacological

inhibition of phosphoinositide 3-kinase (PI3K) blocks ERK1/2 activation in EGF-treated

preglomerular smooth muscle cells (4, 45). Furthermore, various isoforms of Protein

Kinase C (PKC) have been shown to mediate intracellular ERK1/2 activation in response

to different ligands (45, 46, 70, 86). It is not clear from these reports, though, if the

mechanism of ERK1/2 activation differs depending upon the receptor activated, or if









ERK1/2 activation occurs simultaneously via multiple independent signaling mechanisms

in response to treatment with the same ligand.

Here, we sought to determine whether AT1 receptor-generated ERK1/2 activation

occurs via Src kinase-independent signaling in addition to Src kinase dependent

signaling. Specifically, the effect of PDGFR, EGFR, PI3K, PKC, Raf or MEK inhibition

on Ang II-induced ERK1/2 activation was first investigated in order to identify a

mechanism whereby ERK1/2 activation occurs in a Src kinase-independent manner. In

addition, the effect of heterotrimeric G protein and PKC( inhibition on Ang II-induced

ERK1/2 activation was examined. An attenuation of Src kinase-independent ERK1/2

activation only occurred when heterotrimeric G protein, PKC( or MEK activities were

inhibited, suggesting that these proteins mediate Ang II-induced ERK1/2 activation

independent of Src family tyrosine kinases. In addition, it was found that MEK

phosphorylation was dependent upon PKC( activity, identifying a mechanism whereby

PKC( activates ERK1/2 via the upstream activation of MEK. As such, ERK1/2

activation occurs via heterotrimeric G protein/PKC signaling independent of Src family

tyrosine kinases in response to AT1 receptor activation.

It was previously observed that Src kinases mediate about 50% of ERK1/2

activation and a portion of Ang II-induced cell proliferation (Chapter 2). More

importantly, Src kinase-activated ERK1/2 mediates cell proliferation without direct

translocation of ERK1/2 into the nucleus. Previous work has shown that ERK1/2

translocates into the nucleus to modulate gene transcription in response to Ang II (122).

As such, ERK1/2 nuclear translocation must therefore be influenced by Src kinase-

independent signaling events. Therefore, whether heterotrimeric G protein/PKCZ









signaling mediates Ang II-induced ERK1/2 nuclear translocation and cell proliferation

was also assessed.

The effect of inhibition of the heterotrimeric G protein/PKC pathway on ERK1/2

nuclear translocation and cell proliferation was determined using a PKC myristoylated

pseudosubstrate. In contrast to Src kinases, the nuclear translocation of ERK1/2 is

dependent upon PKC( activity as pretreatment with the PKC( MP abolished Ang II-

induced ERK1/2 nuclear translocation. In addition, PKC( inhibition also reduced Ang II-

induced cell proliferation. Interestingly, PKC( inhibition in combination with Src kinase

inhibition completely attenuated Ang II-induced cell proliferation. Therefore, ERK1/2

activation and cell proliferation are controlled through both heterotrimeric G

protein/PKC-dependent and Src kinase-dependent signaling events in response to

angiotensin II. Each of these pathways appears to mediate a portion of this response, but

only heterotrimeric G protein/PKC signaling causes ERK1/2 to directly translocate into

the nucleus. As such, it appears that Ang II-induced ERK1/2 activation is mediated by

two independent signaling cascades operating through distinct mechanisms.

Materials and Methods

Cell Culture

WT/AT1 and SYF/AT1 cells were cultured as described in Chapter 2. CHO/AT1

and CHO/ATi-M5 cells were cultured in F-12 media supplemented with 10% fetal

bovine serum, 2 mM L-glutamine, 10 units/mL penicillin, 10 [tg/mL streptomyocin, 1

mM sodium pyruvate, and 10 mM HEPES. WT/AT1 and SYF/ATi were growth arrested

in serum-free DMEM for 48 h prior to experiments. CHO cells were growth arrested in

the same manner for 24 h. All cell culture reagents were obtained from Invitrogen.









Pharmacological Inhibitors

AG1295, AG1478, GDP-PS, G66976, G66983, LY294002, PD98059, PP2, Rafl

kinase Inhibitor 1 and Rotlerrin were all obtained from Calbiochem and used at

concentrations found to have maximum inhibitory effect (32, 46, 74, 85, 89, 117, 122,

140, 148). Sodium fluoride (SIGMA), chelerythrine (LKT Labs), and the PKC(

myristoylated pseudosubstrate (BioMol) were also used at previously determined

concentrations (58, 124, 164). SYF/AT1 cells were permeabilized with 5 nM saponin

(USB) before treatment with GDP-PS (103). All other reagents were obtained from

SIGMA or Fisher. Cells were pretreated with inhibitor for the indicated time and

stimulated with 100 nM Ang II as described.

siRNA Treatment of WT/ATi Cells

siRNA reagents were purchased from Santa Cruz Biotechnology. Cells were

grown in 100 mm culture plates (Corning) to 80% confluency. Adherent cells were

trypsinized and resuspended in serum-containing medium without antibiotics. Cells and

medium were centrifuged at 500 x g for 5 min, and pelleted cells were resuspended in

fresh serum-containing medium without antibiotics. Cells were transferred to 6 well

culture plates (Corning) and grown to 40-50% confluency. Transfection reagents were

prepared as described in the online protocol (http://www.scbt.com/support/protocols)

with the exception that the concentration of siRNA used was increased four fold. Cells

were next transfected for 48 hrs at 37C with either control siRNA or PKC-specific

siRNA in serum-containing medium without antibiotics. Cells were serum-starved for 48

hours and treated with 100 nM Ang II for 0, 5 and 10 min. Cells were lysed and whole

cell protein lysates were prepared. Cell lysates were separated on a 10% SDS-PAGE gel,









transferred to a nitrocellulose membrane and Western blotted with the indicated

antibodies.

Immunoprecipitation, Western Blotting and Densitometric Analysis

Cells were immunoprecipitated as described in Chapter 2. Proteins were detected

using enhanced chemiluminescence as described in Chapter 2. The anti-MEK1/2, the

anti-MEK1/2(P) and the anti-PKC antibodies used for Western blotting were from Santa

Cruz Biotechnology. Western blots were scanned and densitized using UnScanIt Gel

Analysis (Silk Scientific) as described in Chapter 2.

Immunofluorescence and Quantification of Fluorescence

Cells were transfected with GFP-tagged ERK2 and ERK2 movement examined via

immunofluorescence as described in Chapter 2. Cells were pretreated with either 1 PM

PKC( myristoylated pseudosubstrate or DMSO for 1 hour prior to stimulation with 100

nM Ang II. Nuclear or cytoplasm fluorescence was quantified using the Image J Progam

(NIH) as described in Chapter 2.

Cell Count

Cell counts were performed using a hemacytometer as described in Chapter 2.

Cells were pretreated with either 1 pM PKC myristoylated pseudosubstrate or DMSO

for 1 hour prior to stimulation with 100 nM Ang II.

Statistical Analysis

Data were analyzed by two-way ANOVA. All data passed a Normality Test as

well as Equal Variance Test. Pairwise comparisons were made following the Holm-

Sidak method. All data are expressed as mean +/- SEM. = p<0.05, ** = p<0.01.









Results

Src Kinase Independent ERK1/2 Activation Does Not Require EGF Receptor,
PDGF Receptor or PI3K Activity

A mechanism for c-Src/Yes/Fyn-independent ERK1/2 activation in response to

Ang II was first identified through pharmacological inhibition of candidate proteins. A

brief literature search identified proteins which have previously been implicated in

ERK1/2 activation in response to various activating ligands (Table 3-1).

Pharmacological inhibitors for each of these proteins were then obtained, and SYF/AT1

cells were treated for the indicated time with concentrations of these inhibitors previously

found to suppress protein function, in order to identify proteins that mediate ERK1/2

activation independent of c-Src/Yes/Fyn. Cells were stimulated with 100 nM Ang II and

whole cell lysates were Western blotted with phospho-ERK /2 antibodies to identify

changes in active ERK1/2 levels.

Table 3-1. Pharmacological inhibition of Src kinase-independent ERK1/2 inhibition.
Inhibitor Treatment
Protein Inhibitor Final Concentration Time (min)
EGFR AG1478 30 gM 30
Heterotrimeric G proteins GDPI3S 2 mM 20
MEK PD98059 50 gM 60
Rafl Rafl kinase 1 30 gM 60
inhibitor
PDGFR AG1295 30 gM 60
PKC Chelerythrine 30 gM 60
PI3K LY294002 30 gM 60

The involvement of growth factor receptor transactivation on c-Src/Yes/Fyn-

independent ERK1/2 activation was first examined. SYF/AT1 cells were pretreated with

either PDGFR (AG1295) or EGFR (AG1478) selective inhibitors, and then stimulated

with 100 nM Ang II. No reduction in Ang II-induced ERK1/2 activation was observed










A SYF/AT1

Ang II (min) 0 5 0 5
AG1295 + +
-36kD ERK1(P)
36 kDa -- -- ERK2(P)
IB: aERK1/2(P)-pAbs
36 kDa--m .. 4 ERK2

IB: aERK1/2-Abs
B
SYF/AT1
Ang II (min) 0 5 10 0 5 10
AG1478 + + +
36 kDa- .,- -- ,, 4-- ERK2(P)
IB: aERK1/2(P)-pAbs
36 kDa- 4o w w*ERK2
IB: aERK1/2-Abs

C
SYF/AT1
Ang II (min) 0 5 10 0 5 10
LY294002 + + +
36 kDa EERK2 P
IB: aERK1/2(P)-pAbs
36 kDa- -- ERK2

IB: aERK1/2-Abs


Figure 3-1. ERK1/2 activation in SYF/AT1 cells does not require transactivation of the
PDGFR or the EGFR or activation of PI3K. A C: SYF/AT1 cells were
pretreated with the indicated pharmacological inhibitors at the concentrations
and for the treatment times listed in Table 1. Cells were stimulated with 100
nM Ang II for indicated times, and active ERK1/2 levels were assessed by
Western blotting with anti-ERK1/2(P) antibodies (top panels). Total ERK
protein loading was demonstrated by stripping the membrane and reprobing
with anti-ERK1/2 antibodies (bottom panels). All Westerns are representative
of three independent experiments. This figure is used with permission from
(42).









in the PDGFR inhibitor-treated SYF/AT1 cells compared to non-treated controls (3-1A).

Similarly, treatment of SYF/AT1 cells with the EGFR selective inhibitor had no effect on

Ang II-induced ERK1/2 activation (Figure 3-1B). Collectively, these data indicate that

Ang II-induced ERK1/2 activation occurring through c-Src/Yes/Fyn-independent

mechanisms does not require EGFR or PDGFR transactivation.

