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Jak2 Tyrosine Kinase: New Insights Regarding Structure, Function, and Pharmacology


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Jak2 TYROSINE KINASE: NEW INSIGHTS REGARDING STRUCTURE, FUNCTION, AND PHARMACOLOGY By ERIC M. SANDBERG 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 2004

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Copyright 2004 by Eric M. Sandberg

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This dissertation is dedicated to my parents, for their constant love and support.

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ACKNOWLEDGMENTS I would like to thank, first and foremost, my adviser, Dr. Peter Sayeski. Dr. Sayeski has an enthusiasm for science that is inspiring. His flexibility allowed me to successfully pursue my own research interests, while his seemingly inborn mentoring skills never let me flounder. I feel lucky to have been Dr. Sayeskis first graduate student. He took the time to teach me research techniques, and also how to write scientific papers, grantsmanship, and professionalism in science. He has been a mentor and a friend. Second, I want to thank my graduate committee. Drs. Mohan Raizada, Colin Sumners, and Peggy Wallace have been very supportive; their technical input has been extrememtly useful, and their encouragement has been invaluable. Third, I want to thank all of the members of the Sayeski Lab, and everyone else who trained and/or helped me along the way. Danielle Vonderlinden and Melissa Johns kept the lab running smoothly, and their technical assistance significantly accelerated our time to publication. Tiffany Wallace, Mike Godeny, Issam McDoom, and Dr. Xianyue Ma have been both collaborators and friends. I also want to thank Drs. Michael Katovich and Hideko Kasahara for serving on my examination committee, and Tim Vaught, for his generous assistance with optical microscopy. Additionally, I want to thank Dr. David Ostrov, who was an essential collaborator throughout my graduate studies. Without him, our structure-function and inhibitor studies would not have been possible. iv

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Finally, I want to thank my family and friends who supported me throughout graduate school. In particular, my parents instilled in me a passion for learning and science from an early age. With a mom who is a nurse, and a dad who drove his 1972 Datsun until it had precisely 176,000 miles on the odometer (so he could brag that he had driven his car for 1 light second), it is no surprise that I pursued medical science as a career. I also want to thank Mia DeBarros for her constant love, support, and technical insight. v

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION TO THE Jak-STAT PATHWAY..................................................1 History of the Jak-STAT Pathway................................................................................1 Initiation of Jak-STAT Signaling.................................................................................4 Physiology and Pharmacology of Jak2.........................................................................6 Target Genes and Regulation of Signal Transduction Pathways..........................6 Regulation of Jak2 Signaling.................................................................................7 Jak2 in Cancer.....................................................................................................10 Jak2 in Cardiovascular Disease...........................................................................11 Jak2 Pharmacology.....................................................................................................14 Structure-Function Relationship of Jak2....................................................................16 2 EXPERIMENTAL METHODS.................................................................................23 Cell Culture.................................................................................................................23 Vaccinia Virus Transfection/Infection.......................................................................23 Immunoprecipitation/Western Blotting......................................................................23 Immunocytochemistry................................................................................................24 In Vitro Kinase Assays...............................................................................................24 Site-Directed Mutagenesis..........................................................................................25 Luciferase Assays.......................................................................................................25 Molecular Modeling...................................................................................................25 Analysis of DNA Laddering.......................................................................................26 Hoechst 33342 Staining..............................................................................................26 Propidium Iodide Staining..........................................................................................26 Analysis of Mitochondrial Membrane Integrity.........................................................26 vi

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3 MEANS OF CIRCUMVENTING PROBLEMS WITH AG490...............................27 Introduction.................................................................................................................27 Primary Rat Aortic Smooth Muscle Cells..................................................................27 2A Cell Line..............................................................................................................27 4 Jak2 REGULATES ANGIOTENSIN II-DEPEDENDENT ERK2 SIGNALING.....28 Introduction.................................................................................................................28 Results.........................................................................................................................29 ERK2 Activity is Sustained in 2A/AT 1 Cells Compared to 2A/AT 1 +Jak2 Cells after Angiotensin II Treatment.........................................................................29 AG490 Suppresses Angiotensin II-Dependent ERK2 Activation Independent of Jak2 Inhibition.................................................................................................31 There is a Marked Difference in the Angiotensin II-Induced Nuclear Accumulation Pattern of Activated ERK2 between 2A/AT 1 and 2A/AT 1 +Jak2 Cells........................................................................................34 ERK2-Dependent Gene Transcription in the 2A/AT 1 and 2A/AT 1 +Jak2 Cells34 Jak2 is Essential for Angiotensin II-Induced MKP-1 Expression and Co-association of MKP-1 with ERK2...................................................................36 MPK-1 is Required for Angiotensin II-Dependent Inactivation of ERK2..........38 Discussion...................................................................................................................42 5 Jak2 PROMOTES OXIDATIVE STRESS-INDUCED APOPTOSIS IN VASCULAR SMOOTH MUSCLE CELLS..............................................................46 Introduction.................................................................................................................46 Results.........................................................................................................................47 Jak2 Activation by Hydrogen Peroxide is Suppressed in RASM-DN Cells.......47 Jak2 Activation is Required for Oxidative Stress-Induced Apoptosis................47 Quantification of Jak2-Mediated Apoptosis........................................................50 Jak2 Activation by Oxidative Stress Mediates Bax Expression..........................51 Jak2 Activation by Oxidative Stress Promotes Mitochondrial Dysfunction.......53 Jak2 is Required for Caspase-9 Cleavage During Oxidative Stress....................55 Discussion...................................................................................................................56 6 Jak2 RESIDUES GLU 1024 AND ARG 1113 FORM HYDROGEN BONDS, AND ARE ESSENTIAL FOR Jak-STAT SIGNAL TRANSDUCTION............................61 Introduction.................................................................................................................61 Results.........................................................................................................................62 Molecular Modeling Identified a Putative Interaction between Jak2 Residues Glu 1024 and Arg 1113....................................................................................62 Mutation of Jak2 Residue Glu 1024 or Arg 1113 Abolishes Jak2 Kinase Activity63 Individual Mutations of W1020G or E1024A Render Jak2 Dominant Negative65 vii

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Jak2-R1113E is Unable to Become Tyrosine Phosphorylated by Angiotensin II66 Jak2-R1113E is Unable to Tyrosine Phosphorylate STATs in Response to Angiotensin II..................................................................................................69 Jak2-R1113E is unable to mediate angiotensin II-dependent gene expression...70 Arg 1113 is conserved in different Jak kinase family members and among species expressing Jak2....................................................................................71 Discussion...................................................................................................................72 7 IDENTIFICATION OF A NOVEL Jak2 INHIBITOR..............................................75 Introduction.................................................................................................................75 Results.........................................................................................................................76 Homology Modeling and Target Pocket Identification.......................................76 Database Screening to Identify Potential Small-Molecule Inhibitors of Jak2....76 Compound 7 Inhibits Jak2 Autophosphorylation................................................78 Compound 7 Inhibits Jak2 Autophosphorylation in a Time-Dependent Manner78 Compound 7 Inhibits Jak2 Autophosphorylation in a Dose-Dependent Manner81 Compound 7 is Non-Cytotoxic at Concentrations that Maximally Inhibit Jak2 Tyrosine Autophosphorylation........................................................................82 Discussion...................................................................................................................82 8 CONCLUSIONS AND PERSPECTIVES.................................................................86 Role of Jak2 in Angiotensin II-Dependent ERK2 Signaling......................................86 Role of Jak2 during Oxidative Stress.........................................................................87 Jak2 Structure-Function..............................................................................................88 Identification of a Novel Jak2 Inhibitor......................................................................88 LIST OF REFERENCES...................................................................................................90 BIOGRAPHICAL SKETCH...........................................................................................106 viii

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LIST OF FIGURES Figure page 1-1 Differences in the mechanism of Jak-STAT signaling through cytokine receptors and GPCRs.................................................................................................4 1-2 Summary of major Jak2 domains and amino acids critical to Jak2 tyrosine kinase activity.......................................................................................................17 4-1 ERK2 activity is sustained in 2A/AT 1 cells compared to 2A/AT 1 +Jak2 cells after angiotensin II treatment...................................................................................30 4-2 In vitro kinase assay confirms that ERK2 activity is sustained in 2A/AT 1 cells compared to 2A/AT 1 +Jak2.....................................................................................31 4-3 AG490 suppresses angiotensin II-dependent ERK2 activation independent of Jak2 inhibition......................................................................................................33 4-4 Difference in the angiotensin II-dependent nuclear accumulation of phospho-ERK2 between 2A/AT 1 and 2A/AT 1 +Jak2 cells....................................35 4-5 No difference in angiotensin II-dependent ERK mediated gene transcription between 2A/AT 1 and 2A/AT 1 +Jak2 cells..............................................................36 4-6 Jak2 is essential for angiotensin II-induced MKP-1 expression..............................39 4-7 Angiotensin II-dependent inactivation of ERK2 requires Jak2 and MKP-1............41 4-8 Proposed model of the mechanism by which Jak2 mediates ERK2 inactivation after angiotensin II treatment...................................................................................44 5-1 Hydrogen peroxide-induced Jak2 activity is suppressed in RASM-DN cells.........48 5-2 Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular smooth muscle cells..............................................................................................................49 5-3 Hoechst staining to detect nuclear condensation......................................................51 5-4 Quantification of apoptosis in RASM-Control and RASM-DN cells......................52 5-5 Jak2 mediates hydrogen peroxide-induced up regulation of Bax expression..........54 ix

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5-6 Jak2 is essential for hydrogen peroxide-induced mitochondrial membrane dysfunction...............................................................................................................55 5-7 Pixel intensity of each of 4 photographs from each condition in Fig. 5-6 was determined, and intensity of green and red staining was determined for each........56 5-8 Jak2 is required for oxidative stress-induced caspase-9 cleavage............................57 6-1 Molecular modeling of the Jak2 kinase domain suggested a putative interaction between Glu 1024 and Arg 1113..............................................................................62 6-2 Mutation of Glu 1024 or Arg 1113 abolishes the ability of Jak2 to autophosphorylate....................................................................................................64 6-3 The Jak2-W1020G and E1024R mutations render Jak2 dominant negative...........67 6-4 Jak2-R1113E mutant cannot become tyrosine phosphorylated in response to angiotensin II............................................................................................................68 6-5 Angiotensin II-dependent Jak2/AT 1 receptor co-association does not occur in cells expressing Jak2-R1113E..................................................................................68 6-6 Jak2-R1113E is unable to activate STAT1 in response to angiotensin II................70 6-7 Jak2-R1113E is unable to activate STAT-mediated gene transcription in response to angiotensin II.........................................................................................71 6-8 Arg 1113 is conserved in Jak2 among species and in different Jak family members...............................................................................................................72 7-1 SPHGEN identified 49 exposed pockets on the surface of the Jak2 protein...........77 7-2 Compound 7 inhibits Jak2 autophosphorylation......................................................79 7-3 Maximal Jak2 inhibition requires 16 h of incubation with Compound 7.................80 7-4 Compound 7 inhibits Jak2 in a dose-dependent manner..........................................81 7-5 Compound 7 is not cytotoxic at a dose of 100 M..................................................83 x

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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 TYROSINE KINASE: NEW INSIGHTS REGARDING STRUCTURE, FUNCTION, AND PHARMACOLOGY By Eric M. Sandberg December 2004 Chair: Peter P. Sayeski Major Department: Physiology and Functional Genomics The kinase Jak2 is a member of the Janus family of non-receptor tyrosine kinases. One major impediment to understanding the role that Jak2 plays in physiology and pathophysiology is the lack of a specific Jak2 inhibitor. We used several strategies to circumvent this problem. First, using Jak2 -/cells, we examined the role of Jak2 in regulating angiotensin II-dependent ERK2 activity. We found that, contrary to previously published work, Jak2 is required for inactivation of ERK2 after angiotensin II treatment. In response to angiotensin II, Jak2 induces expression of MAP kinase phosphatase-1 (MKP-1), a protein that dephosphorylates and inactivates ERK2. Second, using stable expression of a Jak2 dominant negative mutant that specifically suppresses endogenous Jak2 kinase activity, we found that Jak2 mediates oxidative stress-induced apoptosis of vascular smooth muscle cells. In response to hydrogen peroxide treatment, Jak2 induces expression of the pro-apoptotic Bax protein. xi

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This causes a loss of mitochondrial transmembrane potential, cleavage of Caspase-9, and subsequent apoptosis. Third, we attempted to identify novel Jak2 inhibitors. For this, we used homology modeling to analyze the structure of the Jak2 kinase domain, and we identified a previously unknown amino acid interaction that is required for activation of Jak2. This interaction, consisting of two distinct hydrogen bonds between Jak2 residues Glu 1024 and Arg 1113, may be a suitable target for drug design aimed at disabling Jak2 function. Additionally, we used high-throughput compound docking in silico to identify a novel Jak2 inhibitor. This compound, cyclohexane-1,2,3,4,5,6-hexabromo(designated Compound 7) potently inhibits Jak2 autophosphorylation in a timeand dose-dependent manner. In conclusion, using Jak2 -/cells, stable expression of a Jak2 dominant negative mutant, and structure-function studies, we successfully circumvented the problems that lack of a Jak2-specific inhibitor pose. In doing so, we identified novel roles for Jak2 kinase function in regulation of angiotensin II-dependent ERK2 signaling and in oxidative stress-induced apoptosis of vascular smooth muscle cells. Additionally, we improved our understanding of Jak2 structure by identifying a previously unknown amino acid interaction within the Jak2 kinase domain that is required for Jak2 kinase function, and identified a novel Jak2 inhibitor. xii

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CHAPTER 1 INTRODUCTION TO THE Jak-STAT PATHWAY History of the Jak-STAT Pathway In 1990, the first member of the Jak family of cytoplasmic tyrosine kinases was cloned and sequenced (1). The gene, termed Tyk2, was unique compared to previously identified tyrosine kinases in that it had a kinase-like, or pseudokinase domain, immediately N-terminal to a highly conserved protein tyrosine kinase domain. The tandem manner by which these two domains adjoined one another was reminiscent of Janus, Roman God of two opposing faces. As such, Tyk2 was classified as the first member belonging to the Janus associated kinase family of protein tyrosine kinases (or more simply) the Jaks. Other groups independently cloned the cDNAs encoding Jak1, Jak2, and Jak3 (2-6). Because some of the genes were cloned from hematopoietic tissues, it was hypothesized that the Jak kinases played a critical role in cytokine-mediated signal transduction. This hypothesis was largely correct. In 1992, Wilks and colleagues (3) were the first to clone and publish the Jak2 cDNA sequence. The gene encoded a protein of about 130 kDa in mass. Like the two previously cloned Jaks (Tyk2 and Jak1) the predicted amino acid sequence of Jak2 contained the kinase and pseudokinase domains adjoining one another on the carboxyl half of the protein. These regions are termed the Jak homology 1 (JH1) and Jak homology 2 (JH2) domains, respectively. Wilks and colleagues also identified five other domains that encompassed the amino half of the molecule. These were designated as the JH3, JH4, JH5, JH6, and JH7 domains. The C-terminal half of the JH4 domain and the 1

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2 entire JH3 domain were thought to encode a primitive SH2 domain spanning amino acids 412-480. This was significant because Jak family members lack any canonical SH2 or SH3 domains. Aside from this putative SH2 domain, the remaining domains did not possess the characteristics of any other known proteins. Wilks and colleagues found that, like Tyk2 and Jak1, Jak2 was expressed in almost every tissue examined. In contrast, Jak3 is expressed predominantly in hematopoietic cells (5, 7). Interestingly, subsequent work showed that Jak2 homologs exist in animals as diverse as zebrafish (Danio rerio) and fruitflies (Drosophila melanogaster) (8, 9). Collectively, these studies showed that a new family of cytoplasmic protein tyrosine kinases existed in animals. These family members shared properties that were unique only unto them. Of these genes, Jak2 is expressed in numerous tissues and in evolutionarily diverse species. The importance of Jak2 in cellular signaling was realized when it was discovered that Jak2 appeared to be a critical mediator of cytokine-dependent signal transduction (10-15). Subsequent work quickly identified a correlation between activation of Jak2 in the cytoplasm and increased gene transcription in the nucleus. This observation suggested that a specific class of cytokine-responsive transcription factors was mediating this transcriptional effect. This hypothesis was proven correct when concurrent studies identified a new class of cytokine-responsive transcription factors, termed the Signal Transducers and Activators of Transcription (STAT) proteins (16, 17). These proteins (when tyrosine phosphorylated by Jak2 in the cytoplasm) translocate to the nucleus and mediate gene transcription. Thus, within 2 years, the broad framework of the Jak-STAT signaling paradigm was elucidated.