ERK1/2 activation has also previously been reported to occur through PI3K-

dependent signaling mechanisms (4, 45, 86). The effect of PI3K inhibition on Ang II-

induced ERK1/2 activation was next determined in SYF/AT1 cells using the PI3K

selective inhibitor, LY294002. Control treated SYF/ATi cells exhibited the typical

ERK1/2 activation response after 5 min of Ang II treatment and no decrease in ERK1/2

activation was observed in LY294002-treated cells (Figure 3-1C). In fact, ERK1/2

activation increased slightly when LY294002 was added to SYF/ATi cells. Thus, PI3K

does not regulate Ang II-induced ERK1/2 activation independent of c-Src/Yes/Fyn.

Src Kinase Independent ERK1/2 Activation Is Dependent on MEK1/2, But Not Rafl

ERK1/2 activation typically occurs through the activation of a MAPK signaling

cascade, in which a MAPK-kinase-kinase (MAPKKK) phosphorylates a MAPK-kinase

(MAPKK), which in turn phosphorylate and activate MAPKs such as ERK1/2. The

typical MAPKKK involved in ERK1/2 activation is Rafl, while MEK1/2 are the

MAPKK which dually phosphorylate ERK1/2. It was next investigated whether

pharmacological inhibition of either Rafl or MEK1/2 would effect Ang II-induced

ERK1/2 activation in SYF/ATi cells. SYF/ATi cells pre-treated with the Rafl kinase

inhibitor exhibited no decrease in ERK1/2 activation when compared to untreated cells

(Figure 3-2A). In contrast, Ang II-induced ERK1/2 activation was completely absent

from WT/AT1 cells and SYF/AT1 treated with the MEK1/2 inhibitor (Figure 3-2B).









Therefore, Ang II-induced ERK1/2 activation occurring via c-Src/Yes/Fyn-independent

mechanisms (i.e., in the SYF/AT1 cells) does not require Rafl. However, all Ang II-

induced ERK1/2 activation is dependent upon MEK activation.

A
SYF/AT1
Ang II (min) 0 5 10 0 5 10
Rafll + + +
,.- 4- ERK1
36 kDa- ERK2P)
IB: aERK1/2(P)-pAbs
36 kDa- -- a ERK2
IB: aERK1/2-Abs

B
WT/AT1 SYF/AT1
Ang II (min) 0 5 10 0 5 10 0 5 10 0 5 10
PD98059 ++ + + + +
4-- ERK1(P)
36 kDa .- ERK2(P)
IB: aERK1/2(P)-pAbs
,,- -, -. -- 14- ERK1
36 kDa e m ERK2
IB: aERK1/2-Abs

Figure 3-2. ERK1/2 activation in SYF/AT1 cells requires MEK1/2 activation, but not
Rafl activation. A: WT/AT1 or SYF/AT1 cells were pretreated with the
indicated pharmacological inhibitors at the concentrations and for the
treatment times listed Table 1. Cells were stimulated with 100 nM Ang II for
the indicated times, and active ERK1/2 levels were assessed by Western
blotting with anti-ERKl/2(P) antibodies (top panels). Total ERK protein
loading was demonstrated by stripping the membrane and reprobing with anti-
ERK1/2 antibodies (bottom panels). All Westerns are representative of three
independent experiments. This figure is used with permission from (42).

In summary, ERK1/2 activation in Ang II-treated SYF/AT1 cells was not affected

when cells were treated with EGFR, PDGFR, PI3K or Rafl selective pharmacological

inhibitors. However, Ang II-induced ERK1/2 activation was attenuated in SYF/AT1 cells









treated with the MEK1/2 inhibitor. Intracellular ERK1/2 activation occurring

independent of c-Src/Yes/Fyn requires upstream MEK1/2 activation in order to dually

phosphorylate ERK1/2, but does not depend on AT1 receptor transactivation of the EGF

and PDGF receptors or the phosphorylation of PI3K and Rafl. As such, other proteins

must therefore be acting upstream of MEK1/2 in order to activate ERK1/2 in a c-

Src/Yes/Fyn-independent manner.

Heterotrimeric G Proteins Mediate A Portion of ERK1/2 Activation In A Src
Kinase-independent Manner

Heterotrimeric G proteins have previously been shown to activate ERK1/2 in

response to Ang II (122). Whether the remaining 50% of Ang II-induced ERK1/2

activation in the SYF/AT1 cells was mediated entirely by heterotrimeric G protein

signaling was next examined. SYF/AT1 cells were permeabilized using saponin, and then

pretreated with the heterotrimeric G protein inhibitor, GDP-PS (103). The P-phosphate

group of this compound has been replaced with a sulfate group, hindering the ability of

heterotrimeric G proteins to exchange GDP for GTP and subsequently become activated.

Permeabilized SYF/AT1 cells treated with vehicle control served as controls. Cells were

then stimulated with 100 nM Ang II, and ERK1/2 activation assessed via Western blot.

SYF/AT1 cells permeabilized with saponin and treated with vehicle control still

demonstrated ERK2 activation in response to Ang II (Figure 3-3A). However, ERK2

activation was attenuated in GDP-PS treated cells since there was no increase in ERK2

phosphorylation levels in Ang II-stimulated cells compared to untreated controls. These

data suggest that Ang II-induced activation of ERK1/2 occurring independent of c-

Src/Yes/Fyn requires heterotrimeric G protein activation.









The effect of ligand-independent activation of heterotrimeric G proteins on ERK1/2

activation in the SYF/AT1 cells was next tested. SYF/AT1 cells were pretreated with

NaF, which causes heterotrimeric G proteins to become constitutively activated

independent of exogenous ligand addition (58). Cells were then stimulated with 100 nM

Ang II, and ERK1/2 activation assessed via Western blot. Basal ERK1/2 activation was

significantly increased in SYF/ATi cells treated with NaF when compared to untreated

cells (Figure 3-3B). Furthermore, the addition of Ang II did not further increase ERK1/2

activation levels, suggesting that Ang II activates ERK1/2 in SYF/ATi cells via a

mechanism that is dependent upon heterotrimeric G proteins. The addition of EGF,

which activates ERK1/2 in a c-Src/Yes/Fyn dependent manner (10), further increased

ERK1/2 activation in NaF treated cells (data not shown). These data therefore suggest

that Ang II-induced ERK1/2 activation occurring independent of c-Src/Yes/Fyn requires

heterotrimeric G protein activation.

To further demonstrate that heterotrimeric G proteins mediate all c-Src/Yes/Fyn-

independent ERK1/2 activation in response to Ang II, a previously generated CHO cell

line stably transfected with a mutant ATi receptor was utilized. This mutant ATi

receptor contains specific mutations in the carboxyl terminus preventing heterotrimeric G

proteins from coupling to the receptor and becoming activated (28). These cells are

denoted as CHO/ATi-M5 cells here, and retain their ability to activate tyrosine kinases

such as c-Src/Yes/Fyn. CHO cells stably expressing wild type ATi receptor with similar

affinity and abundance (dentoted as CHO/ATi) were used as controls. Both cell types

were stimulated with 100 nM Ang II, and ERK1/2 activation assessed via Western Blot.

Ang II-induced ERK1/2 activation in CHO/ATi cells reached maximum levels after 5










A
SYF/AT1
Ang II (min) 0 5 10 0 5 10
GDP-pS + + +
+ 4-ERK2(P)
36 kDa -
WB: aERK1/2(P)-pAbs

36 kDa --- ERK2
WB: aERK1/2-Abs

B
SYF/AT1
Ang II (min) 0 5 10 0 5 10
NaF + + +
--ERK1
36 kDa-, -- E-ERK2(P)
WB: aERK1/2(P)-pAbs
*--ERK1
36 kDa-h w ow --ERK2
WB: aERK1/2-Abs

C
CHO/AT1 CHO/AT1-M5
Ang II (min) 0 5 10 0 5 10 0 5 10
PP2 + + +

36 kDa- -- ERK2(P)
WB: aERK1/2(P)-pAbs
36 kDa- MMw ERK2

WB: aERK1/2-Abs


Figure 3-3. Ang II-induced ERK1/2 activation is partially dependent upon heterotrimeric
G proteins. A and B: SYF/AT1 cells were pretreated with either 2 mM GDP-
j3S for 20 min or 2 mM NaF for 1 hr, respectively, and stimulated with 100
nM Ang II for 0, 5, and 10 min. C: CHO/AT1 and CHO/ATi-M5 cells were
first pretreated with either 30 pM PP2 or DMSO for 1 hour before Ang II
treatment. ERK2 phosphorylation was determined via Western blot using the
indicated antibodies. A-C: Total ERK protein loading was demonstrated in
each case by stripping the membrane and reprobing with the anti-ERK1/2
antibodies (bottom panels). All Westerns are representative of three
independent experiments. This figure is used with permission from (42).









min of Ang II treatment (Figure 3-3C). ERK1/2 activation also occurred in CHO/AT1-

M5 cells in response to Ang II, but maximum ERK1/2 activation was significantly

reduced compared to CHO/AT1 cells. Therefore, Ang II-induced ERK1/2 activation is

partially dependent upon heterotrimeric G protein activation. Furthermore, pretreatment

of CHO/ATi-M5 cells with PP2 completely blocked Ang II-induced ERK1/2 activation,

indicating that the remaining 50% of ERK1/2 activation is dependent upon Src kinases.

Protein Kinase C 4 Mediates ERK1/2 Activation In A Src Kinase-independent
Manner

Protein kinase C isoforms have been shown to be activated downstream of

heterotrimeric G proteins and mediate ERK1/2 activation (45, 46, 70, 86). The specific

PKC isoforms responsible for activating ERK1/2 in a c-Src/Yes/Fyn independent manner

were next identified. SYF/AT1 cells were first treated with the broad range PKC

inhibitor, chelerythrine. Cells were then stimulated with 100 nM Ang II, and ERK1/2

activation assessed by Western blot. ERK1/2 activation was eliminated in SYF/AT1 cells

treated with chelerythrine relative to DMSO-treated controls (Figure 3-4A). As such,

these data confirm that Src kinase-independent ERK1/2 activation in response to Ang II

specifically requires PKC.

Over twelve different isotypes of PKC have been identified to date, and a number

of PKC isoforms have already been linked to ERK1/2 activation in response to

stimulation by various ligands (46, 70, 86). Therefore, the specific PKC isoforms

mediating Ang II-induced ERK1/2 activation in the SYF/AT1 cells were next identified.