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3 More recently, it was discovered that in addition to mediating cytokine signal transduction, Jak2 also mediates signaling through G protein coupled receptors (GPCRs) (18). In fact, the number of cytokines and GPCR agonists that activate Jak-STAT signaling has grown steadily since the discovery of the pathway. The cytokines currently known to activate Jak2 include IL-2, IL-3, IL-5, IL-6, IL-11, IL-12, granulocyte macrophage colony-stimulating factor, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, granulocyte colony-stimulating factor, interferon-, growth hormone, prolactin, erythropoietin, thrombopoietin, and leptin (10-15, 19-27). The GPCR agonists that activate Jak2 include angiotensin II, bradykinin, endothelin, platelet activating factor, -melanocyte stimulating hormone, isoproterenol, and phenylephrine (28-32). The mechanism of Jak2 activation by these two receptor subtypes differs, but the downstream effects of Jak2 activation are similar. In both cases, Jak2 acts a critical link in coupling ligand binding at the cell surface with gene transcription in the nucleus. Key differences in Jak-STAT signaling through cytokine receptors and GPCRs are shown in Fig. 1-1 and discussed in the next section. In addition to cytokines and GPCR agonists, Jak2 can also be activated in response to a number of cellular stressors. These include mechanical cell stretch, ischemia-reperfusion, and hydrogen peroxide (33-36). The upstream activators of Jak2 and the downstream effects of Jak2 activation by these stimuli are not well characterized. In Chapter 5, we elucidate the role that Jak2 plays in hydrogen peroxide-induced signaling in vascular smooth muscle cells.

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4 Jak2 -Y--Y Jak2 Ligand P P P STAT PSTAT STAT Ligand Ligand Jak2 Jak2 Jak2 Jak2 STAT STAT STAT -YP P STAT P P STAT P Y P Jak2 Jak2 -Y--Y Jak2 Jak2 LigandLigand P P P P P P STAT P PSTAT STAT STAT Ligand Ligand Jak2 Jak2 Jak2 Jak2 Jak2 Jak2 Jak2 Jak2 STAT STAT STAT STAT STAT STAT -YP P P P STAT STAT P P P P STAT STAT P P Y PY P P Ligand Figure 1-1. Differences in the mechanism of Jak-STAT signaling through cytokine receptors (Top) and GPCRs (Bottom) Initiation of Jak-STAT Signaling Jak2 signaling through cytokine receptors is initiated by the binding of an extracellular cytokine to its cognate monomeric receptor on the cell surface, resulting in receptor dimerization. This oligomerization of two distinct receptors results in the

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5 activation of Jak2 molecules bound non-covalently to the receptors before ligand binding. An activated Jak2 then phosphorylates specific tyrosine residues on the cytoplasmic tails of the receptors creating docking sites for SH2 domain-containing proteins, such as the STATs. Once bound to the receptors, STATs are themselves phosphorylated by Jak2 on tyrosine residues. The tyrosine phosphorylated STATs then dissociate from the receptor to form active homoand hetero-dimer protein complexes. The STAT complexes then translocate into the nucleus where they bind specific DNA sequences in gene promoter elements and modulate gene transcription. Interestingly, the increased tyrosine phosphorylation, nuclear translocation, and DNA binding activity of the STATs occurs in the presence of cycloheximide, suggesting that this signaling pathway uses a post-translational modification of existing proteins and does not require de-novo protein synthesis (37). Thus, Jak2 is capable of transducing a signal from the cell surface to the nucleus through a tyrosine phosphorylation signal transduction cascade. While the downstream consequences of Jak2 signaling through GPCRs are similar to Jak2 signaling through cytokine receptors, there are key differences in how Jak2 signaling is initiated in these two pathways. Similar to Jak2 signaling through cytokine receptors, Jak2 signaling through GPCRs is initiated by binding of an extracellular ligand to its cognate receptor on the cell surface. However, Jak2 is not ubiquitously bound to GPCRs. Instead, it becomes activated in the cytoplasm after ligand binding (18). The exact mechanism by which this occurs has not been elucidated. In the case of angiotensin II (a Jak2-activating GPCR ligand), Jak2 is recruited to the angiotensin II type I receptor (AT 1 R) after its activation. Then Jak2 acts as a molecular bridge, linking STAT proteins

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6 to the AT 1 R. Jak2 phosphorylates the STAT proteins, and the signal cascade proceeds similar to cytokine-mediated Jak2 signaling (38). Physiology and Pharmacology of Jak2 Jak2 signaling plays a critical role in normal physiological processes, including development, regulation of other signaling pathways, and cellular stress responses. In addition, Jak2 signaling has been implicated in the pathophysiology of cancer and heart disease. The role that Jak2 plays in physiology and pathophysiology will be discussed in this section. Target Genes and Regulation of Signal Transduction Pathways Since its discovery, Jak2 activation has been linked to mediation of gene expression. It accomplishes this through activation of the STAT family of transcription factors. Despite this, the identities of downstream target genes of the Jak-STAT pathway are largely unknown. Additionally, Jak2 has been shown to directly regulate other signal transduction pathways. In fact, data in the results section show that Jak2 can indirectly regulate angiotensin II-induced extracellular signal regulated kinase 2 (ERK2) signaling by mediating expression of an ERK2 regulatory protein. Jak2 can influence other angiotensin-dependent signaling events as well. For example, when Jak2 is activated by angiotensin II, Jak2 recruits the Src family tyrosine kinase Fyn to the Jak2-based signaling complex (39). Jak2 then activates Fyn, by binding the SH2 domain of Fyn with very high affinity (K d = 2.36 nM). This strong interaction results in a conformational change within Fyn that allows the Fyn kinase domain to become accessible to substrate. Thus, Jak2 serves as a potent activator of Fyn kinase. Additionally, Jak2 has been shown to regulate ERK2 activity in response to angiotensin II (40). Jak2 was found to be essential for activation of ERK2. Despite this,

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7 our study (Chapter 4) suggests that these findings may be an artifact of using a nonspecific inhibitor of Jak2 kinase function in the studies. Other molecules that are recruited into Jak2-based signaling complexes include c-Src, Grb2, PI3 kinase, PP2A, Yes, Raf-1, Shc, Syp, and FAK (41-47). Furthermore, a review of the literature found that more than 50 different cellular proteins associate with Jak2 in some manner. While the precise relationship of each of these proteins to Jak2 is not known, it would seem that each interaction is occurring for a specific cellular and biochemical reason. As such, these studies demonstrate a role for Jak2 as a cellular headquarters for the recruitment, modification, and modulation of numerous signaling pathways. While Jak2 clearly can regulate other intracellular signal transduction pathways, the Jak2 pathway itself is tightly regulated within the cell. Regulation of Jak2 Signaling For Jak2 to initially become activated, a single tyrosine within the Jak2 activation loop must be phosphorylated. Site-directed mutagenesis studies showed that phosphorylation of Tyr 1007 is required for ligand-induced Jak2 activation and subsequent phosphorylation of several STATs (48). Activation of Jak2 may depend on its interaction with ancillary molecules such as SH2Band SHP-2. The mere expression of SH2Bin the same cell as Jak2 increases Jak2 tyrosine phosphorylation levels and its catalytic activity (49). It is known that SH2Bdirectly binds Jak2, however exactly how SH2Bbiochemically modifies Jak2 to promote its activation has not been determined. The tyrosine phosphatase SHP-2 is a more controversial Jak2-regulatory protein; there are conflicting data regarding whether this protein acts as an activator or an inhibitor of Jak2 function. The discrepancies are due to several factors, including the specific ligand

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8 and cell types used in each experiment. For example, when growth hormone binds to its receptor on transfected COS-7 cells, SHP-2 augments the level by which Jak2 increases c-fos expression (50). Jak2 shows higher tyrosine phosphorylation levels in these cells and it is thus believed that SHP-2 augments Jak2 function by elevating Jak2 tyrosine kinase activity. In contrast, when fibroblasts are treated with interferons, SHP-2 inhibits Jak2 function (51). Equally important to the activators of Jak2 are the inhibitors, that serve to terminate Jak2-dependent signaling. These inhibitors work at different levels of the signal transduction cascade to attenuate Jak2 signaling. In addition to providing a certain level of redundancy in this inhibitory process, the inhibitors also appear to act in a temporal or sequential manner. Since the Jak2 activation state is dependent on its tyrosine phosphorylation levels, one obvious mechanism of inactivation is tyrosine dephosphorylation of Jak2. This is accomplished by protein tyrosine phosphatases, including SHP-1. The binding of ligands such as growth hormone, erythropoietin, IL-2, and IL-4 to their cognate receptors promotes the binding of SHP-1 to the Jak2-based receptor-signaling complex. The SH2 domain of SHP-1 binds a phosphotyrosine residue on Jak2, thus inhibiting Jak2 activity (52). Not surprisingly, loss of SHP-1 expression in cells lead to a variety of transformed phenotypes, owing to the growth-promoting actions of Jak2 (53, 54). Suppressors of Cytokine Signaling (SOCS) also act as potent inhibitors of Jak kinase function. They act in a classical negative feedback mechanism. Cytokine inducible SH2 domain-containing protein (CIS) was the first SOCS family member to be cloned. It was initially identified as a gene that was rapidly induced by IL-3 (55). Based

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9 on sequence homology, subsequent groups cloned and characterized seven additional SOCS family members, termed SOCS1-SOCS7. Accumulation of SOCS mRNA is rapidly induced by a variety of cytokines and growth factors including IL-2, IL-4, IL-6, leukemia inhibitory factor, granulocyte colony stimulating factor, interferon-, growth hormone, prolactin, erythropoietin, and leptin. There are multiple mechanisms by which SOCS proteins suppress Jak2 function. For instance, SOCS1 binds Jak2 via a direct interaction between the SH2 domain of SOCS1 and phosphotyrosine residue 1007 on Jak2. Structure-function studies have shown that this interaction is required for suppression of Jak2 kinase activity, as deletion of the SOCS1 SH2 domain results in the inability of SOCS1 to reduce Jak2 kinase function (56). However, the exact biochemical modification(s) that occur on Jak2 after co-association with SOCS1 is not known. A final group of Jak-STAT inhibitory proteins are termed the Protein Inhibitors of Activated STATs (PIAS). The four members identified so far are PIAS-1, PIAS-3, PIAS-X, and PIAS-Y (57, 58). They differ from protein tyrosine phosphatases and SOCS in that they bind STATs and not Jaks. While they share homology among themselves, they have no previously characterized protein domains. These proteins are constitutively expressed in numerous tissues, and do not appear to be highly specific for which STATs they bind. Unfortunately, the biochemical and cellular mechanisms by which PIAS proteins suppress STAT function are not well understood. Specific activators and inhibitors of Jak2 signaling have been identified. While the activators allow maximal Jak2 activation and kinase function, the inhibitors work in concert to suppress Jak2 signaling at different levels of the Jak-STAT pathway. These regulatory proteins control the magnitude and duration of Jak-STAT signaling. Although

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10 Jak2 activity is tightly controlled within cells, aberrant regulation of this pathway contributes to the pathological progression of certain diseases. Jak2 in Cancer In 1995, studies of the Drosophila Jak2 homolog, hopscotch, were the first to implicate Jak2 in tumorigenesis (59). Specifically, a Glu 695 to Lys mutation in the Hop T42 protein increased the intrinsic tyrosine kinase activity of Jak2, and led to malignant neoplasia of Drosophila blood cells. When the same mutation was introduced into the JH2 domain of mammalian Jak2, the mutant protein similarly had significantly increased kinase activity, and hyperactivated the mammalian Jak-STAT signaling pathway (60). Concurrent work by Meydan and colleagues (61) was the first to implicate Jak2 in human cancer, as they reported an inhibition of acute lymphoblastic leukemia via treatment with the Jak2 pharmacological inhibitor AG490. Collectively, these works suggested a direct link between activated Jak2 and neoplastic cell growth. Tel-Jak2 fusion proteins result from a translocation event between the kinase domain of Jak2 and the HLH domain of Tel. The first reports describing Tel-Jak2 fusion proteins came from a child with early B-precursor acute lymphoid leukemia, and an adult with atypical chronic myeloid leukemia (62, 63). The tumors of these 2 patients are differ because of distinct translocations within the Jak2 and Tel genes, which then give rise to distinct chimeras. Nonetheless, it appears that all Tel-Jak2 fusions confer constitutive Jak-STAT activity. They have been shown to increase NF-B signaling and induce growth factor-independent proliferation in Ba/F3 hematopoietic cells (64, 65). More importantly, the creation of Tel-Jak2 transgenic mice revealed a causal relationship

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11 between the Tel-Jak2 gene product and leukemogenesis, as over expression of this fusion protein stimulated development of T-cell leukemia in these animals (66). While many of the mechanisms involving Jak2 activation during cancer are incompletely understood, striking data have surfaced implicating Jak2 activation in human hepatocellular carcinoma (HCC) cells. Methylation of CpG islands within the SOCS-1 gene, which results in reduced SOCS-1 expression, was directly responsible for constitutive Jak2 activity. Furthermore, restoration of SOCS-1 expression and/or treatment with AG490 reduced Jak2 tyrosine kinase activity and the growth rate of these cells (67). Therefore, increased activation of Jak2 was triggered by inactivation of its inhibitor, and this finding identified a new area of therapeutic research. Increased Jak2 tyrosine kinase activity is associated with many other types of cancers as well. Specifically, Jak2 has been shown to activate ErbB-2, whose oncogenic activity is associated with various human breast cancers (68-70). Additionally, BRCA1 over expression enhanced the ability of Jak2 to activate STAT3 in human prostate cancer cells (71). Jak2 in Cardiovascular Disease Jak2 plays a pivotal role in various cardiovascular signaling systems. Therefore, it is not surprising that activation of this protein has been implicated in the molecular events of certain cardiovascular disease states including cardiac hypertrophy, ischemia-reperfusion injury, and heart failure. A review of the literature presents strong evidence that Jak2 plays a role in the cellular signaling processes associated with these and other pathological disease states. Cardiac hypertrophy (or the increase of cardiac muscle mass) is a natural defense for coping with cardiovascular diseases, and is a major cause of mortality in the United

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12 States (72-74). Cardiac hypertrophy occurs in response to an increased workload on the heart and/or the secretion of certain humoral factors (75-85). Interestingly, cardiac hypertrophy-inducing stimuli also activate Jak2. For example, acute pressure overload in rats increases Jak2 tyrosine phosphorylation levels by causing autocrine/paracrine secretion of angiotensin II (86, 87). Jak2 is also activated by cardiotrophin-1, another potent activator of cardiomyocyte hypertrophy (88, 89). Cardiotrophin-1, a member of the interleukin-6 related cytokine family, induces cardiomyocyte hypertrophy by increasing expression of angiotensinogen mRNA through the activation of STAT3. Activated STAT3 forms dimer complexes that translocate into the nucleus and bind ST-domains within the angiotensinogen gene promoter (90). Jak2 was implicated in this pathological process, as AG490 treatment suppressed the cardiotrophin-induced STAT3 binding to the angiotensinogen promoter and subsequent gene activation (90). Thus, Jak2 appears to have a key role in the signaling system leading to cardiotrophin-1 induced cardiac hypertrophy via increased expression of the angiotensinogen gene. Heart failure is another disease that has been linked to Jak2, but in a different fashion. In short, heart failure is defined as inadequate cardiac output. The progression of heart failure is dependent on a balance between cardiomyocyte hypertrophy and apoptosis (91). Podewski et al. (92) examined signaling events associated with one disease that leads to heart failure, dilated cardiomyopathy (DCM). They showed that Jak2 tyrosine phosphorylation levels were decreased in patients with DCM, while levels of the Jak2 inhibitor, SOCS1, were increased. Based on this and subsequent work, they proposed a model in which decreased Jak2 tyrosine phosphorylation levels (attributable to an increase in SOCS1 activity) result in decreased phosphorylated STAT3. The result

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13 of this is that STAT3 fails to increase expression of cardioprotective genes that would save the heart from failure. What makes this intriguing is that during DCM, reduced Jak2 activity is pathological; while during cardiac hypertrophy, increased Jak2 activity is pathological. Clearly, future work will need to better define the signaling pathways leading to cardiac hypertrophy and heart failure. Presently, these and other results show that either too much, or too little Jak2 activity, can have negative consequences. Jak2 activation is also associated with cardiac injury during ischemia-reperfusion (34). Ischemia-reperfusion is a pathological condition characterized by impeded blood flow to an area of tissue followed by the reestablishment of circulation to that same area. It has been shown that treatment with AG490 leads to a reduction in cardiac infarct size and a reduction in apoptotic cell death of cardiomyocytes after ischemia-reperfusion in an isolated perfused rat heart (34). Furthermore, ischemia-reperfusion leads to STAT5a and STAT6 binding to the angiotensinogen gene promoter. Treatment with either AG490 or the AT 1 receptor antagonist losartan resulted in the loss of STAT/St-domain complex formation, and a subsequent reduction in angiotensinogen mRNA levels. Thus, a positive feedback model in which Jak2 activates Stat5a and Stat6 which bind to the angiotensinogen gene promoter, resulting in an increase in angiotensinogen mRNA, and subsequent angiotensin II production and activation of the AT 1 receptor. Recent evidence has linked Jak2 to vascular injury. Jak2 and STAT3 protein expression levels are increased after balloon injury of rat carotid arteries, and increased activity of the Jak-STAT pathway is involved in the ensuing vascular smooth muscle cell proliferation and neointima formation seen in this model of vascular injury (93). In addition, oxidative stress in the form of hydrogen peroxide potently activates Jak2 in

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14 vascular smooth muscle cells, suggesting a possible role for Jak2 during oxidative stress (35). Evidence supporting a pro-apoptotic role for Jak2 during oxidative stress in vascular smooth muscle cells is given in Chapter 5. In conclusion, reports have shown a clear relationship between Jak2 and neoplastic transformation, and between Jak2 and cardiovascular disease. Researchers are now trying to determine the cellular and biochemical mechanisms by which aberrant Jak2 activity leads to cancerous cell growth; and to determine the precise role that Jak2 plays in cardiovascular disease progression. These results show that in the near future, Jak2 may be an attractive target for pharmacological inhibition during cancer and heart disease. Jak2 Pharmacology Based on the role that Jak2 plays in cancer and cardiovascular disease, pharmacological inhibition of Jak2 may soon hold therapeutic promise. This section reviews research that highlights Jak2 as a therapeutic target. Many studies have used the commercially available Jak2 inhibitor, tyrphostin AG490, to demonstrate the benefits of inhibiting this signaling pathway during certain disease states. For instance, AG490 suppressed growth of human hepatocellular carcinoma cells (67). Jak2 is constitutively activated in these cells because of methylation and transcriptional silencing of the SOCS-1 gene, a negative regulator of Jak2 signaling. AG490 prevented constitutive Jak2 activation, and induced apoptosis in these cells (67). Similarly, AG490 sensitized metastatic breast cancer cells to chemotherapy-induced apoptosis (68), induced apoptosis in myeloblastic cells (94), and blocked growth of acute lymphoblastic leukemia cells in vitro and in vivo by inducing apoptosis (61). AG490 also prevented Jak2-mediated constitutive tyrosine

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15 phosphorylation of ErbB-2 and DNA synthesis in breast cancer cells (68), and abrogated growth of human B-precursor leukemic cells (95). In addition to the potentially therapeutic effects of inhibiting Jak2 in various types of cancer, Jak2 inhibition via AG490 was shown to be therapeutic in several cardiovascular disease models. For instance, AG490 reduced neointima formation in the carotid artery of rats after balloon injury (92). In cultured cardiomyocytes, AG490 attenuated leukemia inhibitory factor-induced hypertrophy and myofilament reorganization (96). Additionally, AG490 inhibited several signaling pathways rapidly induced after myocardial infarction that are thought to contribute to diastolic dysfunction and arrythmogenicity in the post-myocardial infarcted heart (97). Finally, AG490-treated hearts showed a reduction in myocardial infarct size and in the number of cardiomyocytes undergoing apoptosis after ischemia-reperfusion (34). It is apparent from these studies that inhibition of Jak2 in various types of cancer, heart, and vascular disease holds therapeutic promise. While AG490 has been used extensively to study the Jak-STAT pathway in health and disease, and has been instrumental in identifying Jak2 as a therapeutic target, it suffers from a lack of specificity. For instance, AG490 inhibits activation of cyclin dependent kinases and causes growth arrest of cells in G1 phase (98). It inhibits calf serum inducible cell growth and DNA synthesis (98), and is a partial blocker of c-Src activity (99). Most critically, AG490 inhibits epidermal growth factor receptor autophosphorylation more potently than it inhibits Jak2 activity (100, 101). Moreover, no assay is available for quantifying tissue AG490 concentrations, and no data exist

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16 describing the in vivo degradation rate of AG490 (93). Thus, the issue of specificity of AG490 for Jak2 is a major concern. Lack of a specific Jak2 inhibitor has made study of the Jak-STAT pathway difficult. Additionally, Jak2 knockout mice die embryonically, further complicating research efforts (102, 103). Therefore, the identification or development of new, Jak2-specific pharmacological inhibitors would provide useful research tools and potentially therapeutic drugs. As such, investigation into the structural characteristics of the Jak2 protein as they relate to kinase function may be an important step towards achieving this goal. Structure-Function Relationship of Jak2 When the Jak family of protein kinases was first discovered, their unique structural characteristics were quickly noted. The proteins consist of seven novel protein domains now termed Jak homology (JH) domains. Most of these JH domains have a distinct role in controlling Jak2 function. Figure 1-2 is cartoon summarizing the major domains of Jak2. This section reviews the structural characteristics of the Jak2 protein, and recent research that advanced our understanding of the structure-function relationship of Jak2. The carboxyl terminus of Jak2 contains the JH1 and JH2 domains. The JH1 domain is the highly conserved kinase domain, which contains the ATP-binding region and the activation loop. Most of the structure-function data available for Jak2 concerns this domain. Studies have shown the requirement for the JH1 domain for Jak2 tyrosine kinase function (104). In addition, a number of individual amino acids within the JH1 domain have bee identified that are critical to this function. Two adjacent tyrosines, Tyr 1007 and Tyr 1008, were shown to be phosphorylation sites within the

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17 1129Unique N Terminus Conserved C Terminus JH7 JH6 JH5 JH4 JH3 JH2 JH1 Kinase Pseudokinase FERM SH2 1 130 283 307 450543827 Amino Acid Function 2 240 Critical for Jak2/GHR co association 231 235 Critical for Jak2/AT 1 R co association Lys 882 Critical for Jak2 autophosphorylation 994 1024 Try 1007 Tyr 1008 Trp 1020 Glu 1024 Glu 1046 Activation loop Phosphorylated upon Jak2 activation Phosphorylated upon Jak2 activation Critical for Jak2 autophosphorylation Critical for Jak2 autophosphorylation bond with Trp Stabilizes activation loop via H 1020 Figure 1-2. Summary of major Jak2 domains and amino acids critical to Jak2 tyrosine kinase activity Jak2 activation loop. The tyrosine at position 1007 is required for Jak2 tyrosine kinase activity, while the tyrosine at position 1008 seems to be dispensable for the tyrosine kinase function of Jak2 (48). Additionally, Tyr 1007 interacts with a Jak binding protein (JAB), also known as suppressor of cytokine signaling 1 (SOCS1), a protein that negatively regulates the kinase activity of Jak2 (56). Also, mutation of the invariant Lys 882 in the JH1 domain rendered Jak2 catalytically inactive (104, 105). In 1994, a Jak2 containing mutations at both Trp 1020 and Glu 1024 (W1020G/E1024A) was shown to not only be catalytically inactive, but also dominant negative (106). We recently showed that mutation of either of these amino acids

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18 individually rendered Jak2 catalytically inactive (107). Moreover, we elucidated the requirement for Trp 1020 for Jak2 kinase activity, by demonstrating that Trp 1020 forms a hydrogen bond with Glu 1046, an amino acid that previously had not been shown to be required for Jak2 function. The importance of this interaction is that it appears to stabilize the three dimensional structure of the Jak2 activation loop. More recently, we elucidated the requirement for Glu 1024. These data are presented in Chapter 7. The JH2 domain of Jak2 is the pseudokinase domain. While it shares conserved motifs with other protein kinases, this region is catalytically inactive. In fact, the active site and activation loop are modified compared to traditional protein tyrosine kinase activation loops (108). These modifications are, however, conserved amongst Jak family members, suggesting a regulatory role for this pseudokinase domain (109). Other lines of evidence point to a critical role for the JH2 domain. In Drosophila, a mutation in the JH2 domain of the Jak homolog Hop, rendered the protein hyperactive and caused hematopoietic hyperplasia (59). This was the first evidence of a negative regulatory role for the JH2 domain. Subsequent work by Saharinen et al. showed that the JH2 domain of mammalian Jak2 interacts with the JH1 kinase domain and suppresses the activity of Jak2 at the basal level (109). Following this work, another group constructed a homology model of the JH1 and JH2 domains. Using this model, they predicted two major interactions between the JH1 and JH2 domains of Jak2. The first interaction is between the two helices of the two domains. The second interaction occurs between a loop that lies between two stands of the JH2 domain and the activation loop of the JH1 domain (110). Incidentally, another group, Chen et al., showed a similar role for the JH2 domain in Jak3, suggesting functional conservation amongst Jak family members (111).

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19 In addition, Saharinen et al. showed that the JH2 domain is also required for the ability of Jak2 to become maximally activated in response to cytokine stimulation (112). This same group further clarified the regulatory role of the JH2 domain by identifying three specific regions within this domain that are involved in the auto-inhibition of Jak2, which are termed IR1, IR2, and IR3 (113). IR3, which spans amino acid residues 758 to 807, directly inhibited the JH1 kinase domain, while IR1 spanning residues 619 to 670 and IR2 spanning residues 725 to 757, enhanced IR3-mediated inhibition of the JH1 domain. In this work, the authors present an explanatory model for the function of JH2 in regulating JH1. In it, they suggest that under basal conditions, JH2 is bound to JH1. Upon cytokine binding and receptor aggregation, the inhibitory JH1-JH2 interaction is displaced, possibly through homotypic interaction of the JH1 domains of the two Jak2 molecules. Why the JH2 domain is required for maximal cytokine-induced Jak2 activation still remains to be determined. The amino terminal region of Jak2 comprises the JH3 through JH7 domains. This region is largely responsible for receptor interactions and may be responsible for interactions between Jak2 and regulatory molecules. Interestingly, although the Jak family members have traditionally been considered absent of a canonical SH2 domain, it was noted that in Tyk2, the second half of the JH4 domain plus the whole of the JH3 domain weakly resembles an SH2 domain (114). Upon cloning of murine Jak2, it was similarly noted that the sequence GLYVLRWS bore weak homology to the core sequence element of SH2 domains (FLVRES) (3). Using multiple sequence alignments and secondary and tertiary structure predictions, Kampa et al. presented for the first time a three dimensional view of a putative Jak family SH2 domain spanning amino acids 412

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20 480 of Jak3. Furthermore, this group demonstrated binding of phosphorylated proteins to the putative SH2 domain of Jak3, thus proving an SH2-like function for this domain (115, 116). Despite these results, Giordanetto and Kroemer recently published a predicted Jak2 structure comprising JH domains 1 through 7, whereby they contended that the putative SH2 domain of Jak2 might not be fully functional (117). Comparing the SH2 domain of p56 lck with the putative Jak2 SH2 domain, they demonstrated several key differences at amino acids involved in phosphotyrosine binding between the two that may preclude the Jak2 SH2 domain from being fully functional. They stated that despite conservation between the two SH2 domains of one arginine known to interact with phosphate groups (Arg 426 of Jak2, Arg 134 of p56 lck ), another highly conserved arginine known to interact with phosphate groups is replaced by a methionine at the corresponding position in Jak2 (Met 406 of Jak2, Arg 154 of p56 lck ). This, they point out, would abolish interaction with a phosphate group. Furthermore, a lysine within the putative Jak2 SH2 domain corresponds to a highly conserved serine residue in other SH2 domains (Lys 430 of Jak2, Ser 158 of p56 lck ). The authors contend that the longer side chain of lysine would present difficulties in allowing it to interact with a phosphotyrosine residue. Finally, they show that there are key differences in the phosphotyrosine binding pockets of the two SH2 domains. Specifically, Glu 157 of p56 lck is replaced by Pro 429 in Jak2. In the predicted model, this proline appears to restrict access to the phosphotyrosine binding pocket, because of the rigid side chain of proline. Whether an SH2 domain exists within Jak2 and whether it is fully functional may be clarified in the future by determining the crystal structure of Jak2.

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21 Adjacent to the putative SH2 domain of Jak2 lays the FERM domain, which spans from the middle of the JH4 domain through the JH7 domain (118). This domain is present in a number of other proteins including the band 4.1 protein from erythrocytes, some protein tyrosine phosphatases, and the focal adhesion kinases (119-121). Often, this domain is involved in stable association with membrane bound proteins (122). In fact, the amino terminal region of Jak2, especially the JH6 and JH7 domains (comprising part of the FERM domain), has been shown to be crucial for Jak2/receptor interactions. For example, deletion of amino acids 2-239, which deletes the JH7 domain and part of the JH6 domain, abrogated Jak2 association with the growth hormone receptor (123). It was then shown, though, that neither the JH6 and JH7 domains alone, nor the entire Jak2 FERM domain (comprising the JH6 and JH7 domains with the JH5 domain and half of the JH4 domain) were sufficient for Jak2 association with the growth hormone receptor. This suggested that multiple JH domains must interact to allow Jak2 association with cytokine receptors (124). Additionally, amino acids 1-294 were found to be essential for Jak2 binding to the granulocyte-macrophage colony stimulating factor (GM-CSF)c receptor (125). More recently, our lab found that the YRFRR motif within the JH6 domain of Jak2, spanning amino acids 231-235, was essential for both Jak2 association with the angiotensin II type 1 receptor (AT 1 receptor) and STAT1 translocation to the nucleus in response to angiotensin II treatment (126). Similar requirements for the amino terminal region for receptor association have been shown for the other Jak family members (127, 128). The Jak2 FERM domain interacts with cytokine receptors via a proline rich box1 motif on the cytoplasmic chain of the cytokine receptor (129, 124). A recent paper describing a homology model of Jak2 comprising the

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22 JH1-7 domains highlighted hydrophobic amino acids within the FERM domain predicted to be essential for Jak2 association with the box1 motif of cytokine receptors (117). The analysis predicted that Met 181, Phe 236, Phe 240, and Iso 223 within the Jak2 FERM domain would mediate interaction with receptors. These results elucidated the requirement of the FERM domain for receptor interaction, but mutational studies on the predicted amino acids are required for confirmation of these predictions. Collectively, structure-function studies involving the Jak family of non-receptor tyrosine kinases identified seven conserved Jak homology domains in each protein. Studies have described essential functions for each of these domains, including kinase activation for the JH1 domain, autoregulation for the JH2 domain, and interactions with substrates, regulatory proteins, and membrane-bound receptors for the JH3-7 domains. Future structure-function studies will be critical in advancing our understanding of Jak2, and may lead to new ways to control its function, including Jak2-specific pharmacological inhibition.