SYF/AT1 cells were pretreated with pharmacological inhibitors specific for a number of

different PKC isoforms (Figures 3-4B, 3-4C and 3-4D). Cells were then stimulated with







49



A E
SYF/AT,
Ang II (min) 0 5 10 0 5 10
Chelerythrine + + +
36 kDa-_ -__ ERK(P)
WB: aERK1/2(P)-pAbs n
W 100%
36 kDa -ir m I ERK2 Ii
WB: aERK1/2-Abs |
B >< 50%o Rottlerin: 6
SYF/AT1 G66976: c, P, y
Ang II (min) 0 5 10 0 5 10 0% G66983: ca, 3, 5,
G66983 R + + + Chelerythrine: Broad PKC inhibition
36kD'.- ^ERKi-,P, +
WB: aERK1/2(P)-pAbs
36k H ,- -ERK2
WB: aERK1/2-Abs
C
SYF/AT1
Ang II (min) 0 5 10 0 5 10
G66976 + + +
i *ERK1 (P)
36 kDa- ERK2(P)
WB: aERK1/2(P)-pAbs
36 kH-3 -m+E-ERK2
WB: aERK1/2-Abs
D
SYF/AT1
Ang II (min) 0 5 10 0 5 10
Rottlerin + + +
4--E RK I:P:
36k- --- -ERKi P2P
WB: aERK1/2(P)-pAbs
.--ERK1
36 H-3 4-ERK2
WB: aERK1/2-Abs

Figure 3-4. Ang II-induced ERK1/2 activation is partially dependent upon PKC. A:
SYF/AT1 cells were pretreated with either DMSO or 30 [M Cheleryrthine
(A), 30 [M G66983 (B), 30 pM G66979 (C) or 30 pM Rottlerin (D) for 60
min, and then stimulated with 100 nM Ang II for 0, 5, and 10 min. ERK1/2
activation was assessed via Western blotted using the indicated antibodies (top
panel). Total ERK1/2 protein loading was demonstrated by stripping the
membrane and reprobing with the indicated antibodies (bottom panels).
These data are representative of three independent experiments. E: The effect
of inhibitor treatment on the percent maximum Ang II-induced ERK2
activation. Maximum Ang II-induced ERK2 activation was quantified from A
D in the presence (+) or absence (-) of inhibitor. Phospho-ERK2 bands
were densitized. Values were then normalized to ERK2 activation in Ang II-
stimulated cells not pretreated with inhibitor, and multiplied by 100. G66983
and chelerythrine pretreatment significantly reduced ERK1/2 activation
compared to vehicle-treated controls. This figure is used with permission
from (42).









100 nM Ang II, and ERK1/2 activation assessed via Western blot. A common trend was

observed in that Ang II-induced ERK1/2 activation was only reduced by pretreatment

with compounds that inhibited a subset of PKC isoforms that included PKC( (Figures 3-

4E).

The effect of PKC inhibition on Ang II-induced ERK1/2 activation was next

assessed using a PKC( myristoylated pseudosubstrate (MP), a potent and specific

inhibitor for PKC( (164). WT/AT1 and SYF/AT1 cells were first pretreated with either

PKC( MP or vehicle control. Both cell types were then stimulated with 100 nM Ang II,

and ERK1/2 activation assessed via Western blot. ERK1/2 activation in WT/AT1 cells

was significantly reduced with the addition of the PKC( MP, while ERK1/2 activation in

SYF/AT1 cells treated with PKC( MP was significantly reduced to levels found in non-

ligand treated cells (Figures 3-5A and 3-5B). Interestingly, treatment of WT/AT1 cells

with PKC( MP reduced Ang II-induced ERK1/2 activation to levels present in Ang II-

stimulated SYF/AT1 cells. As such, PKC( appears to mediate Ang II-induced ERK1/2

activation independent of Src kinases.

In order to reconfirm that roughly half of Ang II-induced ERK1/2 activation is

mediated by PKC(, PKC-specific siRNA was utilized. WT/AT1 cells were transfected

with either a scrambled siRNA control or a PKC-specific siRNA. The cells were then

stimulated with 100 nM Ang II, and ERK1/2 activation was then assessed via Western

blot. It was found that ERK1/2 activation occurred normally in response to Ang II

stimulation in WT/AT1 cells transfected with control siRNA (Figure 3-6A). ERK1/2

activation was significantly reduced in PKC-siRNA transfected cells when compared to






51


cells transfected with control siRNA. This reduction equated to about a 50% decrease in

ERK1/2 activity (Figure 3-6B), and was the maximum reduction in active ERK1/2

A
WT/AT1 SYF/AT1
Angll(min) 0 5 10 0 5 10 0 5 10 0 5 10
PKC MP + + + + + +
36 kDa ERK2(P
WB: aERK1/2(P)-pAbs
36 kDa -i- --ERK2
WB: aERK1/2-Abs
B

C 5 I
4.5 NS
n, 3.5
U 3 WT/AT,
6 2.5 SYF/AT
C 1S 1.5
0---

CD 0
Ang II + + + +
PKC MP + +

Figure 3-5. PKC mediates Ang II-induced ERK1/2 activation independent of c-
Src/Yes/Fyn. A: WT/ATi and SYF/ATi cells were pretreated with 1 [M
PKC MP for 1 hr, and then stimulated with Ang II for 0, 5 and 10 min.
ERK1/2 activation was then assessed via Western blot analysis with the
indicated antibodies (top panel). Total ERK1/2 protein loading was
demonstrated by stripping the membrane and reprobing with the indicated
antibodies (bottom panel). B: Phosphorylated ERK2 amounts from A (5 min
Ang II treatment) were quantified via densitometric analysis and expressed as
a fold change relative to unstimulated controls. This figure is used with
permission from (42).

capable of being observed without causing cell lethality (data not shown). Reprobing 3-

6A with anti-ERK1/2-Abs confirmed that total ERK1/2 protein levels were similar in

control siRNA and PKC( siRNA transfected cells. Additionally, a knockdown of PKC(

was only observed in cells transfected with PKC-specific siRNA, and did not affect

other PKC isoforms including PKCa. These data further demonstrate that PKC( partially







52



A
WT/AT1
Angll (min) 0 5 10 0 5 10
PKCi siRNA + + +
Control siRNA + + + -
36 kDa- ---ERK2(P)
WB: aERK1/2(P)-DAbs
36 kDa-- m mw4--ERK2
WB: aERK1/2-Abs
61 kDa-- 4-.- PKCi
WB: aPKCi-mAb
79 kDa-, ..Jw. .---- --PKCa
WB: aPKCa-mAb
B

Sc 120
U 100 -- T/AT,
E 80
E 60
40
=- 20
a_ 0
Ang II + +
Control siRNA +
PKCi siRNA +


Figure 3-6. PKC-specific siRNA attenuated Ang II-induced ERK1/2 activation in
WT/AT1 cells. A: WT/AT1 cells were transfected with either PKC( siRNA
or control siRNA. All cells were then treated with 100 nM Ang II for 0, 5, 10
min, and ERK1/2 activation assessed via Western blot analysis with the
indicated antibodies (first panel). Total ERK1/2 protein loading was
demonstrated by stripping the membrane and reprobing with the indicated
antibodies (second panel). PKC( protein knockdown was confirmed by
Western blotting with the indicated antibody (third panel). PKCao protein
levels were demonstrated by reprobing with the indicated antibody (fourth
panel). D: The percentage of maximum ERK2 phosphorylation from C was
determined by dividing by ERK2 activation in Ang II and control siRNA
treated cells, and multiplying by 100. This figure is used with permission
from (42).

mediates Ang II-induced ERK1/2 activation.

PKC4 Mediates MEK1/2 Activation Independent of Src Kinases

Finally, whether PKC( is acting upstream of MEK1/2 in order to activate ERK1/2

independent of c-Src/Yes/Fyn was investigated. SYF/AT1 cells were pretreated with

PKC( MP and then stimulated with Ang II for 0, 5 and 10 min. Whole cell lysates were









then Western blotted with anti-phosphospecific MEK1/2 antibodies in order to assess

MEK1/2 activation. SYF/AT1 cells not pre-treated with the PKC( MP exhibited an Ang

II-dependent increase in MEK2 activation (Figure 3-7). However, Ang II-dependent

MEK2 activation was absent from SYF/AT1 cells treated with PKC pseudosubstrate.

Thus, PKC appears to be acting upstream of MEK in order to activate ERK1/2 in

SYF/ATi cells.

SYF/AT,

Angll(min) 0 5 10 0 5 10
PKC MP + + +
49kDa -- MEK2(P)
49 kDa-
IB: aMEK1/2(P}pAbs

49 kDa- MEK2
IB: aMEK1/2-pAbs

Figure 3-7. MEK phosphorylation is dependent upon PKC Cells were pretreated with
either 1 pM PKC( MP or DMSO for 1 hr, and then stimulated with 100 nM
Ang II. Whole cell lysates were Western blotted with anti-phospho-MEK1/2
antibodies. The membrane was then stripped and reprobed with anti-MEK1/2
antibodies in order to demonstrate equal protein loading. This Western is
representative of three independent experiments. This figure is used with
permission from (42).

ERK1/2 Nuclear Translocation Is Dependent Upon PKC4 In Response to
Angiotensin II

Collectively, these data thus far indicate that Ang II-induced ERK1/2 activation

occurs via c-Src/Yes/Fyn-dependent and heterotrimeric G protein/PKC-dependent

signaling. Currently, the functional consequence of having two independent mechanisms

of ERK1/2 activation in response to Ang II-induced activation of the ATi receptor is not

well understood. Previous reports have shown that ERK1/2 translocates into the nucleus

and initiates gene transcription of early response genes via the phosphorylation of

specific transcription factor targets (98). Other work has shown that ERK1/2 nuclear









translocation is dependent upon heterotrimeric G protein signaling in response to Ang II

(122). In Chapter 2, it is demonstrated that a loss of c-Src/Yes/Fyn does not affect Ang

II-induced ERK1/2 nuclear translocation, but results in a reduction in Ang II-induced cell

proliferation. ERK1/2 nuclear translocation may therefore be controlled by

heterotrimeric G protein/PKC signaling, leading to the induction of cell proliferation.

The effect of blocking heterotrimeric G protein/PKC signaling on both Ang II-induced

ERK1/2 nuclear translocation and cell proliferation was next examined through inhibition

of the PKC-dependent signaling pathway using the PKC( MP.

Ang II-dependent ERK1/2 nuclear translocation was examined in SYF/AT1 cells in

the presence or absence of PKC( MP. These cells are only able to activate ERK1/2 and

induce cell proliferation in a Src kinase independent manner (Chapter 2), allowing one to

test the effect of PKC( inhibition on Src kinase-independent ERK1/2 nuclear

translocation and Ang II-induced cell proliferation. SYF/AT1 cells were transfected with

a GFP-ERK2 plasmid in order to track ERK2 movement in response to Ang II treatment.

Cells were pretreated with either DMSO or PKC( MP for 1 hour, and then stimulated

with 100 nM Ang II. Cells were then fixed, and DAPI stained to visualize the nucleus.