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CHAPTER 2 EXPERIMENTAL METHODS Cell Culture All cells were cultured at 37 o C in a 5% CO 2 humidified atmosphere and were maintained in DMEM containing 10% fetal bovine serum. Ample stocks of these cells are stored in liquid nitrogen. Vaccinia Virus Transfection/Infection BSC-40 cells were transfected in serum free media with Jak2 cDNA cloned into the pRC-CMV plasmid. After 4 h of transfection, cells were infected with 1.0 MOI of vaccinia virus vTF7-3. After 1 h of incubation with the virus, complete media was put on the cells to stop transfection, and the cells were allowed to become infected with virus for 16 h. The virus expresses a T7 RNA polymerase, which drives Jak2 expression from a T7 promoter upstream of the Jak2 cDNA in the pRC-CMV plasmid, allowing overexpression of the Jak2 protein. Immunoprecipitation/Western Blotting To prepare lysates, cells were washed with two volumes of ice-cold PBS containing 1 mM Na 3 VO 4 and lysed in 1.0 mL of ice-cold RIPA buffer containing protease inhibitors. The samples were sonicated and incubated on ice for 30 min. Samples were spun at 16,000 x g for 5 min at 4C, and supernatants were normalized for protein content using the Bio-Rad D c assay. Normalized lysates (approx. 400 g/mL) were immunoprecipitated for 2-4 h at 4C with 2 g of antibody and 20 l of Protein A/G Plus agarose beads (Santa Cruz Biotechnology). After centrifugation, protein complexes were 23

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24 washed 3 times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100) and resuspended in SDS sample buffer. Bound proteins were boiled, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking membrane for 1 h in 5% dry milk/TBST (100 mM Tris, pH 7.5, 0.9% NaCl and 0.05% Tween 20) at room temperature, nitrocellulose membranes were probed with primary antibody for 1-2 h at room temperature in 5% milk/TBST. Blots were washed with TBST and proteins were visualized using enhanced chemiluminescence (ECL) following the manufacturers instructions (Amersham). Immunocytochemistry Cells were grown on microscope slides. After treatment, cells were washed twice with K + free PBS and fixed for 60 min at room temperature with 4% paraformaldehyde. After fixation, cells were washed 4 times with K + free PBS, permeabilized for 10 min at room temperature with 0.2% Triton X-100 in K + free PBS (vol/vol), washed an additional 4 times, and then blocked with 5 mg/mL BSA in K + free PBS for 4 h at room temperature. After blocking, cells were incubated with primary antibody overnight at 4C in K + free PBS containing 5 mg/mL BSA. The next day, cells were washed 5 times and incubated with secondary antibody conjugated to Texas Red for 4 h at room temperature. The cells were then dehydrated through increasing concentrations of ethanol, dipped into xylene, and mounted. The next day, the cells were visualized with a fluorescent microscope. In Vitro Kinase Assays ERK2 immunoprecipitates were washed twice with wash buffer, followed by two washes in kinase reaction buffer (25 mM HEPES pH 7.4 and 20 mM MgCl 2 ). The precipitates were resuspended in 50 l of the same kinase buffer containing 50 M ATP,

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25 2 Ci 32 P -ATP, and 5 g myelin basic protein (MBP). The samples were incubated for 15 min at 30 o C. Reactions were terminated by adding sample buffer. The samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to autoradiography. Site-Directed Mutagenesis Mutations of Jak2 amino acids were generated using the QuikChange site-directed mutagenesis system (Stratagene). All mutations were confirmed by DNA sequence analysis. Luciferase Assays 100 mm dishes of 70% confluent COS-7 cells were transfected with 10 g c-fos/luciferase in 12 l Lipofectin for 5 h. The cells were then trypsinized and seeded into 6-well plates at 4.5 x 10 5 cells per well, and allowed to attach overnight in serum containing medium. The next morning, the cells were washed and placed into serum free DMEM. The next morning, the cells were stimulated with angiotensin II, and luciferase activity was measured from detergent extracts in the presence of ATP and luciferin using the Reporter Lysis Buffer System (Promega) and a luminometer (Moonlight 3010). Molecular Modeling A structural homology model of the Jak2 kinase domain ranging from amino acid 814 to the stop codon (position 1129) was generated using the program Swiss Model. The model was based on the known crystal structure of the kinase domain of the fibroblast growth factor tyrosine kinase receptor. The program HBPLUS Hydrogen Bond Calculator, version 3.15, was used to determine hydrogen bond interactions and bond lengths.

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26 Analysis of DNA Laddering Genomic DNA was isolated using the Easy-DNA kit from Invitrogen. 20 g of DNA was separtated on a 1.8% agarose gel and stained with ethidium bromide. Laddering was analyzed under ultraviolet light using the GelDoc system. Hoechst 33342 Staining Cells were grown on microscope slides, serum starved for 48 h, and treated. Cells were washed twice with PBS and incubated for 30 min at room temperature with 50 g/mL Hoechst 33342 nuclear stain. Cells were then washed, fixed, and mounted as described in the immunocytochemistry section. The cells were visualized using a fluorescent microscope with appropriate filters. Apoptotic cells were counted as those showing condensed and/or fragmented nuclei. Propidium Iodide Staining Cells were grown on microscope slides and stained with 1 g/mL propidium iodide for 10 min at 37 o C. Live cells were examined using confocal microscopy. Same field images were captured under phase contrast and fluorescent conditions. Analysis of Mitochondrial Membrane Integrity Mitochondrial membrane integrity was analyzed using the MitoCapture Apoptosis Detection Kit from BioVision. Cells were treated with 1 mM hydrogen peroxide for 2 h and stained according to the manufacturers protocol. Live cells were then visualized using appropriate filters on a confocal microscope. Predominant green staining occurs in cells with a disrupted mitochondrial membrane potential, while predominant red staining occurs in cells with an intact mitochondrial membrane potential.

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CHAPTER 3 MEANS OF CIRCUMVENTING PROBLEMS WITH AG490 Introduction As discussed in Chapter 1, one of the central problems facing researchers studying Jak2 tyrosine kinase function is the lack of a specific Jak2 inhibitor. While AG490 is a useful research tool, results acquired using AG490 must be corroborated using other methodology. Here we will briefly discuss in vitro strategies that we use to circumvent the problems with using AG490. Primary Rat Aortic Smooth Muscle Cells To study the role that Jak2 tyrosine kinase plays in VSMC physiology, we use primary rat aortic smooth muscle cells (RASM cells) stably transfected with a Jak2 dominant negative mutant (RASM-DN). As controls, we use the same cells expressing a neomycin resistant cassette (RASM-Control). Creation of these cells has been described previously (39). In the RASM-DN cells, Jak2 tyrosine kinase function is suppressed by the Jak2 dominant negative mutant, allowing us to reliably study the function of Jak2. 2A Cell Line To study the role that Jak2 plays in intracellular Ang II-dependent signaling, we used 2A cells, which are human fibrosarcoma cells that were gamma-irradiated and screened to identify Jak2-deficient cells. The cells were then stably transfected with the angiotensin II type I receptor (2A/AT 1 ). Controls are 2A/AT 1 cells stably transfected with Jak2 cDNA to reconstitute Jak2 signaling (2A/AT 1 +Jak2) (130). 27

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CHAPTER 4 Jak2 REGULATES ANGIOTENSIN II-DEPEDENDENT ERK2 SIGNALING Introduction Extracellular signal regulated kinase 2 (ERK2) is a member of the mitogen-activated protein (MAP) kinase family of serine/threonine protein kinases. These proteins become activated by phosphorylation on tyrosine and threonine residues in response to a variety of ligands binding their cognate receptors at the cell surface (131-134). Angiotensin II is one such ligand; it exerts many of its mitogenic effects by binding to the angiotensin II type 1 (AT 1 ) receptor and activating ERK1/2. This occurs via rapid angiotensin II-dependent phosphorylation of the dual specificity kinase MEK, which in turn phosphorylates ERK1/2 on both tyrosine and threonine residues (135). ERK activity is tightly regulated. The duration of ERK activation is regulated by the intracellular signals that phosphorylate and dephosphorylate it (136). While ERKs are activated by ligand binding at the cell surface, they are inactivated by several dual-specificity phosphatases (137). One of these, MAP kinase phosphatase 1 (MKP-1), associates with and is phosphorylated by activated ERK2, and thus protected from proteasomal degradation. The phosphorylated MKP-1 then dephosphorylates ERK2, thereby inactivating it (138). Evidence shows that Jak2 forms a membrane complex with the intermediate signaling molecules Ras and Raf1, and may therefore play a role in the regulation of ERK activity (44, 139-140). In fact, previous work suggested that inhibition of Jak2 using the pharmacological compound AG490, blocks the angiotensin II-dependent activation of 28

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29 ERK2 (18). One problem, though, with using AG490 to study the function of Jak2 is its lack of specificity for Jak2. To examine the role that Jak2 plays in the regulation of Ang II-induced ERK2 signaling, we used the 2A cells described in Chapter 3. Briefly, these are Jak2 -/cells that stably express the AT 1 receptor (2A/AT 1 ). Controls cells are the 2A cells stably transfected with both the AT 1 receptor and Jak2 (2A/AT 1 +Jak2), to reconstitute Ang II-dependent Jak2 signaling. Results ERK2 Activity is Sustained in 2A/AT 1 Cells Compared to 2A/AT 1 +Jak2 Cells after Angiotensin II Treatment We sought to determine the role that Jak2 plays in angiotensin II-dependent ERK2 activity using the 2A-derived cells. 2A/AT 1 and 2A/AT 1 +Jak2 cells were stimulated with 100 nM angiotensin II for 0, 6, 12, 18, 24, and 30 min. The cells were lysed and protein was extracted. The protein extracts were immunoblotted with an anti-ACTIVE-ERK2 antibody that detects phosphorylated ERK2 protein. In the cells lacking Jak2, angiotensin II stimulation resulted in a rapid and sustained increase in ERK2 activation that persisted for 30 min (Fig. 4-1A, Top). However, in the Jak2 cells, angiotensin II caused an increase in ERK2 activity that peaked at 6-12 min and returned to basal levels 18-24 min after angiotensin II stimulation. The membrane was then stripped and reprobed with an anti-ERK2 antibody to show constant ERK2 expression at all time points (Fig. 4-1A, Bottom). The 3 different membranes representing Fig. 4-1A were scanned for densimetric analysis, and ERK2 phosphorylation was plotted as a function of angiotensin II treatment (Fig. 4-1B). The graph shows that in the cells expressing Jak2, ERK2 phosphorylation transiently increased, peaking 6 min after angiotensin II treatment. However, in cells

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30 A Ang II (min) 0 6 12 18 24 30 0 6 12 18 24 302A/AT1 2A/AT1+Jak2 ACTIVE-ERK2Total ERK2 36 36 An g II ( min ) 05101520253035ERK2 Phosphorylation: fold increase over unstimulated 0123456 AT1 AT1 + Jak2 ***BA Ang II (min) 0 6 12 18 24 30 0 6 12 18 24 302A/AT1 2A/AT1+Jak2 ACTIVE-ERK2 ACTIVE-ERK2Total ERK2 Total ERK2 36 36 36 36 An g II ( min ) 05101520253035ERK2 Phosphorylation: fold increase over unstimulated 0123456 AT1 AT1 + Jak2 ***B An g II ( min ) 05101520253035ERK2 Phosphorylation: fold increase over unstimulated 0123456 AT1 AT1 + Jak2 ***B Figure 4-1. ERK2 activity is sustained in 2A/AT 1 cells compared to 2A/AT 1 +Jak2 cells after angiotensin II treatment A) 2A/AT 1 and 2A/AT 1 +Jak2 cells were treated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2 (Top). The membrane was then stripped and reprobed with anti-ERK2 antibody to confirm total ERK2 protein levels (Bottom). B) The three membranes representing Fig. 5-1A were subjected to densimetric analysis. Anti-phospho-ERK2 signal was plotted as a function of both angiotensin II treatment and Jak2 expression. Values are expressed as the mean +/SD. p<0.01, **p<0.05 (Students t test). Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1960, Fig. 3A and 3B with permission from publisher. lacking Jak2, ERK2 phosphorylation was significantly elevated 30 min after angiotensin II stimulation. Thus, the data in Fig. 4-1 suggest that loss of Jak2 expression via a null mutation results in sustained ERK2 phosphorylation in response to angiotensin II. This is

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31 contrary to previously published data, which suggested that inhibiting Jak2 kinase function using AG490 results in diminished angiotensin II-dependent ERK2 phosphorylation (18). To verify our results using an alternate protocol, in vitro kinase assays were performed using myelin basic protein (MBP) as a substrate for ERK2 phosphorylation. In 2A/AT 1 cells, phosphorylation of MBP remained elevated 20 min after angiotensin II treatment, suggesting that ERK2 was catalytically active at this time point (Fig. 4-2). Ang II (min) 0 5 10 15 20 0 5 10 15 202A/AT1 2A/AT1+Jak2 32P MBP 13 Ang II (min) 0 5 10 15 20 0 5 10 15 202A/AT1 2A/AT1+Jak2 32P MBP 32P MBP 13 13 Figure 4-2. In vitro kinase assay confirms that ERK2 activity is sustained in 2A/AT 1 cells compared to 2A/AT 1 +Jak2 2A/AT 1 cells and 2A/AT 1 +Jak2 cells were treated with 100 nM angiotensin II for the indicated times. Lysates were prepared and immunoprecipitated with anti-ERK2-mAb and then resuspended in kinase reaction buffer. Phosphorylation of myelin basic protein was detected by autoradiography. Shown is one of three independent results. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1960, Fig. 3C with permission from publisher. However, in 2A/AT 1 +Jak2 cells, angiotensin II stimulated ERK2 activity peaked at 5-10 min. After 15-20 min, the 32 P-labeled MBP signal was similar to that of basal conditions. Collectively, the results in Fig. 4-1 and 4-2 clearly show that loss of Jak2 expression via a Jak2 null mutation results in enhanced ERK2 activation in response to angiotensin II when compared to Jak2-expressing control cells. AG490 Suppresses Angiotensin II-Dependent ERK2 Activation Independent of Jak2 Inhibition We next wanted to determine whether treating the Jak2 expressing cells with AG490 could recapitulate the observations seen in Fig. 4-1. In other words, could we

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32 reproduce the enhanced ERK2 activation phenomenon seen in the Jak2 deficient cells, by treating the Jak2 expressing cells with AG490? For this, 2A/AT 1 +Jak2 cells were treated with either AG490 or the inert control compound, AG9. The cells were then treated with angiotensin II and phospho-ERK2 levels were directly measured via Western blot analysis. First, 2A/AT 1 cells were treated with angiotensin II to reproduce the enhanced ERK2 activation seen 30 min after angiotensin II treatment (Fig. 4-3A, lanes 1-3). In 2A/AT 1 +Jak2 cells pretreated with the AG9 control compound, Ang II-induced ERK2 phosphorylation levels were high at 5 min, but returned to basal levels by 30 min, similar to that shown in Fig. 4-1A (Fig. 4-3A, lanes 4-6). However, in 2A/AT 1 +Jak2 cells pretreated with AG490, the ability of angiotensin II to induce ERK2 phosphorylation appeared to be lost (Fig 4-3A, lanes 7-9). As such, this result indicates that pharmacological suppression of Jak2 via AG490 does not recapitulate the effect of the Jak2 null mutation and suggests that AG490 is blocking angiotensin II-dependent ERK2 activation via a mechanism that is independent of Jak2. Recent work suggests that one mechanism by which G protein coupled receptors activate ERK1/2 is via transactivation of the epidermal growth factor receptor (EGFR) (14-143). This is important because, as indicated in Chapter 1, AG490 inhibits epidermal growth factor receptor autophosphorylation more potently that it inhibits Jak2 kinase activity (100, 101). To determine whether this mechanism of action is responsible for the angiotensin II-dependent activation of ERK2 in the 2A-derived cells, 2A/AT 1 +Jak2 cells were treated with the EGFR tyrosine kinase inhibitor, AG1478, before measuring angiotensin II-dependent ERK2 activation. AG1478 treatment, at a dose that has

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33 previously been shown to fully suppress EGFR kinase activity (144, 145), failed to inhibit the angiotensin II-mediated activation of ERK2 in 2A/AT 1 +Jak2 cells, when Ang II (min) 0 5 30 0 5 30 0 5 302A/AT12A/AT1+Jak2 ACTIVE-ERK236 AG-9AG-490 AB ControlAG1478 2A/AT1+Jak2Ang II (min) 0 5 30 0 5 30 Active ERK2 36 Ang II (min) 0 5 30 0 5 30 0 5 302A/AT12A/AT1+Jak2 ACTIVE-ERK2 ACTIVE-ERK236 36 AG-9AG-490 AB ControlAG1478 2A/AT1+Jak2Ang II (min) 0 5 30 0 5 30 Active ERK2 Active ERK2 36 36 Figure 4-3. AG490 suppresses angiotensin II-dependent ERK2 activation independent of Jak2 inhibition A) Cells were pretreated for 16 hrs with either 100 M AG9 or 100 M AG490 and then stimulated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and then Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2 protein. B) Cells were pretreated for 1 hr with 10 M AG1478 and then stimulated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and then Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2 protein. Shown is one of three representative results for each. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1961, Fig. 4A and 4B with permission from publisher. compared to similarly treated control cells (Fig. 4-3B). This result suggests that in the 2A-derived cells, angiotensin II mediates activation of ERK2 via a mechanism that is independent of EGFR tyrosine kinase activity. In summary, the data in Fig. 4-3A show that AG490 blocks the angiotensin II-dependent activation of ERK2 via a mechanism that is independent of Jak2. The data in Fig. 4-3B show that this nonspecific effect of AG490 is not because of reduced EGFR tyrosine kinase activity, as direct suppression of EGFR tyrosine kinase activity with

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34 AG1478 does not inhibit angiotensin II-dependent activation of ERK2. Collectively, the data suggest that AG490 is inhibiting ERK2 activation via nonspecific suppression of a tyrosine kinase that is not the EGFR. There is a Marked Difference in the Angiotensin II-Induced Nuclear Accumulation Pattern of Activated ERK2 between 2A/AT 1 and 2A/AT 1 +Jak2 Cells ERK2, when activated by angiotensin II, accumulates in the nucleus of cells and modulates the expression of a variety of genes by activating nuclear transcription factors such as AP-1 (136). We next wanted to determine if a difference in nuclear accumulation of activated ERK2 existed between 2A/AT 1 and 2A/AT 1 +Jak2 cells. For this, both cell types were immunostained with an anti-ACTIVE-ERK2 antibody. The cells were then visualized using a fluorescent microscope to measure angiotensin II-dependent ERK2 nuclear accumulation, as a function of Jak2 expression (Fig. 4-4). There was a marked difference in the nuclear accumulation pattern of phospho-ERK2 between the two cell types. Specifically, in the cells lacking Jak2, angiotensin II treatment facilitated a rapid nuclear accumulation of activated ERK2. This staining persisted for at least 30 min, but also appeared to be perinuclear in nature at this time point (arrows). For the Jak2 expressing cells however, angiotensin II treatment promoted a transient nuclear accumulation of activated ERK2 that was visible at 5 min, but virtually gone at 30 min. Collectively, these data suggest that the sustained levels of phospho-ERK2 seen in Fig. 4-1 correlate with increased phospho-ERK2 immunoreactivity in the nucleus of the cell. ERK2-Dependent Gene Transcription in the 2A/AT 1 and 2A/AT 1 +Jak2 Cells To determine if differences in ERK2-mediated gene expression existed between the 2A/AT 1 and 2A/AT 1 +Jak2 cells, we measured angiotensin II-dependent, ERK2

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35 0 min5 min30 min2A/AT1+Jak22A/AT1 0 min5 min30 min2A/AT1+Jak22A/AT1 Figure 4-4. Difference in the angiotensin II-dependent nuclear accumulation of phospho-ERK2 between 2A/AT 1 and 2A/AT 1 +Jak2 cells 2A/AT 1 cells and 2A/AT 1 +Jak2 cells were treated with 100 nM angiotensin II for either 0, 5 or 30 min. The cells were incubated with anti-phospho-ERK2-pAb and immunostained with goat-anti-rabbit antibody conjugated to Texas Red. The cells were visualized using a fluorescent microscope to detect nuclear accumulation of activated ERK2 protein. Arrows indicate apparent perinuclear staining. Shown is one of three independent results. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1962, Fig. 5 with permission from publisher. mediated gene transcription in the two cell types. This was accomplished using a synthetic promoter containing seven copies of the AP-1 binding element upstream of a luciferase reporter. After transfection with this construct, the cells were treated with 100 nM angiotensin II for the indicated times and then lysed. Angiotensin II treatment elicited a marked increase in luciferase activity in both cell types (Fig. 4-5). Although the 2A/AT 1 cells repeatedly generated higher fold changes in luciferase activity

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36 compared to the Jak2-expressing control cells, the difference failed to reach statistical significance at any time point. Jak2 is Essential for Angiotensin II-Induced MKP-1 Expression and Co-association of MKP-1 with ERK2 The data in Fig. 4-1 and 4-2 show that in the cells lacking Jak2, ERK2 has sustained levels of activation when compared to the Jak2-expressing cells. We hypothesized that the signal transduction pathways leading to the induction of ERK2 100 nM An g I I ( hrs) 051015202530AP-1 luciferase: fold increase over unstimulated 01234567 AT1 AT1 + Jak2 Figure 4-5. No difference in angiotensin II-dependent ERK mediated gene transcription between 2A/AT 1 and 2A/AT 1 +Jak2 cells 2A/AT 1 and 2A/AT 1 +Jak2 cells were transfected with 10 g of a luciferase reporter whose expression is driven by a synthetic promoter containing seven copies of the AP-1 binding element to measure ERK-mediated gene transcription. The cells were treated with 100 nM angiotensin II for the indicated times. Lysates were prepared and luciferase activity was measured. Shown is one of three independent results. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1963, Fig. 6 with permission from publisher.

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37 specific phosphatases were different in the two cell types. Specifically, we hypothesized that in the cells lacking Jak2, there would be little to no angiotensin II-mediated induction of the phosphatases that inactivate ERK2. We therefore tested the ability of angiotensin II to induce gene expression of two such phosphatases, PP2A and MKP-1, in both cell types. Quiescent cells were treated with 100 nM angiotensin II for 0, 15, 30, or 60 min, and protein lysates were prepared. The whole cell lysates were first immunoblotted with an anti-PP2A antibody (Fig. 4-6A). The results show that there was no marked difference in PP2A protein expression between the 2A/AT 1 and 2A/AT 1 +Jak2 cells in response to angiotensin II. Based on these results, we concluded that angiotensin II was not modulating gene expression of PP2A. We next immunoblotted similarly prepared protein extracts with an anti-MKP-1 antibody, and found that in 2A/AT 1 cells, angiotensin II induced very little MKP-1 expression (Fig 4-6B). In the 2A/AT 1 +Jak2 cells however, angiotensin II induced marked MKP-1 expression, demonstrating that maximal angiotensin II-induced MKP-1 protein expression requires Jak2. MKP-1 associates with ERK2 in response to angiotensin II treatment, and is activated by ERK2. MKP-1, in turn dephosphorylates ERK2 (138). We next investigated if Jak2 is required for co-association of MKP-1 with ERK2. 2A/AT 1 and 2A/AT 1 +Jak2 cells were treated with 100 nM angiotensin II for 0, 15, and 30 min. The cells were lysed and cellular protein was extracted. The protein extracts were immunoprecipitated with anti-ERK2 antibody and then immunoblotted with anti-MKP-1 antibody. The results show that no increase in co-association of ERK2 and MKP-1 was

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38 observed in 2A/AT 1 cells, whereas in 2A/AT 1 +Jak2 cells, angiotensin II induced a substantial increase in co-association of ERK2 and MKP-1 (Fig. 4-6C). The data in Fig. 4-6B suggest that Jak2 is playing a key role in the angiotensin II-dependent increased expression of MKP-1. Previous work has shown that ERK2 itself can also be a critical mediator of MKP-1 gene expression (137, 138). To determine the relative contribution of ERK2 and Jak2 in mediating angiotensin II-dependent MKP-1 gene expression, both cell types were treated with angiotensin II in the presence or absence of the MEK specific inhibitor, PD98059 (Fig. 4-6D). For the cells lacking Jak2, ligand treatment only modestly induced MKP-1 expression (lanes 1-3), and this was completely blocked with PD98059 (lanes 4-6). However, for the cells expressing Jak2, ligand treatment again induced marked MKP-1 expression (lanes 7-9), and this was partially blocked with PD98059 (lanes 10-12). Thus, the data indicate that there is both a Jak2-dependent component and an ERK2-dependent component to the angiotensin II-mediated induction of MKP-1, as maximal MKP-1 expression is only attained when cells have both functional Jak2 and ERK2. Collectively, the data in Fig. 4-6 suggest that Jak2 is not only required for induction of MKP-1 expression in response to angiotensin II, but also for co-association of MKP-1 and ERK2. Additionally, the data indicate that that both Jak2 and ERK2 are required for maximal angiotensin II-mediated MKP-1 protein expression. MPK-1 is Required for Angiotensin II-Dependent Inactivation of ERK2 The data in Fig. 4-6 show that Jak2 is required for angiotensin II-dependent induction of MKP-1, but not PP2A. However, the data clearly show that both proteins are present in the cell. To determine the extent to which these two phosphatases regulate

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39 Ang II (min) 0 15 30 60 0 15 30 60 A2A/AT1 2A/AT1+Jak2PP2A 24 C Ang II (min) 0 15 30 0 15 302A/AT12A/AT1+Jak2 IP:ERK2-mAbIB:MKP1-pAbMKP-1 36Ang II (min) 0 30 60 0 30 60 0 30 60 0 30 602A/AT1 2A/AT1+Jak2 ControlControlPD98059PD98059 MKP-1 DMKP-1 36B2A/AT1 2A/AT1+Jak2 Ang II (min) 0 30 60 0 30 60 Ang II (min) 0 15 30 60 0 15 30 60 A2A/AT1 2A/AT1+Jak2PP2A PP2A 24 24 C Ang II (min) 0 15 30 0 15 302A/AT12A/AT1+Jak2 IP:ERK2-mAbIB:MKP1-pAbMKP-1 MKP-1 36 36Ang II (min) 0 30 60 0 30 60 0 30 60 0 30 602A/AT1 2A/AT1+Jak2 ControlControlPD98059PD98059 MKP-1 MKP-1 DMKP-1 MKP-1 36 36B2A/AT1 2A/AT1+Jak2 Ang II (min) 0 30 60 0 30 60 Figure 4-6. Jak2 is essential for angiotensin II-induced MKP-1 expression 2A/AT 1 and 2A/AT 1 +Jak2 cells were treated with angiotensin II for the indicated times and cell lysates were prepared. A) Whole cell lysates were Western blotted with anti-PP2A-mAb to detect PP2A expression. B) Whole cell lysates were Western blotted with anti-MKP-1-mAb to detect MKP-1 expression. C) Lysates were immunoprecipitated with anti-ERK2-mAb and Western blotted with anti-MKP-1-mAb to detect co-association of MKP-1 with ERK2. D) Cells were pre-treated for 60 min with either DMSO or 50 M PD98059 and then stimulated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and then Western blotted with anti-MKP-1-mAb to detect MKP-1 protein. Shown is one of four (A) or three (B, C, D) independent results for each. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1964, Fig. 7 with permission from publisher.

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40 the dephosphorylation of ERK2, angiotensin II-mediated ERK2 phosphorylation was measured in the presence, or absence, of PP2A and MKP-1 specific inhibitors. To inhibit PP2A, which is a serine/threonine specific phosphatase, 2A/AT 1 and 2A/AT 1 +Jak2 cells were pretreated with the PP2A specific inhibitor, okadaic acid (146). The cells were then treated with angiotensin II and ERK2 phosphorylation was measured via Western blot analysis (Fig. 4-7 A). In the 2A/AT 1 cells, before ligand treatment, ERK2 was already phosphorylated, possibly because of the presence of a phosphatase inhibitor. Addition of angiotensin II did not significantly increase the signal at either the 5 or 30 min time points, thereby suggesting that the signal was at, or near, maximal phosphorylation levels before ligand treatment. Additionally, the data suggest that angiotensin II treatment did not induce the necessary cellular factors, such as Jak2, that are required for dephosphorylating ERK2, since ERK2 remained phosphorylated 30 min after ligand treatment. For the 2A/AT 1 +Jak2 cells, ERK2 was also basally phosphorylated before ligand treatment. In contrast however, 30 min of angiotensin II treatment promoted its relative dephosphorylation. This observation suggests two important things. First, it indicates that the 2A/AT 1 +Jak2 cells contain the necessary component(s) to promote the dephosphorylation of ERK2 (ie. Jak2). Second, the data suggest that this ligand dependent dephosphorylation does not require PP2A since the dephosphorylation occurs in the presence of okadaic acid. To inhibit MKP-1, which is a threonine/tyrosine dual specificity phosphatase, 2A/AT 1 and 2A/AT 1 +Jak2 cells were pretreated with the MKP-1 inhibitor, vanadate (147). The cells were then treated with angiotensin II and ERK2 phosphorylation was again measured via Western blot analysis with anti-ACTIVE-ERK2 antibody (Fig. 4-7B).

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41 In the 2A/AT 1 cells, before ligand treatment, ERK2 showed some basal phosphorylation. Addition of angiotensin II modestly increased the signal at both the 5 and 30 min time points. These cells were once again unable to dephosphorylate ERK2 after 30 min of ligand treatment. Similarly, in the 2A/AT 1 +Jak2 cells, ERK2 was basally phosphorylated before ligand treatment and 5 min of angiotensin II treatment A Okadaic AcidAng II (min) 0 5 30 0 5 3036 2A/AT12A/AT1+Jak2 B VanadateAng II (min) 0 5 30 0 5 30 Active ERK2 36 2A/AT12A/AT1+Jak2Active ERK2 A Okadaic AcidAng II (min) 0 5 30 0 5 3036 2A/AT12A/AT1+Jak2 B VanadateAng II (min) 0 5 30 0 5 30 Active ERK2 Active ERK2 36 2A/AT12A/AT1+Jak2Active ERK2 Figure 4-7. Angiotensin II-dependent inactivation of ERK2 requires Jak2 and MKP-1 A) 2A/AT 1 and 2A/AT 1 +Jak2 cells were pretreated for 1 hr with 500 nM okadaic acid and then stimulated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and then Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2 protein. B) 2A/AT 1 and 2A/AT 1 +Jak2 cells were pretreated for 1 hr with 100 M sodium ortho-vanadate and then stimulated with 100 nM angiotensin II for the indicated times. Whole cell lysates were prepared and then Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2 protein. Shown is one of three independent results for each. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1965, Fig. 8 with permission from publisher. modestly increased its signal. However, unlike the previous experiments in the Jak2 expressing cells, ERK2 phosphorylation levels remained elevated at the 30 min time point. These data thereby suggest that MKP-1 is critical for mediating the angiotensin II-dependent dephosphorylation of ERK2, as vanadate treatment of these cells blocks the

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42 angiotensin II-dependent dephosphorylation of ERK2. Collectively, the data in Fig. 4-7 suggest that PP2A and MKP-1 play distinct roles in the dephosphorylation of ERK2; PP2A appears to be largely responsible for the basal phosphorylation state of ERK2 while MKP-1 appears to regulate angiotensin II-dependent dephosphorylation. Moreover, this ligand dependent dephosphorylation of ERK2 by MKP-1 requires Jak2. Discussion Here, we report several new observations. First, lack of Jak2 signaling in a cell increases the duration of ERK2 activity after angiotensin II stimulation. This was shown by both Western blot analysis and in vitro kinase assays. This observation is contrary to what has been reported previously in studies using AG490 to study the role that Jak2 plays in ERK activation. One possible reason for this discrepancy is that AG490 has been shown to inhibit other tyrosine kinases nonspecifically (98-101). A second observation that we report is that angiotensin II induces rapid induction of the MKP-1 gene. We report that angiotensin II not only causes activation of ERK2, but also simultaneously induces upregulation of MKP-1, a phosphatase that inactivates ERK2. This is an interesting example of the tight regulation of ERK signaling within a cell. Furthermore, we report that angiotensin II-induced upregulation of MKP-1 gene expression is dependent on Jak2 expression. We believe that this is the reason that in 2A/AT 1 cells lacking Jak2, ERK2 activity is sustained after angiotensin II stimulation compared to 2A/AT 1 +Jak2 cells. Without Jak2 present, angiotensin II is simply unable to upregulate MKP-1 expression, and thus the cell is slower to inactivate ERK2. Interestingly, in the 2A/AT 1 cells lacking Jak2, after 30 min of angiotensin II treatment the active ERK2 showed predominantly perinuclear localization. There was

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43 not as much nuclear localization of active ERK2 after 30 min of treatment compared to 5 min of treatment. Therefore, despite the sustained ERK2 activation in the 2A/AT 1 cells lacking Jak2, there is not a sustained ERK2 nuclear localization. This is probably the reason that no significant difference in gene transcription between the two cell types was observed. Furthermore, it suggests that ERK2 nuclear localization is not dependent only upon ERK2 activation. Instead, ERK2 nuclear localization appears to be controlled in a temporal manner, so that even though a cell may have sustained ERK2 activity, it may not show sustained ERK2 nuclear localization and ERK2-mediated gene transcription. This presents an interesting possibility. Perhaps in cells with sustained ERK2 activity, the cytosolic actions, rather than the nuclear actions, of ERK2 are sustained. This could be particularly relevant in cancer cells showing constitutive ERK activity. Further testing is needed to determine this. Fig. 4-8 shows a model depicting what we believe is occurring in normal cells. Upon angiotensin II stimulation, ERK2 becomes phosphorylated on threonine and tyrosine residues. Simultaneously, Jak2 becomes tyrosine phosphorylated and associates with the AT 1 receptor. Jak2 induces expression of MKP-1, presumably through activation of one or more STAT proteins. ERK2 also acts on the MKP-1 promoter to increase its expression. The MKP-1 protein that is generated then associates with and inactivates ERK2. Previous work has shown that the duration of ERK activation is critical for determining cell fate (148-150). In some cell signaling systems for instance, transient activation of ERK2 is a common feature of cell proliferation. Sustained activation on the other hand, is associated with very different cellular events such as apoptosis or

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44 senescence. Previously published work has shown that Jak2 can promote both cellular proliferation and apoptosis (151, 152). How Jak2 elicits such different cellular responses is presumably dependent on the specific cell type and ligand used in each experiment. However, the exact cellular and biochemical mechanism(s) by which Jak2 accomplishes this is not fully known. Our work here shows that Jak2 plays an important role in determining whether ERK2 activation is transient or sustained. As such, we may have Jak2 ERK2 P P (+) MKP-1 Promoter Nucleus (+) (-)AT1 Receptor Jak2 ERK2 P P P P (+) MKP-1 Promoter Nucleus (+) (-)AT1 Receptor Figure 4-8. Proposed model of the mechanism by which Jak2 mediates ERK2 inactivation after angiotensin II treatment Angiotensin II binding to its type 1 receptor activates ERK2, while simultaneously activating Jak2. Jak2, probably through the action of STAT proteins, increases expression of MKP-1. ERK2 also increases MKP-1 expression. The expressed MKP-1 protein then associates with and dephosphorylates ERK2, thus inactivating the signal. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1966, Fig. 9 with permission from publisher.