In the absence of Ang II, GFP-ERK2 was distributed fairly evenly between the nucleus

and cytoplasm of the SYF/AT1 cells (Figure 3-8A). DAPI counterstain of figures 3-8A

and merging of the GFP and DAPI images confirmed these findings (Figure 3-8F). In

contrast, ERK1/2 accumulation was markedly increased in the nucleus of SYF/ATi cells

treated with Ang II (Figure 3-8B & 3-8C), and this finding was confirmed by DAPI

counterstain (Figure 3-8G and 3-8H). ERK2 nuclear translocation was blocked in

SYF/AT1 cells pretreated with PKC( MP (Figure 3-8D and 3-8E), and merging of GFP









and DAPI images confirmed this reduction in Ang II-induced ERK2 nuclear translocation

(Figure 3-81 and 3-8J). Quantification of nuclear fluorescence relative to cytoplasmic

fluorescence revealed that Ang II-induced GFP-ERK2 nuclear translocation was blocked

in the presence of PKC MP (Figure 3-8K). Thus, ERK1/2 nuclear translocation in

response to Ang II is influenced by heterotrimeric G protein/PKC signaling, while c-

Src/Yes/Fyn also mediate ERK1/2 activation but do not influence ERK1/2 nuclear

translocation.

Cell Proliferation Is Attenuated Through Inhibition of PKC4 Signaling In Response
to Angiotensin II

Ang II-induced ERK1/2 activation has been shown to initiate cell proliferation (33,

89, 90, 133). It has primarily been thought that this occurs through the translocation of

ERK1/2 into the nucleus and the subsequent initiation of growth response gene

transcription (122). It has been demonstrated that Ang II-induced ERK2 nuclear

translocation is unaffected by the loss of c-Src/Yes/Fyn-mediated ERK1/2 activation

(Chapter 2). The effect of eliminating heterotrimeric G protein/PKC signaling on Ang

II-induced cell proliferation was next investigated, independent of the ability of ERK1/2

to translocate into the nucleus.

Both WT/AT1 and SYF/AT1 cells were pretreated with PKC( MP and then

stimulated with 100 nM Ang II. Changes in cell number were assessed via a direct cell

count. Ang II-induced cell proliferation was reduced in WT/ATi cells pretreated with

PKC( MP when compared to DMSO-treated controls (Figure 3-9). Interestingly, Ang II-

induced cell proliferation was not significantly different in PKC( MP pretreated WT/ATi

cells and SYF/AT1 cells stimulated with Ang II, suggesting that PKC-dependent

signaling mediates the portion of Ang II-induced cell proliferation not controlled by Src









kinases. Finally, Ang II-induced cell proliferation was completely blocked in SYF/ATi

cells pretreated with PKCL MP. These data collectively suggest that both PKCL and c-

Src/Yes/Fyn mediate Ang II-induced cell proliferation through the activation of ERK1/2,

though the mechanisms by which this occurs appear to be different since ERK1/2

SYFIAT1
Ang II + + + +
PKCC MP + +


I-,
E 50-
a8 I



&0

Ang I -
PKQ MP -


I I
I Iu SY-FIAT-


ii
I
4. .4-
-I-


Figure 3-8. Nuclear translocation of active ERK2 is controlled by PKCQ-dependent
signaling. A F: SYF/ATi cells were transfected with a GFP-ERK2 plasmid
and then stimulated with 100 nM Ang II for 0 and 10 min. Nuclear
translocation of ERK2 was assessed by fluorescent microscopy. A C: GFP-
ERK2 images in cells treated with 100 nM Ang II for 0 and 10 min. D & E:
GFP-ERK2 images in cells pretreated with 1 uM PKC( MP or DMSO (1 hr),
then stimulated with 100 nM Ang II. F J: Merging of images A E
respectively with DAPI stained images. K: Nuclear fluorescence from A E
was quantified and normalized to cytoplasmic fluorescence. All images are
representative of the entire field and were taken at 40X magnification. Bar
represents 15 microns. Shown is one of two independent results. This figure
is used with permission from (42).











**
4 I I **
3.5 NS

E 2.5 .] WT/AT1
z 2 SYF/AT1
0 1.5-

U- 0.5


Ang II + + + +
PKCC MP + + + +


Figure 3-9. Ang II-induced cell proliferation is completely attenuated by blocking c-
Src/Yes/Fyn and PKC(-dependent signaling. WT/ATi and SYF/ATi cells
were stimulated with Ang II for 0 and 24 hr. Some cells were pretreated with
1 pM PKC MP for 1 hr. All cells were then detached and counted using a
hemacytometer. All data are the mean of three independent experiments.
This figure is used with permission from (42).

translocates into the nucleus in response to activation by PKC( and remains in the

cytoplasm when activated by c-Src/Yes/Fyn.

Discussion

A diverse set of signaling pathways have been implicated in ERK1/2 activation, but

the precise mechanisms of ERK1/2 activation in response to Ang II are not fully

understood (4, 45, 69, 70, 85, 86, 89, 109, 115, 117, 140, 153). It appears that ERK1/2

activation occurs via multiple signaling mechanisms. However, the cellular outcome

associated with the activation of ERK1/2 via different signaling cascades is in question.

Are these signaling pathways functionally redundant, or does the activation of ERK1/2

by one pathway result in a different cellular outcome than when ERK1/2 is activated by

another signaling cascade?









Here, SYF/AT1 MEF cells were utilized in order to identify the mechanism

underlying Src kinase-independent ERK1/2 activation in response to Ang II as well as a

functional consequence for activating ERK1/2 in this manner. The advantage of utilizing

these cells is that ERK1/2 activation was examined in a Src kinase-deficient background,

eliminating all Src kinase function and the possibility that ERK1/2 activation can be

mediated via any of these very similar family members. It was found that while c-

Src/Yes/Fyn tyrosine kinases do play a role in the activation of ERK1/2 as previously

reported (54, 115, 140), ERK1/2 activation is not completely dependent on these proteins

and persists at reduced levels in their absence. Interestingly, c-Src/Yes/Fyn are capable

of activating only about 50% of intracellular ERK1/2. This seems to be a generalized

phenomenon in other cells types as well, including CHO and RASM cells. An

explanation for these results is that the remaining 50% of intracellular ERK1/2 are

activated by c-Src/Yes/Fyn-independent mechanisms. This was subsequently confirmed

since it was found that about 50% of Ang II-induced ERK1/2 activation involved

heterotrimeric G protein and PKC( signaling. In summary, Ang II-induced ERK1/2

activation occurs via two specific mechanisms that work independent of one another.

While both pathways activate an equal portion of ERK1/2 and contribute to cell

proliferation, the mechanism whereby each pathway independently mediates this effect

appears to be different. It had previously been thought that ERK1/2 must translocate into

the nucleus in order to initiate events necessary for cell proliferation to occur, including

the transcription of early response genes such as c-fos (14, 15, 109). Interestingly, the

results in this Chapter indicate that the loss c-Src/Yes/Fyn had no effect on the ability of

ERK1/2 to translocate into the nucleus. ERK1/2 was able to enter the nucleus in the









absence of c-Src/Yes/Fyn; however, cell proliferation was still markedly reduced. An

explanation for these findings is that ERK1/2 activated via c-Src/Yes/Fyn-dependent

signaling acts upon cytoplasmic proteins to mediate proliferation, while ERK1/2

activated via PKC-dependent signaling translocates into the nucleus to directly mediate

transcriptional events (14). A mechanism describing how c-Src/Yes/Fyn-activated

ERK1/2 initiates Ang II-induced cell proliferation is described in the next chapter.

The utility of having two mechanisms that dually activate ERK1/2 in response to

stimulation of the AT1 receptor by Ang II is nonetheless intriguing. Both c-Src/Yes/Fyn

and heterotrimeric G protein-dependent signaling appear to occur simultaneously in

response to Ang II, and exhibit an additive effect on ERK1/2 activation and subsequent

cell proliferation in response to Ang II. Within adult mammalian systems, Ang II-

induced cell proliferation is associated with abnormal cell proliferation during

cardiovascular diseases and cancer, and to date has not been implicated in cell growth

and proliferation during a non-disease state (24, 140, 145, 157-159). As such, this study

may have therapeutic merit since local inhibition of both heterotrimeric G protein/PKCL

signaling as well as c-Src/Yes/Fyn-dependent signaling may be necessary in order to

completely block Ang II-induced cell proliferation during disease states.














CHAPTER 4
ERK1/2 REGULATES ANGIOTENSIN II-DEPENDENT CELL PROLIFERATION
VIA THE CYTOPLASMIC ACTIVATION OF RSK2 AND NUCLEAR ACTIVATION
OF ELK1

Introduction

In the previous chapters, ERK1/2 activation is shown to be mediated independently

by either Src family tyrosine kinase-dependent signaling or heterotrimeric G

protein/PKC-dependent signaling in two different cell types. Both of these signaling

mechanisms accounted for roughly 50% of Ang II-induced ERK1/2 activation.

Interestingly, heterotrimeric G protein/PKC signaling regulates ERK1/2 nuclear

translocation, while c-Src/Yes/Fyn-dependent signaling influences cytoplasmic ERK1/2

activation in response to Ang II. Furthermore, these two pathways were both implicated

in Ang II-induced cell proliferation, with inhibition of both signaling cascades necessary

in order to achieve complete attenuation of Ang II-induced cell proliferation. These data

were very striking since previous reports had shown that ERK1/2 nuclear translocation

was a critical step in initiating the transcription of early response genes such as c-fos,

which promotes cell proliferation (109). However, it appears here that cytoplasmic

ERK1/2, under the control of c-Src/Yes/Fyn, also mediates Ang II-induced cell

proliferation independent of nuclear ERK1/2.

One explanation for how cytoplasmic ERK1/2 can influence early response gene

transcription is that it phosphorylates cytoplasmic substrates, which in turn translocate

into the nucleus and regulate transcriptional activity. Members of the Ribosomal S6

kinase (RSK) family of proteins are well-known cytoplasmic targets of ERK1/2, and









have been shown to promote the transcription and translation of selected mRNAs

important for cell growth (57). Previous reports have shown that ERK1/2 activates RSK

family proteins, however it is not clear if these proteins represent a possible pathway by

which Ang II-activated ERK can initiate the events leading to cell proliferation (110, 128,

132).