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45 determined that one mechanism by which Jak2 influences cell fate is by altering the duration of ERK2 activation via induction of MKP-1. Clearly, further studies are needed to fully address this issue.

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CHAPTER 5 Jak2 PROMOTES OXIDATIVE STRESS-INDUCED APOPTOSIS IN VASCULAR SMOOTH MUSCLE CELLS Introduction In 1998, it was demonstrated that Jak2 is activated in response to oxidative stress (36). While the signaling cascade responsible for oxidative stress-induced Jak2 activity was at least partially elucidated in vascular smooth muscle cells, no physiological role has been ascribed to this pathway. Runges group (35) showed that Jak2 activation by oxidative stress caused up regulation of heat-shock protein 70, a protein that can protect cells from oxidative stress. These data were generated using AG490 to inhibit Jak2 function. Based on these data, they suggested that Jak2 might help vascular smooth muscle cells adapt to oxidative stress (35). Oxidative stress in vascular smooth muscle cells can cause proliferation, contraction, or apoptosis (153-155). How the same stimulus can result in such opposing endpoints is unknown, but is probably dependent on oxidant dose. Furthermore, the signaling proteins that mediate these different responses are unknown. Oxidative stress-induced apoptosis of vascular smooth muscle cells contributes to the progression of a number of vascular pathologies, including atherosclerosis and restenosis (156, 157). Identifying the mediators of oxidant-induced apoptosis may therefore uncover novel therapeutic targets. Interestingly, both proand anti-apoptotic roles have been ascribed to Jak2 tyrosine kinase activity in a variety of signaling systems (34, 97, 158). We therefore 46

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47 sought to determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis in vascular smooth muscle cells. Results Jak2 Activation by Hydrogen Peroxide is Suppressed in RASM-DN Cells To study the role that Jak2 activation plays in oxidative stress-induced apoptosis, we used rat aortic smooth muscle cells stably expressing a Jak2 dominant negative protein (RASM-DN). We used the same cells, expressing only a neomycin resistant cassette, as controls (RASM-Control). To show that Jak2 activation by hydrogen peroxide is inhibited in RASM-DN cells, we treated RASM-Control and RASM-DN cells with 0.2, 0.5, or 1.0 mM hydrogen peroxide for 0, 5, or 10 min (Fig. 5-1, Top). Cellular lysates were immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Jak2 antibody, to detect tyrosine phosphorylated Jak2. Aliquots from the cellular lysates were Western blotted with anti-Jak2 antibody to demonstrate equal protein loading amongst all samples (Fig. 5-1, Bottom). The results show that Jak2 is strongly activated by hydrogen peroxide in RASM-Control cells, but Jak2 activation is greatly reduced in RASM-DN cells. This shows that RASM-Control and RASM-DN cells are good models for studying the physiological role of Jak2 during oxidative stress in vascular smooth muscle cells. Jak2 Activation is Required for Oxidative Stress-Induced Apoptosis To determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis, RASM-Control and RASM-DN cells were treated with 1 mM hydrogen peroxide for 24 h. We assessed apoptosis by analyzing genomic DNA isolated from each cellular condition (Fig. 5-2A). Genomic DNA isolated from RASM-Control cells that were treated with hydrogen peroxide was fragmented into approximately 200 base pair bands,

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48 Jak2(P)H2O2 (min) RASM-ControlRASM-DN 11105010510 555510101010 111 TotalJak2 0.2 mM0.5 mM1.0 mM0.2 mM0.5 mM1.0 mM Jak2(P)H2O2 (min) RASM-ControlRASM-DN 111 11105010510 555510101010 111 111 TotalJak2 0.2 mM0.5 mM1.0 mM0.2 mM0.5 mM1.0 mM Figure 5-1. Hydrogen peroxide-induced Jak2 activity is suppressed in RASM-DN cells RASM-Control and RASM-DN cells were treated with 0.2 mM, 0.5 mM, or 1.0 mM hydrogen peroxide for 0, 5, or 10 min. Cellular lysates were immunoprecipitated with anti-phosphotyrosine-mAb antibody and immunoblotted with anti-Jak2-pAb antibody to detect tyrosine phosphorylated Jak2 (Top). An aliquot from each lysate was Western blotted with anti-Jak2-pAb to demonstrate equal Jak2 expression amongst all samples (Bottom). Shown is one of three representative experiments. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34548, Fig. 1 with permission from publisher. characteristic of apoptosis. In contrast, genomic DNA from RASM-DN cells treated with hydrogen peroxide showed no evidence of DNA fragmentation. Furthermore, AG490 prevented genomic DNA fragmentation in RASM-Control cells treated with hydrogen peroxide. Next, to show that RASM-DN cells could undergo apoptosis in response to another pro-apoptotic stimulus, genomic DNA was isolated from RASM-DN cells treated for 4 h with 5 M staurosporine, a potent activator of the intrinsic apoptosis pathway (Fig. 5-2B). The banding pattern characteristic of fragmented DNA was clearly seen in these cells. Finally, to determine the effect of different doses of hydrogen peroxide on vascular smooth muscle cell apoptosis, we treated RASM-Control and RASM-DN cells with 0.2, 0.5, or 1.0 mM hydrogen peroxide for 24 h and examined genomic DNA for fragmentation (Fig. 5-2C). The results show that, as expected no DNA fragmentation

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49 occurs in RASM-DN cells. In RASM-Control cells, 1.0 mM hydrogen peroxide was required to induce DNA fragmentation. Collectively, these results show that Jak2 activation is required for oxidative stress-induced apoptosis in vascular smooth muscle cells. A LadderUntreatedH2O2AG490+H2O2UntreatedH2O2AG490+H2O2 RASM-DNRASM-Control B UntreatedStaurosporine RASM-DN RASM-DNRASM-Control LadderUntreated0.2 mM0.5 mM1.0 mMUntreated0.2 mM0.5 mM1.0 mMH2O2 (24h)CA LadderUntreatedH2O2AG490+H2O2UntreatedH2O2AG490+H2O2 RASM-DNRASM-Control RASM-DNRASM-Control B UntreatedStaurosporine UntreatedStaurosporine RASM-DN RASM-DNRASM-Control LadderUntreated0.2 mM0.5 mM1.0 mMUntreated0.2 mM0.5 mM1.0 mMH2O2 (24h)C Figure 5-2. Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular smooth muscle cells A) RASM-DN and RASM-Control cells were either left untreated, or treated with 1 mM hydrogen peroxide for 24 h, or with 1 mM hydrogen peroxide for 24 h after 16 h of pretreatment with 100 M AG490. Genomic DNA was isolated and separated on a 1.8% agarose gel. The gel was stained with EtBr and visualized under U.V. light to detect genomic DNA laddering. B) RASM-DN cells were either left untreated or treated with 5 M staurosporine for 4 h. Genomic DNA was isolated and separated on a 1.8% agarose gel. The gel was stained and visualized to detect genomic DNA laddering. C) RASM-Control and RASM-DN cells were treated with 0.2 mM, 0.5 mM, or 1.0 mM hydrogen peroxide for 24 h. Genomic DNA was isolated and separtated on a 1.8% agarose gel. The gel was stained with EtBr and visualized under U.V. light to detect genomic DNA laddering. Shown is one of four (A and B) or three (C) representative experiments for each. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34548, Fig. 2 with permission from publisher.

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50 Quantification of Jak2-Mediated Apoptosis We next quantified the amount of apoptosis occurring in RASM-Control and RASM-DN cells treated with hydrogen peroxide. The cells were grown on microscope slides and treated with 1 mM hydrogen peroxide for either 0 or 24 h. The cells were then stained with the nucleus-specific Hoechst 33342 dye. Representative photomicrographs of RASM-Control and RASM-DN cells either left untreated, or treated with hydrogen peroxide, or hydrogen peroxide after pretreatment with the Jak2 inhibitor AG490, are shown (Fig. 5-3). RASM-Control cells treated with hydrogen peroxide had shrunken nuclei that fluoresced more intensely than untreated cells, which is indicative of apoptotic cells. In contrast, RASM-Control cells treated with AG490 before hydrogen peroxide treatment appeared similar to untreated cells. Similarly, RASM-DN cells either treated with hydrogen peroxide alone, or treated with both AG490 and hydrogen peroxide showed nuclear staining akin to untreated cells. Four replicate experiments were performed for each condition, and a minimum of 100 cells were counted from each replicate. Cells clearly showing condensed and/or fragmented nuclei were counted as apoptotic. Data are presented as percentage of cells undergoing apoptosis (Fig. 5-4). These data show that untreated RASM-Control and RASM-DN cells showed very low levels of apoptosis (2.4+/-0.54% and 2.9+/-0.85% respectively). RASM-Control cells treated with hydrogen peroxide showed 54.1+/-3.71% of total cells undergoing apoptosis, while only 12.3+/-1.93% of RASM-DN cells treated with hydrogen peroxide were apoptotic. Finally, 5.7+/-0.81% of RASM-Control cells and 4.4+/-0.98% of RASM-DN cells that were pretreated with AG490 before hydrogen peroxide treatment were apoptotic.

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51 UntreatedH2O2RASM-ControlRASM-DN AG490+H2O2UntreatedH2O2RASM-ControlRASM-DN AG490+H2O2 Figure 5-3. Hoechst staining to detect nuclear condensation RASM-Control and RASM-DN cells were grown on microscope slides and treated with 1 mM hydrogen peroxide for 0 or 24 h, or with 1 mM hydrogen peroxide for 24 h after 16 h of pretreatment with 100 M AG490. The cells were then stained with 50 g/mL Hoechst 33342 nuclear stain, fixed, mounted, and visualized using a florescent microscope to detect nuclear condensation. Shown is one of four representative photographs of each condition. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34549, Fig. 3A with permission from publisher. Jak2 Activation by Oxidative Stress Mediates Bax Expression We hypothesized that Jak2 was promoting apoptosis by regulating expression of a pro-apoptotic protein. Since Bax is a pro-apoptotic protein required for oxidative stress-induced apoptosis, we used Western blotting to determine whether Jak2 was required for oxidative stress-mediated up regulation of Bax expression. For this, RASM-Control and RASM-DN cells were treated with 1 mM hydrogen peroxide for 0, 1, 2, or 3 h (Fig. 5-5A). Cellular lysates were Western blotted with anti-Bax antibody (Fig. 5-5A, Top). The data show that in RASM-Control cells, hydrogen peroxide induced rapid and transient induction of Bax expression, which peaked at 1-2 h. In contrast, hydrogen peroxide did not induce Bax expression in RASM-DN cells.

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52 RASM-Control RASM-DN UntreatedH2O2AG490 +H2O2H2O2UntreatedAG490+H2O2 010203040506070% apoptotic cells ** *RASM-Control RASM-Control RASM-DN RASM-DN UntreatedH2O2AG490 +H2O2H2O2UntreatedAG490+H2O2 010203040506070% apoptotic cells ** * Figure 5-4. Quantification of apoptosis in RASM-Control and RASM-DN cells. A minimum of 100 cells from each of 4 replicates for each of the 6 treatment conditions represented in Fig 5-3 was counted. Apoptotic cells were counted as those showing condensed and/or fragmented nuclei. Data are presented as percentage of cells undergoing apoptosis +/S.D. *, p<0.001. Statistical analysis was performed using Students t test. printed with Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34549, Fig. 3B with permission from publisher. We next stripped and re-probed the membrane with anti-BCL-2 antibody to determine whether Jak2 influences expression of theanti-apoptotic BCL-2 protein (Fig. 5-5A, Middle). We found that BCL-2 expression increased slightly in both cell types in response to hydrogen peroxide. Because there was no difference in the two cell types, we concluded that Jak2 does not effect expression of BCL-2. To demonstrate equal protein loading amongst all samples, the membrane was stripped and re-probed with anti-STAT1 antibody (Fig. 5-5A, Bottom). These data show that Jak2 activation is required for induction of the pro-apoptotic Bax protein in response to oxidative stress. To confirm this result, RASM-Control cells were pretreated with either the Jak2 inhibitor AG490 or its inactive analog AG9, and then treated with hydrogen peroxide for 0, 1/2, 1, 2, or 3 h

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53 (Fig. 5-5B). The data show that AG490 attenuates hydrogen peroxide-mediated Bax induction in RASM-Control cells (Fig. 5-5B, Top). The membrane was stripped and reprobed with anti-STAT1 antibody to demonstrate equal loading amongst all samples (Fig. 5-5B, Bottom). Collectively, the data in Fig. 5-5 present strong evidence that Jak2 activation promotes oxidative stress-induced apoptosis by mediating an increase in Bax protein expression levels in vascular smooth muscle cells. Jak2 Activation by Oxidative Stress Promotes Mitochondrial Dysfunction During oxidative stress, the pro-apoptotic Bax protein localizes to the outer mitochondrial membrane and increases mitochondrial membrane permeability (159, 160). This is an essential step in the intrinsic apoptosis pathway (161, 162). Since we showed in Fig. 5-5 that Jak2 tyrosine kinase mediates induction of Bax expression, we hypothesized that activation of Jak2 by hydrogen peroxide may also contribute to mitochondrial dysfunction. To test this, we used the MitoCapture reagent to stain live cells treated with hydrogen peroxide and visualized the cells using confocal microscopy (Fig. 5-6). In healthy, non-apoptotic cells, the dye accumulates predominantly in the mitochondria where it forms aggregates, and fluoresces red. In contrast, in apoptotic cells, because of the change in mitochondrial trasnsmembrane potential, the dye remains predominantly in the cytosol as a monomer, and fluoresces green. The data show that in untreated RASM-Control and RASM-DN cells, the MitoCapture dye fluoresces predominantly red, indicating that these cells are non-apoptotic. After 1 mM hydrogen peroxide treatment for 2 h, the RASM-Control cells show a decrease in red staining and a dramatic increase in green staining, indicative of mitochondrial dysfunction in these cells. In contrast, the RASM-DN cells treated with hydrogen peroxide actually show increased

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54 A 1 mM H2O2(h) RASM-ControlRASM-DNIB: Bax-pAb01/212301/2231 25 IB: STAT1-pAb 80B1 mM H2O2(h) AG9AG490IB: Bax-pAbIB: STAT1-pAb01/2121/223130 25 80 IB: BCL2-mAb 25A 1 mM H2O2(h) RASM-ControlRASM-DNIB: Bax-pAb01/212301/2231 25 25 IB: STAT1-pAb 80 IB: STAT1-pAb 80 80B1 mM H2O2(h) AG9AG490IB: Bax-pAbIB: STAT1-pAb01/2121/223130 25 80 B1 mM H2O2(h) AG9AG490IB: Bax-pAbIB: STAT1-pAb01/2121/223130 25 25 80 80 IB: BCL2-mAb 25 25 Figure 5-5. Jak2 mediates hydrogen peroxide-induced up regulation of Bax expression A) RASM-Control and RASM-DN cells were treated with 1 mM hydrogen peroxide for 0, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-Bax-pAb antibody (Top). The membrane was stripped and reprobed with anti-BCL2-mAb (Middle). Finally, the membrane was stripped and re-probed with anti-STAT1-pAb to demonstrate equal loading amongst all samples (Bottom). B) RASM-Control cells were pretreated with 100 M AG9 or 100 M AG490 for 16 h, followed by treatment with 1 mM hydrogen peroxide for 0, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-Bax-pAb (Top). The membrane was stripped and re-probed with anti-STAT1-pAb to demonstrate equal loading amongst all samples (Bottom). Shown is one of three representative experiments for each. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34550, Fig. 4 with permission from publisher. red staining, and only marginally increased green staining compared to control cells. This indicates that hydrogen peroxide-induced Jak2 activation promotes mitochondrial dysfunction in vascular smooth muscle cells. To quantify these results, we used