In this chapter, the mechanisms whereby ERK1/2 activated by either c-Src/Yes/Fyn

or heterotrimeric G proteins/PKC-dependent signaling generates the proliferative

response associated with AT1 receptor activation will be defined. It was hypothesized

that c-Src/Yes/Fyn-activated ERK1/2 mediates Ang II-induced cell proliferation through

RSK, whereas heterotrimeric G protein/PKC signaling regulates cell proliferation

through control of ERK1/2 nuclear translocation and subsequent elkI activation. To

examine this, WT/AT1 and SYF/ATi cells or CHO/ATi and CHO/ATi-M5 cells were

utilized. Each of these cell lines have been stably transfected with the ATi receptor and

were the same as those utilized in previous chapters. It was found that ERK1/2, activated

via c-Src/Yes/Fyn-dependent signaling, phosphorylates ribosomal S6 kinase 2 (RSK2),

which subsequently translocates into the nucleus and modulates c-fos activity at the

transcriptional and post-translational levels. These events partially mediate cell

proliferation since pretreatment with SL0101, a potent and specific inhibitor of RSK,

significantly attenuated Ang II-induced cell proliferation. ERK1/2 activated by

heterotrimeric G protein/PKC signaling localizes to the nucleus, where it phosphorylates

the transcription factor elkI and regulates c-fos transcription. Together with ERK1/2-

RSK signaling, these events mediate Ang II-induced cell proliferation. As such, this

study demonstrates that the AT1 receptor coordinately utilizes both hetrotrimeric G









protein and Src family tyrosine kinase signaling to achieve a common cellular outcome

via two different mechanisms acting in distinct cellular compartments.

Materials and Methods

Antibodies and Pharmacological Inhibitors

A cocktail of phospho-specific ERK1/2 antibodies (Promega and Santa Cruz

Biotechnologies) were used in order to increase signal to noise ratio. Both of these

antibodies are specific for phospho-threonine 202 and phospho-tyrosine 204 within the

conserved TEY motif. The phospho-specific RSK polyclonal antibody (aRSK(P)-pAb)

was purchased from Cell Signaling Technologies and recognizes the phospho-threonine

356/phospho-serine 360 motif. The phospho-specific SRF antibody [SRF(P)] recognizes

the phosphorylated Ser 103 residue; the phospho-specific elkI [elkl(P)] monoclonal

antibody recognizes phospho-serine 383. Both of these antibodies were purchased from

Cell Signaling Technologies. The RSK1 antibody (aRSKI-pAb) and the RSK2 antibody

(aRSK2-pAb) were obtained from Santa Cruz Biotechnology. A cocktail of ERK1/2

antibodies (aERK1/2-Abs) were used to measure total ERK1/2 protein levels. This

cocktail consisted of ERK1/2 monoclonal and polyclonal antibodies from Santa Cruz

Biotechnology. The ERK1/2 monoclonal antibody was used separately for

immunofluorescence. The aTyr(P) mAb (PY20) was from BD Transduction

Laboratories. The c-fos polyclonal antibody (acfos-pAb) was from Santa Cruz

Biotechnology. The phospho-serine polyclonal antibody [aSer(P)-pAb)] was purchased

from AnaSpec, Inc. The SL0101 compound was purchased from Toronto Research

Pharmaceuticals. The PKC( myristoylated pseudosubstrate (PKC( MP) was purchased

from Biomol Laboratories. PP2 and PD98059 compounds were obtained from

Calbiochem. Leptomycin B (LMB) was purchased from Sigma.









Cell lines and Cell Culture

The Chinese hamster ovary cell lines (CHO/ATi-WT and CHO/ATi-M5 cells)

were a gift from Dr. Kenneth Bernstein (Emory University), and have an equal

abundance of AT1 receptor as well as affinity for Ang II (28). The WT/AT1 and

SYF/AT1 mouse embryonic fibroblast cells are described in Chapter 2. VSMC cells were

cultured in the same media, but without Zeocin. WT/ATi, SYF/ATi, and VSMC were

growth arrested in serum-free DMEM for 48 h prior to experiments.

Cell Lysate Preparation, Immunoprecipitation and Western Blotting

For Westerns, cells were lysed in radioimmune precipitation assay (RIPA) buffer

containing protease inhibitors as described in Chapter 2. Cell lysates were

immunoprecipitated where indicated exactly as described in Chapter 2. Proteins were

detected using enhanced chemiluminescence exactly as described in Chapter 2 (117).

Densitometric Analysis

Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk

Scientific) as described in Chapter 2. The average pixel value minus background was

obtained for each cell type and normalized to the average pixel value for the respective

non-Ang II-treated cells.

Immunofluorescence

WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (Lab-Tek) and

grown to 70% confluency. Cells were serum-starved in serum free DMEM supplemented

with BSA for 48 hr. Following starvation, all cells were ligand-treated with 100 nM Ang

II. Slide chambers were removed from slides, and cells were washed one time with PBS

(pH 7.4). Cells were fixed in 4% paraformaldehyde (Fisher) for 10 min at room

temperature, rinsed in PBS and permeabilized for 3 min in acetone (Fisher) at -20C.









Cells were rinsed three times in PBS, and blocked for 15 min in 3% BSA/PBS at room

temperature in a homemade hydration chamber to prevent evaporation. Cells were

incubated with the primary antibody indicated for each experiment (1:500 in 3%

BSA/PBS) for 1 hr. All cells were next rinsed three times in PBS. Cells were incubated

with the appropriate fluorochrome-conjugated secondary antibody (1:100 in 3%

BSA/PBS) for 1 hour at room temperature in a hydration chamber. In the case of the

aRSK2-pAb, the rabbit IgG-FITC secondary antibody (Sigma) was used. For the

aERK2-mAb and aelkl(P)-mAb, the mouse IgG-Texas Red secondary antibody

(Sigma) was used, while aGoat IgG-FITC secondary antibody (Santa Cruz) was utilized

in conjunction with the aSRF(P)-pAb. Upon completion of incubation with the

appropriate secondary antibody, cells were washed three times in PBS. A coverglass was

mounted to each slide using Vectashield + DAPI mounting medium (Vector Labs). The

edges of the slides were sealed with nail polish sealant (Maybelline LLC) and allowed to

dry. All dry slides were stored at -20C until viewed. Slides were viewed on a Zeiss

Axioplan II Fluorescence microscope.

Quantification of nuclear and cytoplasmic fluorescence

Fluorescence in the nucleus or cytoplasm was quantified using the Image J Progam

(NIH) as described in Chapter 2.

c-fos transcriptional activity

SREw/Luc, mSRF/Luc, mTCF/Luc and TK/Luc plasmids were kindly provided by

Dr. Jessica Schwartz (University of Michigan) (71). WT/AT1 and SYF/AT1 cells were

plated onto four-chambered slides (LabTek) and transiently-transfected with each

individual plasmid using 8 tL lipofectin (Invitrogen). All transfected cells were

incubated for 5 hr at 37C. The transfection was stopped by washing cells in PBS, and









incubating the cells in serum-containing DMEM for 12-16 hrs. Cells were serum-starved

in DMEM + BSA (0.5% wt/vol) for 12-16 hrs, and treated with 100 nM Ang II for the

amount of time indicated in each experiment. Cells were placed in IX Reporter Lysis

Buffer (Promega), and exposed to one -80C freeze/thaw cycle (30 min each) to aid in the

disruption of cell membrane integrity. Cells were placed on a shaker at room temperature

for one additional hour to ensure complete lysis, and then transferred to a microcentrifuge

tube. Cells were centrifuged at 12,000 x g for 2 min at 4C, and 20 tL of cell lysate was

combined with 100 [tL luciferin substrate (Promega). Luciferase activity was measured

in a Monolight 3010 Luminometer (PharMingen) at 10 sec intervals.

Cell Migration Assay

Cell migration assays were performed using a commercially available Cell

Migration Kit (BioLabs). VSMCs were grown in 6 well cell culture plates. Cells were

detached, and placed in the upper chamber of a migration apparatus in serum-free

DMEM. The lower chamber of the apparatus was filled with serum-free DMEM. Cells

were then stimulated with 100 nM Ang II in the presence of SL0101 or vehicle for 24 hr,

and allowed to migrate from the upper chamber through a nylon membrane and into the

lower chamber. The membrane was washed, stained and photographed to visualize

migratory cells. The membrane was then placed in destaining solution and migratory cell

number was quantified indirectly via spectrophotometry at absorption 595 nm.

Cell Count

WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to

80% confluency. Cells were serum-starved and treated with 100 nM Ang II as indicated.

Both cell types were then counted using a hemacytometer as described (122).









Statistical Analysis

Data were analyzed by two-way ANOVA. All data passed a Normality Test as

well as Equal Variance Test. Pairwise comparisons were made following the Holm-

Sidak method. All data are expressed as mean +/- SEM. = p<0.05, ** = p<0.01.

Results

RSK Phosphorylation and ERK1/2-RSK Co-association Are Dependent Upon Src
Kinases in Response to Angiotensin II

The data in Chapter 2 suggest that ERK1/2 activated by c-Src/Yes/Fyn acts upon

cytosolic proteins to promote Ang II-dependent cell proliferation. It was first examined

whether or not the phosphorylation of RSK, a well known cytoplasmic substrate of

ERK1/2 (109, 128, 132), was dependent upon c-Src/Yes/Fyn and ERK1/2 signaling.

WT/AT1 and SYF/AT1 cells were stimulated with Ang II, and RSK activation

assessed via Western blot using a phospho-specific RSK antibody that recognizes

phosphorylated RSK1, RSK2 and RSK3. RSK activation occurred in response to Ang II

in WT/ATi cells and was maximal after 10 min of Ang II treatment and declined

thereafter (Figure 4-1A and data not shown). However, Ang II-induced RSK activation

was completely absent from SYF/ATi cells, indicating that c-Src/Yes/Fyn are necessary

for the activation of RSK in response to Ang II. These results were recapitulated by pre-

treating WT/ATi cells with PP2, a Src kinase inhibitor (Figure 4-1B). In addition, Ang

II-induced RSK phosphorylation in WT/ATi cells was dependent upon ERK1/2 activity

since pretreatment with the ERK activation inhibitor, PD98059, attenuated RSK

activation in these cells (Figure 4-1C). RSK activation in response to Ang II is therefore

mediated by Src kinases and ERK1/2.