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55 morphometric scanning software to determine the ratio of green staining intensity to red staining intensity (Green:Red) (Fig. 5-7). A higher Green:Red staining intensity is indicative of a greater loss of mitochondrial transmembrane potential. The results indicate that the Green:Red staining intensity was significantly higher in RASM-Control Control DN Untreated2 hr H2O2Control DN Untreated2 hr H2O2 Figure 5-6. Jak2 is essential for hydrogen peroxide-induced mitochondrial membrane dysfunction RASM-Control and RASM-DN cells were grown on microscope slides and treated with 1 mM hydrogen peroxide for 0 or 2 h. Cells were stained with the MitoCapture reagent and visualized using a confocal microscope. Predominant red staining is indicative of healthy cells, while predominant green staining is indicative of apoptotic cells. Shown is one of three representative experiments. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34551, Fig. 5A with permission from publisher. cells treated with hydrogen peroxide than in similarly treated RASM-DN cells, showing that relative to RASM-Control cells, RASM-DN cells maintained their mitochondrial transmembrane potential. Jak2 is Required for Caspase-9 Cleavage During Oxidative Stress Caspase-9 is the primary initiator caspase of the intrinsic apoptosis pathway, and is involved in hydrogen peroxide-induced apoptosis. Caspase-9 is cleaved to its active form following disruption of mitochondrial integrity (163). We therefore examined caspase-9

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56 cleavage after hydrogen peroxide treatment in RASM-Control and RASM-DN cells (Fig. 5-8). The two cell types were treated with 1 mM hydrogen peroxide for 0, 1, 2, or 3 h, and cellular lysates were Western blotted with anti-CLEAVED-Caspase-9 antibody (Fig. 5-8, Top). The data show that in RASM-Control cells, hydrogen peroxide treatment causes accumulation of cleaved caspase-9. In contrast, little caspase-9 cleavage occurred 00.10.20.30.40.50.60.70.80.9Green:Red intensityControlControl+H2O2DNDN+H2O2 00.10.20.30.40.50.60.70.80.9Green:Red intensityControlControl+H2O2DNDN+H2O2 Figure 5-7. Pixel intensity of each of 4 photographs from each condition in Fig. 5-6 was determined, and intensity of green and red staining was determined for each. Data are presented as average ratio of green to red pixel intensity +/S.D. for each condition. *, p<0.01. Statistical analysis was performed using Students t test Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34451, Fig. 5B with permission from publisher. in RASM-DN cells. The membrane was stripped and re-probed with anti-STAT1 antibody to show equal protein loading amongst all samples (Fig. 5-8, Bottom). Jak2 tyrosine kinase activity is therefore required for cleavage and activation of caspase-9 in vascular smooth muscle cells during oxidative stress. Discussion Although Jak2 is traditionally considered a mediator of cytokine signaling, other ligands and stimuli can activate this signaling pathway (18, 36). Oxidative stress is one

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57 such stimulus; Jak2 is potently activated by hydrogen peroxide in a number of cell types, yet no physiological endpoint has been attributed to hydrogen peroxide-induced Jak2 activity (35, 36). Additionally, hydrogen peroxide induces apoptosis in vascular smooth muscle cells, yet few intracellular mediators of hydrogen peroxide-induced apoptosis have been identified (164). 1 mM H2O2(h) RASM-ControlRASM-DNIB: CLEAVED-Caspase-9 IB: STAT1-pAb 80 01/21231/22310 Non-specific bandCLEAVED-Caspase-9Non-specific band 1 mM H2O2(h) RASM-ControlRASM-DNIB: CLEAVED-Caspase-9 IB: STAT1-pAb 80 80 01/21231/22310 Non-specific bandCLEAVED-Caspase-9Non-specific band Figure 5-8. Jak2 is required for oxidative stress-induced caspase-9 cleavage RASM-Control and RASM-DN cells were treated with 1 mM hydrogen peroxide for 0, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-CLEAVED-Caspase-9-pAb to detect Caspase-9 activation (Top). The membrane was stripped and re-probed with anti-STAT1-pAb to demonstrate equal loading amongst all samples (Bottom). Shown is one of three representative experiments. Adapted from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34551, Fig. 5C with permission from publisher. Here, we report that Jak2 activation by oxidative stress in the form of hydrogen peroxide mediates apoptosis of vascular smooth muscle cells. We demonstrated fragmentation of genomic DNA, a characteristic of apoptosis, in control rat aortic smooth muscle cells treated with hydrogen peroxide (RASM-Control), but that fragmentation was non-existent in the same cells expressing a Jak2 dominant negative protein (RASM-DN), or in RASM-Control cells treated with the Jak2 inhibitor AG490. We observed a significant decrease in the percentage of cells undergoing apoptosis in response to hydrogen peroxide in the RASM-DN cells compared to RASM-Control cells. Moreover, pretreatment with AG490 reduced the percentage of cells undergoing hydrogen peroxide

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58 induced apoptosis nearly to the level of untreated cells, in both cell types, again suggesting a critical role for Jak2 in apoptosis. Finally, we provided evidence of the mechanism by which this occurs. In RASM-Control cells, expression of the pro-apoptotic Bax protein was rapidly induced. There was no such Bax induction in the RASM-DN cells. Furthermore, in RASM-Control cells, AG490 attenuated hydrogen peroxide-mediated Bax induction. This is the first evidence that Jak2 mediates Bax protein expression in response to hydrogen peroxide. During apoptosis, Bax is largely responsible for loss of mitochondrial transmembrane potential and subsequent increase in mitochondrial membrane permeability. This mitochondrial dysfunction allows translocation of macromolecules such as cytochrome c from the inner mitochondrial membrane to the cytosol, leading to cleavage and activation of caspase-9. We found that mitochondrial membrane integrity was compromised and caspase-9 was cleaved in RASM-Control cells, but not in RASM-DN cells, indicating an essential role for Jak2 in these events. This report has therefore identified apoptosis as a physiological endpoint of Jak2 activation by hydrogen peroxide. Moreover, this work shows that Jak2 is a novel mediator of hydrogen peroxide-induced apoptosis in vascular smooth muscle cells. These results could have profound consequences for the treatment of a number of vascular diseases in which oxidative stress-mediated cell death plays a prominent role. As such, this work identifies Jak2 as a potential therapeutic target in vascular diseases associated with oxidative stress. Atherosclerosis is one such disease. During the development of an atherosclerotic plaque, a fibrous cap forms over the plaque. Vascular smooth muscle cells that have migrated from the medial layer of the blood vessel are

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59 found within the fibrous cap. These cells are exposed to large amounts of oxidative stress derived largely from circulating macrophages. This oxidative stress can cause apoptosis of the vascular smooth muscle within the fibrous cap, leading to cap weakening, and accelerating the time to plaque rupture. If Jak2 plays a role in oxidative stress-induced apoptosis in vivo during atherosclerosis, inhibition of Jak2 could stabilize the plaque. Since Jak2 knockout mice die embryonically, investigating the role that Jak2 plays during atherosclerosis is difficult. One possibility is to use vascular smooth muscle cell restricted expression of the Jak2 dominant negative protein to study the in vivo role of Jak2. Mice expressing Jak2 only in vascular smooth muscle cells could then be crossed with apolipoprotein deficient mice, which develop atherosclerosis when fed a high fat diet, to examine the role of Jak2 during atherosclerosis. Interestingly, Jak2 can play either proor anti-apoptotic roles depending on the signaling system and apoptotic stimulus examined. Where, then, is the specificity of the response controlled? One possibility is that the specific STAT proteins that are activated by Jak2 determine whether Jak2 plays a proor anti-apoptotic role. For instance, activation of STAT3 is usually associated with inhibition of apoptosis, while STAT1 activation has been associated with induction of apoptosis. It is possible that STAT1 is the predominant STAT activated during oxidative stress. Studies using dominant negative mutants of the STAT1 and STAT3 proteins could be used to determine which STAT protein is responsible for the pro-apoptotic role for Jak2. Finally, whether Jak2 plays a role in oxidative stress-induced apoptosis in other cell types should be examined. Many cell types are exposed to high oxidative stress, especially during disease. These include endothelial cells and cardiomyocytes. It will be

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60 interesting to determine if the pro-apoptotic role of Jak2 is restricted to vascular smooth muscle cells, or if it is a ubiquitous role.

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CHAPTER 6 Jak2 RESIDUES GLU 1024 AND ARG 1113 FORM HYDROGEN BONDS, AND ARE ESSENTIAL FOR Jak-STAT SIGNAL TRANSDUCTION Introduction Structure-function studies have identified several specific amino acid residues within Jak2 that are essential for its activation. For example, conversion of Lys 882 to Glu (K882E) within subdomain II rendered Jak2 catalytically inactive (104, 105). Similarly, conversion of Tyr 1007 to Phe (Y1007F) prevented ligand-mediated Jak2 activation (48). A double mutation of W1020G/E1024A within subdomain VIII not only inactivated Jak2, but also rendered the molecule dominant negative (106). This double mutant is of interest to our laboratory because we have shown that expression of it in cells inhibits angiotensin II-mediated Jak2 activation, Jak2/AT 1 receptor co-association, STAT1 tyrosine phosphorylation, and ligand-dependent gene transcription (38, 39). We recently showed that mutation of either Trp 1020 or Glu 1024 individually renders Jak2 catalytically inactive (107). We are interested in elucidating the requirement of these two amino acids for Jak2 kinase function. Recently, we showed that Trp 1020 forms a hydrogen bond with Glu 1046 that is critical to maintain the structural integrity of the Jak2 activation loop (107). The role that Glu 1024 plays in Jak2 kinase function, however, is not known. Here, we investigated the requirement for Glu 1024 for Jak2 kinase function using homology modeling of the Jak2 kinase domain and site-directed mutagenesis. Our data indicate that Glu 1024 forms an interaction with an arginine at position 1113, via two 61

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62 distinct hydrogen bonds, and is essential for angiotensin II-dependent activation of the Jak-STAT signaling pathway. Conversion of Arg 1113 to lysine, alanine, or glutamic acid renders Jak2 catalytically inactive. Consequently, this is the first report describing Arg 1113 as being essential for Jak2 kinase activity. Results Molecular Modeling Identified a Putative Interaction between Jak2 Residues Glu 1024 and Arg 1113 Previously, we showed that mutation of Glu 1024 to Ala rendered Jak2 catalytically inactive (107). To understand how Glu 1024 contributes to Jak2 kinase function, we generated a molecular model of the Jak2 kinase domain (Fig. 6-1). Arg 1113 2.51 Arg 1113 2.51 Glu 1024 Glu 1024 Figure 6-1. Molecular modeling of the Jak2 kinase domain suggested a putative interaction between Glu 1024 and Arg 1113 he model was designed using the program Swiss Model and was based on the known crystal structure of the kinase domain of the fibroblast growth factor tyrosine kinase receptor. Shown are bond distances in angstroms. The model was based on the known crystal structure of the basic fibroblast growth factor receptor. Our model indicated that Glu 1024 forms a critical interaction with Arg 1113, an amino acid that thus far has not been shown to be essential for Jak2 function. The

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63 model predicted that the activation loop of the Jak2 kinase domain is maintained in its proper conformation via critical interactions between the oxygen groups on the side chain of Glu 1024 and the terminal amino groups on the side chain of Arg 1113. Using the program HBPLUS Hydrogen Bond Calculator, we determined that these interactions were hydrogen bonds, based on the bond lengths (2.91 angstroms and 2.51 angstroms, respectively) between the atoms involved. In each bond, the amino group is the electron donor, while the oxygen group is the electron acceptor. Furthermore, our model suggested that permutation of Arg 1113 would render Jak2 catalytically inactive. Mutation of Jak2 Residue Glu 1024 or Arg 1113 Abolishes Jak2 Kinase Activity We tested the ability of a Jak2 protein containing point mutations at either Glu 1024 or Arg 1113 to autophosphorylate, to determine if indeed an interaction between these two amino acids, as predicted by our model, exists. For this, we transfected BSC-40 cells with 10 g of Jak2 cDNA containing E1024R, E1024D, R1113E, R1113A, or R1113K mutations. BSC-40 cells transfected with 5 g of wild type Jak2 cDNA served as a positive control, while cells transfected with empty plasmid (pRC) served as a negative control. Cells were lysed and protein was extracted. Protein extracts were immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-phosphotyrosine antibody to detect tyrosine phosphorylated Jak2. Previously, we showed that conversion of Glu 1024 to Ala (E1024A), a neutrally charged amino acid, rendered Jak2 catalytically inactive (107). Here, we show that conversion of Glu 1024 to Asp (E1024D), a negatively charged amino acid, or Arg (E1024R), a positively charged amino acid, similarly abolishes Jak2 tyrosine kinase activity (Fig. 7.2, Top). Likewise, conversion of Arg 1113 to Glu (R1113E), a negatively charged amino acid, Ala

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64 (R1113A), a neutrally charged amino acid, or Lys (R1113K), a positively charged amino acid, rendered Jak2 catalytically inactive (Fig. 2, Top). In addition, a double mutation whereby Glu 1024 was converted to Arg, and Arg 1113 was converted to Glu, thus effectively switching the positions of these two amino acids, also rendered Jak2 catalytically inactive (Fig. 6-2, Top). We confirmed expression of all transfected constructs by Western blotting the same membrane with anti-Jak2 antibody (Fig. 6-2, Bottom). These data show that both Glu 1024 and Arg 1113 are critical for the ability of Jak2 to autophosphorylate, thus supporting the prediction that these two amino acids form a critical interaction. IP: Jak2-pAbIB: Tyr(P)-mAbpRC-Jak2-E1024R/R1113EpRC-Jak2-R1113KpRC-Jak2-R1113ApRC-Jak2-R1113EpRC-Jak2-E1024DpRC-Jak2-E1024RpRC-Jak2-WTpRC IP: Jak2-pAbIB: Jak2-pAb 111111 IP: Jak2-pAbIB: Tyr(P)-mAbpRC-Jak2-E1024R/R1113EpRC-Jak2-R1113KpRC-Jak2-R1113ApRC-Jak2-R1113EpRC-Jak2-E1024DpRC-Jak2-E1024RpRC-Jak2-WTpRC IP: Jak2-pAbIB: Jak2-pAb 111111 Figure 6-2. Mutation of Glu 1024 or Arg 1113 abolishes the ability of Jak2 to autophosphorylate. BSC-40 cells were transfected with 10 g of the indicated plasmids and infected with vaccinia virus clone vTF7-3 to induce high-level protein expression. Lysates were prepared, immunoprecipitated with anti-Jak2-pAb, and Western blotted with anti-phosphotyrosine-mAb to assess the ability of Jak2 containing mutations to autophosphorylate (top). The nitrocellulose membranes used for Western blotting were stripped and reprobed with anti-Jak2-pAb to assess Jak2 protein expression (bottom). Shown is one of three independent experiments.