WTIATI SYFIAT I
Angll 0 5 10 0 5 10
III kDa-
7QkDa.. -0 PSKP)
W B: ctRS K(P)-pAb
.ER~1,
W91- ERK1f2-Abg


WT/AT,
Angll 0 5 10 0 5 10
PP2 + + +
111 kD a-
79 k D ~


III kD~a
-- 4-RSKI '~4a t*-06 PWSK2
WB' itRSKI.pjkb WB: a RSK2.pAb
35kD a-: A W-W-ER -01W-..*ERK2
WB: ctERK112-Abs WffB: &.E RKI2-Abs

E
INTIAT, SY1IAT,
Aug 11 0 5 10 0 5 10
40P-S P)
IP:&~ERK1rAP)-pAb
WB:cR PS kP> Ab
35 kD a.- -4 m~ -E R k2


4 -RSK(P)


W B PSVP> pAb
315 k D a.-g 4
WB: ERK1t2-Abs

W TIAT*
AngI11 0 5 10 0 5 -10
P809- + +

111kD a..
WB: LR S P)- pAb


Whole Cell Lysates
WB: ERK<1/2-Abs


P)


WB: ERK1/2-Abs




Figure 4-1. RSK2 phosphorylation and RSK2-ERK1/2 co-association are decreased in
SYF/AT1 cells. A: WT/AT1 cells and SYF/AT1 cells were stimulated with
100 nM Ang II for 0, 5, and 10 min and total RSK phosphorylation assessed
via Western blot (WB) using the indicated antibody (top). Total protein
loading was demonstrated by stripping the membrane and reprobing for
ERK1/2 (bottom). WT/AT1 cells were pretreated with either 30 [M PP2 (B)
or 50 [M PD98059 (C) for 30 min, and stimulated with 100 nM Ang II for 0,
5, and 10 min. Total RSK phosphorylation was assessed by Western blot
using the indicated antibody (top). Total protein loading was demonstrated by
stripping the membrane and reprobing for ERK1/2 (bottom). D: The presence
or absence of RSK1 and RSK2 in cells was examined by Western blotting
WT/AT1 and SYF/AT1 whole cell lysates with the indicated antibodies (top).
Control MDCK and NIH3T3 whole cell lysates were also run on the same gel.
Total protein loading was demonstrated by stripping the membrane and
reprobing for ERK1/2 (bottom). E: Active RSK2-ERK co-association was
assessed in WT/AT1 and SYF/AT1 whole cell lysates by immunoprecipitating
(IP) and Western blotting with the indicated antibodies (top). Total protein
loading was demonstrated by blotting whole cell lysates with the indicated
antibodies (bottom). All Westerns are representative of at least three
independent blots. This figure is used with permission from (43).









The specific isoform(s) of RSK phosphorylated in response to Ang II within the

WT/AT1 and SYF/AT1 cells was next determined. Of the three known RSK isoforms,

only RSK1 and RSK2 are phosphorylated by ERK1/2 (37). Whole cell lysates from

WT/ATi and SYF/ATi cells were prepared and Western blotted with RSK1 or RSK2

specific antibodies alongside positive control whole cell lysates. RSK1 was not

expressed in WT/ATi cells, but was expressed in SYF/ATi cells (Figure 4-1D). These

expression levels did not change with the addition of Ang II (data not shown). However,

RSK2 was expressed equally in both cell types. Therefore, RSK2 is most likely the

isoform phosphorylated in the presence of c-Src/Yes/Fyn. As an aside, c-Src/Yes/Fyn

may regulate RSK1 protein degradation since RSK1 was present in c-Src/Yes/Fyn

deficient cells, but absent from cells containing these proteins.

Previous reports have shown that active ERK1/2 bind RSK proteins via the ERK

docking site and, once bound, modulate RSK activity (121, 128, 132). It was next

determined whether RSK and ERK1/2 co-association is controlled by c-Src/Yes/Fyn, in

order to determine if the ERK1/2 activated by c-Src/Yes/Fyn binds to RSK. WT/AT1 and

SYF/ATi whole cell lysates were immunoprecipitated with a phospho-specific ERK1/2

antibody, and then Western blotted with the phospho-specific RSK antibody. ERK1/2-

RSK co-association was evident after 5 min of Ang II treatment in WT/ATi cells, but was

completely absent from SYF/ATi cells (Figure 4-IE). As such, these data demonstrate

that ERK1/2 and RSK co-associate in response to Ang II, and this is c-Src/Yes/Fyn-

dependent.









RSK Nuclear Translocation Is Src Kinase Dependent, While ERK1/2 Nuclear
Translocation Is PKC4 Dependent in Response to Angiotensin II

Phosphorylated RSK has been shown to translocate into the nucleus and modulate

the transcriptional activity of target genes in response to stimulation of various cytokine

and growth factor receptors (16, 162). It was next determined whether RSK nuclear

translocation occurs in response to Ang II, and whether this event is regulated by c-

Src/Yes/Fyn. WT/AT1 and SYF/AT1 cells were pretreated with leptomycin B (LMB) to

prevent the nuclear exportation of proteins, and then stimulated with Ang II. RSK

nuclear accumulation was then assessed by immunofluorescence. Translocation of RSK2

into the nucleus occurred in response to Ang II treatment in WT/AT1 cells, and the

nuclear accumulation of RSK2 was confirmed by merging the RSK2 image with DAPI

(Figures 4-2A & 4-2B, 4-2E & 4-2F). However, Ang II-induced RSK2 nuclear

translocation did not occur in the absence of c-Src/Yes/Fyn (Figures 4-2C & 4-2D, 4-2G

& 4-2H). Pretreatment of WT/AT1 cells with PP2, a Src family kinase inhibitor, also

prevented Ang II-induced RSK2 nuclear accumulation (Figures 4-21 & 4-2J, 4-2K & 4-

2L). Quantification of nuclear and cytoplasmic fluorescence confirmed that RSK2

nuclear translocation occurred in WT/ATi cells stimulated with Ang II, but was blocked

in SYF/AT1 cells or in WT/AT1 cells pretreated with PP2 (Figure 4-2M). Collectively,

these data show that Ang II-induced RSK nuclear translocation is regulated by c-

Src/Yes/Fyn.

Ang II-induced ERK1/2 nuclear translocation and RSK2 nuclear translocation

patterns were next assessed in WT/ATi and SYF/AT1 cells. All cells were pretreated







70



WT/AT, SYF/AT,
Ang II + +
LMB +

RSK2
60 ** ** W/AT,
S0H 50D SYF/AT



PP2 + + 20 -

RSK2 [0
Ang +



WT/AT SYF/AT
Ang II + + Z
LMB + + + +
70 ** NSIIWT/AT
RSK2 60- SYF/AT
I 50- T
o 2. 40
ERK1/2 30


RSK2/ERK1&2/ 0 1
DAPI Merged Ang 1 + +




Figure 4-2. RSK2 and ERK1/2 nuclear phosphorylation in response to Ang II in WT/ATi
and SYF/ATi cells. A-L, N-Y: WT/ATi and SYF/ATi cells were retreated
with 0.005 [tg Leptomycin B for 5 min prior to stimulation with 100 nM Ang
II for 10 min. All cells were incubated with antibodies specific for the
indicated proteins (right) and respective fluorochrome-conjugated secondary
antibody. E-H, K, L, V-X: Images were DAPI stained and merged with the
respective fluorescent protein images. I-L: WT/ATi cells were pretreated
with 20 AM PP2 for 1 hr prior to stimulation with 100 nM Ang II. M: Nuclear
fluorescence from A L was quantified and normalized to cytoplasmic
fluorescence. Z: Nuclear fluorescence from R U was quantified and
normalized to cytoplasmic fluorescence. All images are representative of the
entire field and were taken at 40X magnification. Bar represents 15 microns.
This figure is used with permission from (43).

with LMB and then stimulated with Ang II. RSK2 and ERK1/2 nuclear translocation

were then assessed via immunofluorescence. Both RSK2 and ERK1/2 translocated into

the nucleus in response to Ang II in WT/ATi cells (Figures 4-2N & 4-20, 4-2R & 4-2S).

Merging of RSK2 and ERK1/2 images confirmed that RSK2 and ERK1/2 nuclear









translocation patterns were the same in these cells (Figures 4-2V and 4-2W). However,

nuclear translocation of ERK1/2 persisted in SYF/AT1 cells while RSK2 nuclear

translocation was attenuated (Figures 4-2P and 4-2Q, 4-2T and 4-2U). Merging of the

ERK1/2 and RSK2 images confirmed that these two proteins exhibit different patterns of

nuclear translocation in SYF/AT1 cells (Figures 4-2X and 4-2Y). Quantification of

nuclear and cytoplasmic fluorescence revealed that GFP-ERK2 nuclear translocation was

similar in WT/AT1 and SYF/AT1 cells (Figure 4-2Z). As such, these data demonstrate

that ERK1/2 nuclear translocation is regulated by PKC-dependent signaling whereas

RSK2 nuclear translocation is controlled by c-Src/Yes/Fyn.

SRF and TCF Binding Within the c-fos Promoter Are Mediated in A RSK And A
ERK1/2-dependent Manner, Respectively

c-fos transcription is regulated via the binding of specific transcription factors to

the serum response element (SRE) within the c-fos promoter, namely the serum response

factor (SRF) and ternary complex factor (TCF) (71, 78, 84, 162). RSK has been

implicated in the phosphorylation of the SRF while ERK1/2 have shown to phosphorylate

TCF proteins, thereby increasing the activity of these transcription factors (78, 84, 142,

162). However, the roles of the SRF and the TCF during Ang II-induced c-fos

transcription are still unknown.

Whether SRF and TCF activity are regulated by either PKC-dependent or c-

Src/Yes/Fyn-dependent signaling in response to Ang II was next examined. WT/ATi and

SYF/ATi cells were stimulated with Ang II, and SRF or TCF nuclear phosphorylation

assessed via immunofluorescence. Nuclear SRF phosphorylation occurred in response to

Ang II treatment in WT/AT1 cells (Figures 4-3A and 4-3B, 4-3F and 4-3G). Ang II-

induced SRF phosphorylation was completely lost in SYF/ATi cells stimulated with Ang









II (Figures 4-3D and 4-3E, 4-31 and 4-3J). In addition, pretreatment of WT/AT1 cells

with PKC( MP did not affect Ang II-induced SRF phosphorylation (Figures 3C and 3H).

Quantification of nuclear and cytoplasmic fluorescence revealed that Ang II-induced SRF

phosphorylation in WT/AT1 cells was not affected by PKC MP pre-treatment, while

Ang II-induced SRF phosphorylation did not occur in SYF/AT1 cells (Figure 4-3Q).

Thus, SRF phosphorylation is dependent upon c-Src/Yes/Fyn-dependent signaling and

not PKC(.

The nuclear phosphorylation of elk, a TCF activated in response to ERK1/2, was

assessed in the same manner. ElkI phosphorylation occurred in response to Ang II in

WT/AT1 cells (Figures 4-3K and 4-3L, Figures 4-3N and 4-30). In addition, Ang II-

induced elkI phosphorylation persisted in SYF/AT1 cells (data not shown). Pretreatment

with PKC MP attenuated elkI nuclear phosphorylation (Figures 4-3M and 4-3P).

Quantification of nuclear and cytoplasmic fluorescence revealed that Ang II-induced elkI

phosphorylation in WT/AT1 cells was blocked by PKC MP pretreatment (Figure 4-3R).

Thus, TCF phosphorylation appears to be mediated by PKC-dependent signaling and not

by c-Src/Yes/Fyn.










WT/AT,


Ang II
PKC NMP


U .


U.


WT/AT/


100
90
a 80
70
5 60
50
~ 40
- 30
z 20
10
0
Ang II
PKCC MP


** NS
I m I


U


U


SRF(P)


SRF(P)/DAPI Merged


elkl (P)


elkl(P)/DAPI Merged


D WHAT,
n SYF/ATj


NS
A 1 747


100
90 ** r 1
g 80
1= 0 70
S0 60-
S50-
S 40 -
S30 -
20
-10
Ang II + +
PKC MP +


Figure 4-3. Ang II-induced SRF and elkl nuclear phosphorylation in WT/AT1 and
SYF/AT1 cells. A-P: WT/ATi or SYF/ATi cells were stimulated with 100 nM
Ang II for 10 min. All cells were incubated with antibodies specific for the
indicated proteins (right) and their respective fluorochrome-conjugated
secondary antibody. F-J, N-P: Protein fluorescence images were merged with
DAPI stained images. C & H, M & P: Cells were pretreated with 1 [M PKC(
MP for 1 hr prior to Ang II treatment. Q: Nuclear fluorescence from A E
was quantified and normalized to cytoplasmic fluorescence. R: Nuclear
fluorescence from K M was quantified and normalized to cytoplasmic
fluorescence. These results are representative of three independent
experiments. All images are representative of the entire field and were taken
at 40X magnification. Bar represents 15 microns. This figure is used with
permission from (43).

Both the SRF and TCF have been shown to modulate c-fos transcriptional activity

in response to growth hormone treatment (71). Whether SRF and TCF binding of the c-


Ang II
PKCCMP


SYF/AT,


+ +
+









fos SRE occurred in response to Ang II, and, if these events were altered by the absence

of c-Src/Yes/Fyn, were next tested. WT/AT1 and SYF/AT1 cells were transiently

transfected with wild-type or mutated SRE-luciferase plasmids and then stimulated with

Ang II. The wild-type SRE plasmid (SREw/Luc) alone mediated Ang II-induced

luciferase expression in both cell types (Figure 4-4). Ang II-induced luciferase activity

was reduced by about 50% in SREw/Luc transfected SYF/ATi cells relative to

transfected WT/ATi cells, indicating that c-fos transcriptional activity is in part

dependent upon c-Src/Yes/Fyn. Furthermore, mutation of either the SRF or TCF binding

sites only partially blocked Ang II-induced luciferase expression in WT/ATi cells. In

addition, Ang II-induced luciferase expression was completely blocked in SYF/ATi cells

transfected with either the mSRF/Luc or mTCF/Luc plasmids. Note that the reporter

plasmid alone (TK/Luc) consistently failed to respond to Ang II in both cell types. These

data therefore indicate that the SRF and TCF transcription factors both partially modulate

c-fos transcriptional activity in either a c-Src/Yes/Fyn or PKC-dependent manner,

respectively.

c-fos Protein Expression Is Dependent Upon Src Kinase Signaling And PKC4
Signaling

It was next determined if the inhibition of PKC-dependent and/or Src kinase-

dependent signaling reduced c-fos protein levels. WT/AT1 and SYF/AT1 cells were

stimulated with Ang II and c-fos protein levels assessed via Western blot. c-fos protein

exhibited an Ang II-dependent increase in both cell types, and was maximal after 60 min

of Ang II treatment (Figure 4-5A). However, Ang II-induced c-fos protein production

was reduced in SYF/ATi cells relative to WT/ATi cells. Infact, there was about a 50%

reduction in c-fos in SYF/ATi cells compared to WT/AT1 cells (Figure 4-5B).










WT/AT1 SYF/AT1



o 200%
0 *-_ NS NS
150%- --



100%
I50%


SREw/Luc mSRF/Luc mTCF/Luc TK/Luc SREw/Luc mSRF/Luc mTCF/Luc TK/Luc
Ang II + + + + + + + +



Figure 4-4. c-fos transcriptional activity in WT/AT1 and SYF/AT1 cells in response to
Ang II. WT/AT1 or SYF/AT1 cells were transfected with the indicated
plasmid and then stimulated with Ang II for 0 or 5 hr. Data are expressed as
percentage of luciferase activity relative to unstimulated cells. These results
are representative of three independent experiments. This figure is used with
permission from (43).

Pretreatment of WT/AT1 cells with PKC( MP also reduced c-fos protein amounts by

50%, while PKC( MP addition to Ang II-stimulated SYF/AT1 cells resulted in a complete

loss of Ang II-induced c-fos protein production. These data demonstrate that Ang II-

induced c-fos protein synthesis is partially influenced by PKC and partially by Src

kinase-dependent signaling.

c-fos Phosphorylation Is Dependent Upon Src Kinase-RSK Signaling

Previous reports have shown that c-fos activity is not only regulated at the

transcriptional level, but also post-translationally via specific phosphorylation events

(16). Specifically, phosphorylation of c-fos by RSK at Ser residues within the C-terminal

tail increases the stability of c-fos and the subsequent growth-promoting effects

associated with extended c-fos activity (16). Whether c-fos serine phosphorylation is







76


dependent upon c-Src/Yes/Fyn mediated ERK/RSK2 activation was next tested. WT/AT1

A
WT/AT1 SYF/AT1
AngII(min) 0 60 60 0 60 60
PKCi MP + +

49 kDa- 4- c-fos
WB: acfos-pAb
36 kDa- IM mN O- Nt ERK2

WB: aERK2-pAb

B
I]
Sc 100 -
0 0 WT/AT1
E 80- NS 0 SYF/AT1
0 60
S40
-= 20

Ang II + + + +
PKCi MP + +


Figure 4-5. c-fos protein levels in response to Ang II in WT/AT1 and SYF/AT1 cells. A:
WT/ATi or SYF/ATi cells were stimulated with 100 nM Ang II for 0 or 60
min, and c-fos protein production and total protein levels assessed by Western
blot using the indicated antibodies. Some cells were pretreated with 1 tM
PKC( MP for 1 hr as indicated. B: These data are expressed as the percentage
of maximum c-fos protein production in response to Ang II. Protein amounts
were densitized and values were normalized to the amount of c-fos protein in
WT/ATi cells treated with Ang II. These results are representative of three
independent experiments. This figure is used with permission from (43).

and SYF/AT1 cells were stimulated with Ang II and cell lysates were immunoprecipitated

with anti-phosphoserine antibody and Western blotted with a c-fos specific antibody to

assess for changes in c-fos phosphorylation at Ser residues. A marked four-fold increase

in c-fos phosphorylation was observed in WT/ATi cells after 60 min of Ang II treatment

(Figure 4-6A). Phosphorylated c-fos levels remained elevated after 120 min of Ang II

treatment, and began to decline by 240 minutes. In contrast, a comparatively small









increase in c-fos phosphorylation was observed in Ang II-treated SYF/AT1 cells after 60

min, and phosphorylated c-fos levels declined to baseline amounts by 120 min. These

results are displayed graphically, and were normalized to total c-fos protein amounts to

account for differences in total c-fos protein (Figure 4-6B). Collectively, these data

suggest that c-fos phosphorylation is reduced in the absence of c-Src/Yes/Fyn and

subsequent ERK/RSK2 activation in response to Ang II.

Angiotensin II-induced Cell Proliferation Requires RSK And PKC4 Activity

The consequence of selective RSK inhibition on Ang II-induced cell proliferation

was next assessed since RSK activation (under the influence of Src kinase activated

ERK1/2) appears to modulate c-fos levels and activity. Recently, a highly selective and

potent RSK inhibitor, SL0101, was isolated by Lannigan and colleagues (129).

Interestingly, this compound is a natural product derived from theforesta refracta plant

found in the amazon rain forest. SL0101 has already been shown to selectively inhibit

RSK without interfering with upstream activators of RSK like ERK, MEK, EGFR and

PKC (77). Furthermore, this compound has been shown to prevent cell proliferation

in cancer cells and thus has established RSK as a target for therapeutic intervention and

SL0101 as an anti-cancer agent (18). However, a role for RSK in Ang II-induced cell

proliferation remains to be established.

WT/AT1 and SYF/AT1 cells were stimulated with Ang II, and cell proliferation

assessed via a direct cell count. A marked 3-fold increase in cell number was observed in

WT/ATi cells treated with Ang II (Figure 4-7A). Ang II-induced increases in cell

number were reduced by 1.5 fold in SYF/ATi cells relative to WT/ATi cells.

Pretreatment with SL0101 reduced WT/AT1 cell number to levels found in SYF/ATi

cells treated with Ang II. However, SL0101 pretreatment did not affect Ang II-induced










A
WT/AT1 SYF/AT,
Ang II (min) 0 60 120 240 0 60 120 240

49 kDa- W [ 4 -c-fos(P)

IP: aSer(P)-mAb
WB: acfos-pAb

B

coj
0 5
S 3. 5 **
2 4.5*

S3.5
o o 3 -WT/AT1
0 uo z2.5
2 2
S 1.5 ---* -- SYF/AT1

5 0.5
oE 0
0 -0 60 120 240
U-
Ang II (min)



Figure 4-6. Ang II-induced c-fos phosphorylation in WT/AT1 and SYF/AT1 cells.
WT/AT1 or SYF/AT1 cells were stimulated with 100 nM Ang II for 0, 60,
120, or 240 min. A: c-fos phosphorylation was then assessed by
immunoprecipitating and Western blotting with the indicated antibodies. B:
Protein bands were densitized and values were expressed as a fold change in
c-fos phosphorylation relative to unstimulated cells as a function of total c-fos
protein amounts. These results are representative of three independent
experiments. This figure is used with permission from (43).

increases in SYF/ATi cell number, suggesting that Ang II-induced cell proliferation

occurring independent of c-Src/Yes/Fyn is not dependent upon RSK2 and also that

SL0101 exhibits low toxicity. Finally, PKC MP pretreatment completely attenuated

Ang II-induced increases in SYF/ATi cell number but only partially attenuated Ang II-

induced increases in WT/ATi cell number. Furthermore, the addition of PKC MP and

SL0101 to WT/ATi cells blocked all Ang II-induced increases in cell number. These

data therefore demonstrate that PKC( partially regulates Ang II-induced cell proliferation









independent of RSK2. Furthermore, RSK2 mediates Ang II-induced cell proliferation

downstream of c-Src/Yes/Fyn since the addition of SL0101 to SYF/AT1 cells did not

further lower the already reduced amount of Ang II-induced cell proliferation exhibited

by these cells.

To recapitulate these findings in another cell type, the effect of SL0101 addition on

cells devoid of heterotrimeric G protein activation (CHO/ATi-M5 cells) was examined.

A marked 3-fold increase in cell number was observed in CHO/ATi cells treated with

Ang II (Figure 4-7B). Ang II-induced increases in cell number were reduced by about

0.5 fold in CHO/ATi-M5 cells relative to CHO/ATi cells. Pretreatment of CHO/ATi-M5

cells with SL0101 completely blocked Ang II-induced increases in cell number, whereas

SL0101 partially attenuated cell number in CHO/AT1 cells stimulated with Ang II to

levels found in Ang II-treated CHO/ATi-M5 cells. Thus, RSK2 does not regulate Ang II-

induced cell proliferation through heterotrimeric G protein-dependent mechanisms. The

pretreatment of CHO/ATi cells with PKC MP partially reduced Ang II-induced cell

proliferation; however, CHO/ATi-M5 cell number was not affected by the addition of

PKC MP. Therefore, PKC appears to partially mediate Ang II-induced cell

proliferation, and it does so downstream of heterotrimeric G proteins. Finally, all Ang II-

induced cell proliferation in CHO/AT1 cells was blocked by pretreatment with PKC MP

and SL0101. Collectively, these data further demonstrate that Ang II-induced cell

proliferation is regulated by heterotrimeric G protein/PKC and c-Src/Yes/Fyn/RSK-

dependent signaling







80


A

Sa0 WT/AT1
E 3.1 SYF/AT1
z= 3.5
3
0 2.5 NS **
E

co 1.5
S 1 heterotrimeric
0.5 G protein
O 0 ", 0
"L PKC(
Angll -- + + + +-- + + ++
SL0101 + ++ + + ++
PKC(MP - + + + + + + + + ERK1/2


B

.Q
E
z 3.5 CHO/AT1
z 3- **0 CHO/AT1M5
0
S2.5 AT

2 1.5


0 c-Src/Yes/Fyn
u o I n I .. .
Ang ll + + ++ + + + +
SL0101 - + + ++ + + + + ERK1/2
PKCMP - - + + + + ++ + +



Figure 4-7. Ang II-induced cell proliferation in response to RSK and PKC/ inhibition.
A: WT/AT1 and SYF/AT1 cells were stimulated with Ang II for 0 (-) or 24 (+)
hr. Some cells were pretreated with either 30 pM SL0101, 1 pM PKC( MP,
or both inhibitors in combination for 1 hr as indicated. Cells were counted
and fold changes in cell number plotted relative to unstimulated cells (left).
The drawing on the right illustrates the intact endogenous ERK1/2 activation
pathway in the SYF/AT1 cells. B: CHO/AT1 and CHO/ATi-M5 cells were
stimulated with Ang II for 0 (-) or 24 (+) hr. Some cells were pretreated with
either 30 pM SL0101, 1 pM PKC( MP, or both inhibitors in combination for 1
hr as indicated. Cells were counted and fold changes in cell number plotted
relative to unstimulated cells (left). The drawing on the right illustrates the
intact endogenous ERK1/2 activation pathway in CHO/ATi-M5 cells. These
results are representative of three independent experiments. This figure is
used with permission from (43).









Angiotensin II-induced ERK1/2 Activation Is Mediated By Both Src Kinases and
PKC4 in Vascular Smooth Muscle Cells

Thus far, it has been demonstrated that Ang II-induced ERK1/2 activation is

mediated by Src kinase and heterotrimeric G protein/PKC signaling in both MEF and

CHO cells. It was next determined if Ang II-induced ERK1/2 activation is also mediated

by both c-Src/Yes/Fyn and heterotrimeric G protein/PKC signaling in cells which

endogenously express the AT1 receptor. Here, primary cultures of VSMCs isolated from

rat aortas were utilized. c-Src/Yes/Fyn or PKC( activity was then blocked using

pharmacological inhibitors. VSMC were pretreated with either PP2, PKC( MP or PKC(

MP and PP2 in combination. Cells were then stimulated with 100 nM Ang II for 0, 5 and

10 min. Whole cell lysates were prepared and Western blotted with phospho-specific

ERK1/2 antibodies to identify changes in ERK1/2 activation. Ang II-induced ERK1/2

activation occurred after 5 min of Ang II treatment in VSMC (Figure 4-8A). ERK1/2

activation was significantly reduced in VSMC treated with either PP2 or the PKC(

pseudosubstrate alone, and treatment with either of these inhibitors alone resulted in

about a 50% reduction in ERK2 phosphorylation (Figure 4-8B). Furthermore, ERK1/2

activation was completely reduced in cells treated with both PP2 and the PKC(

pseudosubstrate in combination. Collectively, these data confirm that the mechanisms of

intracellular ERK1/2 activation are the same in the AT1 receptor-transfected MEF and

CHO cells as in VSMCs which endogenously express the AT1 receptor. VSMC ERK1/2

activation occurs via c-Src/Yes/Fyn or heterotrimeric G protein/PKC-dependent

signaling in response to angiotensin II.










A
VSMC
Angll(min) 0 5 10 0 5 10 0 5 10 0 5 10
PP2 + ++ + + +
PKC MP - + + + + + +
.-ERK1(P)
36 kDa- ERK2(P)
WB: aERK1/2(P)-pAbs
36 kDa a-- -... ., .... -ERK2
WB: aERK1/2-Abs

B
o 120
w 100 NS
0 80
S60
0(D 40 -
2& 20

Ang II + + + +
PP2 + +
PKC MP + +



Figure 4-8. Ang II-induced ERK1/2 activation is mediated by c-Src/Yes/Fyn and PKC(-
dependent signaling in VSMC. A: VSMCs were pretreated with either
DMSO, 30 [iM PP2, 1 [iM PKC( myristoylated pseudosbstrate, or both PP2
and PKC MP in combination. All inhibitor treatment times were 1 hr.
ERK1/2 activation was then measured via Western blot analysis using the
indicated antibodies (top panel). Total ERK1/2 protein loading was
demonstrated by stripping the membrane and reprobing with the indicated
antibodies (bottom panel). B: Three representative Western blots of A were
scanned and densitized and the percent maximum ERK2 phosphorylation was
calculated by dividing by active ERK2 amounts in non-inhibitor treated cells
after 5 min of Ang II stimulation and multiplying by 100. This figure is used
with permission from (43).

Angiotensin II-induced Cell Migration Is Attenuated in VSMCs Treated With
SL0101

Previous work has demonstrated that aberrant migration and proliferation of

VSMCs is triggered by angiotensin II during cardiovascular diseases such as

atherosclerosis (72). VSMC migration precedes proliferation, and results in the









formation of a fibrous plaque. It was next tested whether Ang II-induced VSMC

migration could be attenuated by pretreating with SL0101.

VSMCs were pretreated for 1 hour with the indicated concentration of SL0101 or

vehicle control, and then stimulated with 100 nM angiotensin II for 24 hours. Cells were

allowed to migrate across a nylon membrane where they were then stained, photographed

and counted via quantification of the stain using a spectrophotometer. Little to no cell

migration occurred in the absence of Ang II; however, cell migration did occur in

response to Ang II (Figure 4-9). Furthermore, Ang II-induced cell migration was

attenuated in a dose-dependent manner through the addition of SL0101. Maximum

reductions in Ang II-induced cell migration were observed with the addition of 100 tM

SL0101. As such, some Ang II-induced cell migration is RSK-dependent.

Discussion

In Chapters 2 and 3, intracellular ERK1/2 activation is shown to be mediated by

both c-Src/Yes/Fyn-dependent and heterotrimeric G protein/PKC-dependent signaling,

and both of these signaling pathways contribute equally to cell proliferation in response

to Ang II (42). Here, these findings are extended by defining the mechanism as to how

this occurs (summarized in Figure 4-10). The key to these findings is that heterotrimeric

G protein/PKC-dependent signaling dictates whether ERK1/2 translocates into the

nucleus and phosphorylates specific transcription factors like elkl, leading to increased c-

fos transcriptional activity. c-Src/Yes/Fyn-signaling, on the other hand, phosphorylates

ERK1/2 in the cytoplasm, where ERK1/2 remains and complexes with RSK2. RSK2

becomes activated, and then translocates into the nucleus to modulate c-fos transcription

and c-fos protein activity. As such, these signaling events coordinately regulate

proliferation in response to Ang II.






84


Effect of RSK inhibition on angiotensin II-induced cell migration







30 xU


p = 0.01
II


- Ang + Ang + Ang + Ang
+ Vehicle + Vehicle 30 itM SL0101 100 M SLOlOl01


Figure 4-9. Angiotensin II-induced cell migration is attenuated through selective RSK
inhibition. Cells were pretreated with the indicated concentration of SL0101
or vehicle control for 1 hour. Cells were then stimulated with 100 nM Ang II
for 24 hr, and allowed to migrate. Migratory cells (purple) were photographed
after Ang II treatment, and cell number was assessed through
spectrophotometry. These data are representative of three independent
experiments. This figure is used with permission from (43).

These findings support the idea that two separate pools of ERK1/2 exist within the cell: a

pool of ERKl/2 which complexes with and activates RSK2 and a pool of ERK1/2 which

translocates directly into the nucleus. However, ERK2 activation resulted in the

dissociation of the ERK2-RSK complex within these cells. Other studies in COS7 cells

ectopically expressing the three RSK isoforms demonstrated that ERK-RSK complexes














Cell membrane


PKCi c-Src/Yes/Fyn

MEK1/2 MEK1/2

ERK1/2 ERK1/2(P) ERK1/2 ERK1/2(P)

RSK2(P)


ERK1/2(P) RSK2(P) RSK2(P)

SR ^^c-fos c-fos(P)


SRE
Nucleus

Cell
Proliferation


Figure 4-10. Mechanistic diagram illustrating how Src kinase and PKC-dependent
ERK1/2 activation pathways dually regulate Ang II-induced cell proliferation.
ERK1/2 activation is separately mediated by Src family tyrosine kinase and
heterotrimeric G protein/PKC signaling in response to Ang II. With regards
to PKC( regulation, ERK1/2 translocates into the nucleus upon stimulation of
the AT1 receptor. Here, ERK1/2 phosphorylates elkl, which binds to the c-fos
SRE and partially regulates c-fos transcriptional activity. c-fos transcriptional
activity is also regulated by binding of the SRF. The SRF is phosphorylated
in response to nuclear RSK2, which translocates into the nucleus after being
phosphorylated by ERK1/2 in the cytoplasm. Cytoplasmic ERK1/2
phosphorylation is regulated by c-Src/Yes/Fyn-dependent signaling,
independent of heterotrimeric G protein/PKC activity. Additionally, nuclear
RSK2 directly phosphorylates c-fos and increases the activity and stability of
this protein. Thus, two independent pathways of ERK1/2 activation
coordinately regulate Ang II-induced cell proliferation by inducing c-fos
transcription and increasing c-fos activity through the post-translational
modification of this protein. This figure is used with permission from (43).