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65 Individual Mutations of W1020G or E1024A Render Jak2 Dominant Negative It is known that double mutation of W1020G/E1024A renders Jak2 dominant negative (106). This means that when these two amino acids are both mutated, the resulting mutant Jak2 can inhibit the ability of wild type Jak2 to autophosphorylate. We recently showed that mutation of either Trp 1020 or Glu 1024 individually renders Jak2 catalytically inactive (107). We therefore sought to determine if mutation of either of these amino acids individually would render Jak2 dominant negative, as well as catalytically inactive. For this, we transfected BSC-40 cells with either 5 g of wild type Jak2 cDNA alone, or with 5 g of Jak2 cDNA plus increasing amounts of mutant Jak2 cDNAs. If the mutation tested does render Jak2 dominant negative, then upon co-transfection with increasing amounts of mutant cDNA, the ability of the wild type Jak2 to autophosphorylate will be inhibited. Addition of wild type plasmid alone results in increased Jak2 tyrosine phosphorylation levels (Fig. 6-3A, Top, lane 2 vs. lane 1). Addition of increasing amounts of the previously characterized Jak2 dominant negative mutant (W1020G/E1024A) inhibits wild type phosphorylation (lanes 3 and 4). Mutation at either Trp 1020 (W1020G) or at Glu 1024 (E1024A) alone is also sufficient to render Jak2 dominant negative in a dose-dependent manner (lanes 5-8). It is clear, though, that the dominant negative that results from mutation of W1020G or E1024A alone is not nearly as potent an inhibitor as the double W1020G/E1024A mutation. For instance, when 5 g of wild type Jak2 was co-transfected with 5 g of the Jak2-W1020G or the Jak2-E1024A mutant, we observed a diminished, but existent degree of Jak2 autophosphorylation. In comparison, when 5 g of wild type Jak2 was co-transfected with 5 g of Jak2 containing the W1020G/E1024A double mutation, the ability of wild

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66 type Jak2 to autophosphorylate was almost completely lost. Therefore, individual mutations at either Trp 1020 or Glu 1024 can render Jak2 both catalytically inactive and dominant negative. The individual mutants, though, do not show as strong a dominant negative character as the double Jak2-W1020G/E1024A mutant. We next tested the ability of the R1113E mutant to render Jak2 dominant negative. Again, we transfected BSC-40 cells with either 5g of wild type Jak2 cDNA alone, or with increasing amounts of the R1113E mutant plasmid. The results show that even when 5g of wild type Jak2 cDNA is co-transfected with 15g of Jak2 containing the R1113E mutation, wild type Jak2 is able to autophosphorylate (Fig. 6-3B). Therefore, the data suggest that mutation of Arg 1113, while disrupting Jak2 kinase function, does not render a dominant negative phenotype. Jak2-R1113E is Unable to Become Tyrosine Phosphorylated by Angiotensin II The data in Figs. 6-2 and 6-3 were generated using ligand-independent experimental conditions. To determine whether Arg 1113 is critical for proper Jak2 function in a ligand-dependent signaling system, we used COS-7 cells to study the role that Arg 1113 plays in angiotensin II-induced activation of the Jak-STAT signaling pathway. We first tested the ability of angiotensin II to activate Jak2 containing the R1113E point mutation. For this, COS-7 cells were transfected with g of AT 1 receptor, and either 10 g of wild type Jak2 cDNA or 12.5 g of Jak2-R1113E cDNA. The cells were treated with 100 nM angiotensin II for either 0, 5, or 10 min. Cells were lysed and protein was extracted. Protein extracts were immunoprecipitated with anti-phosphotyrosine antibody and immunoblotted with anti-Jak2 antibody to detect tyrosine phosphorylated Jak2. The results show that in response to angiotensin II, wild type Jak2

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67 becomes increasingly tyrosine phosphorylated over the time course, while Jak2-R1113E is unable to become activated in response to ligand treatment (Fig. 6-4). pRC-Jak2-WT (g)pRC-Jak2-WT (g)A 00505551551555515pRC-Jak2-WTpRC-Jak2-W1020G/E1024ApRC-Jak2-W1020G/E1024ApRC-Jak2-W1020GpRC-Jak2-W1020GpRC-Jak2-E1024ApRC-Jak2-E1024AIP: Jak2-pAbIB: Tyr(P)-mAbIP: Jak2-pAbIB: Jak2-pAbpRC-Jak2-mutant (g) 111111 550050515 IP: Jak2-pAbIB: Tyr(P)-mAb55B 111pRC-Jak2-R1113E (g) pRC-Jak2-WT (g)pRC-Jak2-WT (g)A 00505551551555515pRC-Jak2-WTpRC-Jak2-W1020G/E1024ApRC-Jak2-W1020G/E1024ApRC-Jak2-W1020GpRC-Jak2-W1020GpRC-Jak2-E1024ApRC-Jak2-E1024AIP: Jak2-pAbIB: Tyr(P)-mAbIP: Jak2-pAbIB: Jak2-pAbpRC-Jak2-mutant (g) 111111 550050515 IP: Jak2-pAbIB: Tyr(P)-mAb55B 111pRC-Jak2-R1113E (g) Figure 6-3. The Jak2-W1020G and E1024R mutations render Jak2 dominant negative BSC-40 cells were transfected with the indicated plasmids and infected with vaccinia virus clone vTF7-3. A) Lysates were prepared, immunoprecipitated with anti-Jak2-pAb, and Western blotted with anti-phosphotyrosine-mAb to assess the ability of Jak2-W1020G or Jak2-E1024A to act as a dominant negative (top). The membrane was stripped and reprobed with anti-Jak2-pAb to assess Jak2 protein expression in each sample (bottom). B) Using the same experimental paradigm, the ability of Jak2-R1113E was tested for its ability to act as a dominant negative. Shown is one of two independent experiments for each.

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68 Ang II (min) 0 5 10 0 5 10 Jak2-WT Jak2-R1113EIP: aTyr(P)-mAbIB: aJak2-pAb111 Ang II (min) 0 5 10 0 5 10 Jak2-WT Jak2-R1113EIP: aTyr(P)-mAbIB: aJak2-pAb111 Figure 6-4. Jak2-R1113E mutant cannot become tyrosine phosphorylated in response to angiotensin II COS-7 cells were transfected with 10 g of AT 1 receptor and either 10 g of wild type Jak2 or 12.5 g Jak2-R1113E cDNA. The cells were treated with 100 nM angiotensin II for the indicated times and lysates were prepared to assess Jak2 tyrosine phosphorylation. Lysates were immunoprecipitated with anti-Tyr(P)-mAb and Western blotted with anti-Jak2-pAb. Shown is one of three independent experiments. AngII (min) 0 3 6 0 3 6 Jak2-WT Jak2-R1113EIP: -mAbIB: Jak2-pAb 111AngII (min) 0 3 6 0 3 6 Jak2-WT Jak2-R1113EIP: -mAbIB: Jak2-pAb 111 Jak2-WT Jak2-R1113EIP: -mAbIB: Jak2-pAb 111 Figure 6-5. Angiotensin II-dependent Jak2/AT 1 receptor co-association does not occur in cells expressing Jak2-R1113E COS-7 cells were transfected with HA-tagged AT 1 receptor and either wild type Jak2 or Jak2-R1113E cDNA. The cells were treated with 100 nM angiotensin II for the indicated times and lysates were prepared to assess Jak2/AT 1 receptor co-association. Lysates were immunoprecipitated with anti-HA-mAb and Western blotted with anti-Jak2-pAb. Shown is one of three independent experiments. Following angiotensin II-induced Jak2 tyrosine phosphorylation, Jak2 associates with the AT 1 receptor (18). To determine if Jak2 containing the R1113E mutation could associate with the AT 1 receptor, we transfected COS-7 cells similar to those described above. The cells were then treated with 100 nM angiotensin II for either 0, 3, or 6 min, lysed, and protein was extracted. Protein lysates were immunoprecipitated with anti-HA antibody to immunoprecipitate the HA-tagged AT 1 receptor, and immunoblotted with anti-Jak2 antibody to detect Jak2/AT 1 receptor co-association (Fig. 6-5). The results show that in response to angiotensin II, wild type Jak2 rapidly associates with the AT 1

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69 receptor, while Jak2-R1113E is unable to do so, thus demonstrating that Arg 1113 is also essential for Jak2/AT 1 receptor co-association. Jak2-R1113E is Unable to Tyrosine Phosphorylate STATs in Response to Angiotensin II Once Jak2 associates with the AT 1 receptor in response to angiotensin II, STAT proteins are recruited to the Jak2/AT 1 receptor complex (38). Jak2 then tyrosine phosphorylates these receptor-bound STATs (18). To examine angiotensin II-dependent STAT activation in cells expressing Jak2-R1113E, we again transfected COS-7 cells similar to those described above. The cells were then treated with 100 nM angiotensin II for either 0, 5, or 10 min, lysed, and protein was extracted. The protein lysates were immunoprecipitated with anti-STAT1 antibody and immunoblotted with anti-Jak2 antibody to detect STAT1/Jak2 co-association (Fig. 6-6, Top). We show that in cells expressing wild type Jak2, angiotensin II induced STAT1/Jak2 co-association, whereas in cells expressing Jak2-R1113E, no STAT1/Jak2 co-association was seen in response to angiotensin II. We stripped the same membrane and reprobed it with anti-phosphotyrosine antibody to detect tyrosine phosphorylated STAT1 protein (Fig. 6-6, Middle). The results show that in cells expressing wild type Jak2, angiotensin II induces marked STAT1 tyrosine phosphorylation, whereas in cells expressing Jak2-R1113E, STAT1 does not become tyrosine phosphorylated in response to angiotensin II. Finally, we stripped the same membrane and reprobed it with anti-STAT1 antibody to demonstrate equal sample loading (Fig. 6-6, Bottom). Collectively, the data in Fig. 6-6 demonstrate that in cells expressing Jak2-R1113E, angiotensin II-dependent activation of STAT1 is disrupted, as measured by reduced STAT1/Jak2 co-association and STAT1 tyrosine phosphorylation.

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70 AngII (min) 00510510IP: STAT1-pAbIB: Jak2-pAb111 IP: STAT1-pAbIB: STAT1-pAb79 IP: STAT1-pAbIB: Tyr(P)-mAb79 Jak2-WTJak2-R1113E AngII (min) 00510510IP: STAT1-pAbIB: Jak2-pAb111 IP: STAT1-pAbIB: STAT1-pAb79 IP: STAT1-pAbIB: Tyr(P)-mAb79 Jak2-WTJak2-R1113E Figure 6-6. Jak2-R1113E is unable to activate STAT1 in response to angiotensin II Transfected COS-7 cells were treated with angiotensin II for the indicated times and lysates were prepared. Lysates were immunoprecipitated with anti-STAT1-pAb and Western blotted with anti-Jak2-pAb to assess Jak2/STAT1 co-association (Top). The membrane was stripped and reprobed with anti-Tyr(P)-mAb to assess STAT1 tyrosine phosphorylation (Middle). The membrane was again stripped, and then reprobed with anti-STAT1-pAb to demonstrate equal sample loading (Bottom). Shown is one of three independent experiments. Jak2-R1113E is unable to mediate angiotensin II-dependent gene expression We tested the ability of cells expressing the Jak2-R1113E mutant to activate STAT-mediated gene transcription. For this, COS-7 cells were transfected with 10 g of AT 1 receptor, 10 g of a luciferase reporter plasmid encoding a Stat1-binding sis-inducible element, and either 10 g of wild type Jak2 cDNA or 12.5 g of Jak2-R1113E cDNA. The luciferase reporter plasmid contains a tandem repeat of a minimal DNA enhancer element, the thymidine kinase TATA-containing promoter, and the firefly luciferase cDNA. Each copy of the DNA enhancer contains a STAT1-inducible SIE element, a serum response element, and an AP-1 binding site. Others and we previously showed that this plasmid is a good indicator of Jak-STAT-mediated gene transcription (108, 126). The cells were serum-starved for 24 h, and then treated with 100 nM angiotensin II for 0, 4, 8, or 12 h. We show that in cells expressing wild type Jak2,

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71 angiotensin II treatment produced a rapid and transient increase in Jak-STAT mediated gene expression that peaked at 4-8 h after angiotensin II treatment (Fig. 6-7). This response was greatly attenuated in cells expressing the Jak2-R1113E plasmid. Thus, these data show that Arg 1113 is also critical for angiotensin II-induced STAT-mediated gene transcription. An g II ( hrs ) 024681012Luciferase activity: fold change overunstimulated 14 0.51.01.52.02.53.03.5 Jak2-WT Jak2-R1113E ***An g II ( hrs ) 024681012Luciferase activity: fold change overunstimulated 14 0.51.01.52.02.53.03.5 Jak2-WT Jak2-R1113E Jak2-WT Jak2-R1113E *** Figure 6-7. Jak2-R1113E is unable to activate STAT-mediated gene transcription in response to angiotensin II. COS-7 cells were transfected with 10 g of AT 1 receptor, 10 g of a luciferase reporter plasmid encoding a STAT1-binding sis-inducible element, and either 10 g of wild type Jak2 or 12.5 g of Jak2-R1113E cDNA. The cells were treated with 100 nM angiotensin II for the indicated times and detergent extracts were prepared. Luciferase activity was measured using the Reporter Lysis Buffer System (Promega). Shown is one of two independent experiments. Values are expressed as the mean +/SD. n=4 for each time point, p<0.05, ** p<0.005 (Students t test). Arg 1113 is conserved in different Jak kinase family members and among species expressing Jak2 The data in Figs. 6-4 through 6-7 show that Arg 1113 is essential for ligand dependent Jak2-mediated signaling. We therefore wanted to determine if this amino acid

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72 was highly conserved throughout the evolutionary history of Jak2. Comparison of the amino acid sequence of the different Janus kinase family members, and the amino acid sequence of Jak2 from various species expressing the gene, indicates that Arg 1113 is a highly conserved residue (Fig. 6-8). The highly conserved nature of Arg 1113 further indicates a critical role for this amino acid in Jak2 function. Mouse Jak2 1102 M T E C W N N N V S Q R P S F R D L S F G WMouse Jak1 1102 M R C C W E F Q P S N R T T F Q N L I E G FMouse Jak3 1102 M Q L C W A P S P H D R P A F G T L S P Q LMouse Tyk2 1102 M Q N C W E T E A S F R P T F Q N L V P I L Mouse Jak2 M T E C W N N N V S Q R P S F R D L S F G WRat Jak2M T E C W N N N V N Q R P S F R D L S L R VPig Jak2 M T E C W N N N V N Q R P S F R D L A L R VHuman Jak2 M T E C W N N N V N Q R P S F R D L A L R VZebra Fish Jak2 M Q E C W D N D P S L R P N F K E L A L R VPuffer Fish Jak2 M E Q C W D N D P Y L R P S F K E L A L S IChicken Jak2 M L S C W A F A P S A R P T F T E L A A R V Mouse Jak2 1102 M T E C W N N N V S Q R P S F R D L S F G WMouse Jak1 1102 M R C C W E F Q P S N R T T F Q N L I E G FMouse Jak3 1102 M Q L C W A P S P H D R P A F G T L S P Q LMouse Tyk2 1102 M Q N C W E T E A S F R P T F Q N L V P I L Mouse Jak2 M T E C W N N N V S Q R P S F R D L S F G WRat Jak2M T E C W N N N V N Q R P S F R D L S L R VPig Jak2 M T E C W N N N V N Q R P S F R D L A L R VHuman Jak2 M T E C W N N N V N Q R P S F R D L A L R VZebra Fish Jak2 M Q E C W D N D P S L R P N F K E L A L R VPuffer Fish Jak2 M E Q C W D N D P Y L R P S F K E L A L S IChicken Jak2 M L S C W A F A P S A R P T F T E L A A R V Figure 6-8. Arg 1113 is conserved in Jak2 among species and in different Jak family members Comparison of the amino acid sequence of the different murine Janus kinase family members, and the amino acid sequence of Jak2 from various species, indicates that Arg 1113 is a highly conserved residue throughout evolution. Discussion We provided evidence that Glu 1024 forms two distinct hydrogen bonds with Arg 1113 that are critical for Jak2 tyrosine kinase activity. As such, these are the first data describing Arg 1113 as being critical for Jak2 kinase function. Molecular modeling studies identified a putative interaction between Glu 1024 and Arg 1113. Using the program HBPLUS Hydrogen Bond Calculator, we determined that this interaction consisted of two distinct hydrogen bonds between the oxygen groups on the side chain of

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73 Glu 1024 and the terminal amino groups on the side chain of Arg 1113. Using site-directed mutagenesis, we showed that E1024R, E1024D, R1113K, R1113A, and R1113E point mutations all rendered Jak2 catalytically inactive. Converting Glu 1024 to Arg (E1024R), and Arg 1113 to either Ala (R1113A) or Glu (R1113E) changed the charge on the amino acid at those positions and thus likely disrupted the ionic interaction between amino acids 1024 and 1113. While the E1024D and R1113K substitutions maintained the charge at those positions, both of these mutations shortened the side chains of the amino acids at their respective positions. Aspartic acid has a side chain that is one carbon shorter than that of glutamic acid; lysine has a side chain that is one amino group shorter than that of arginine. Thus, despite the conservative nature of the R1113K and E1024D mutations, the kinase function of Jak2 was lost. Consequently, we believe that the shorter side chains were not sufficiently long to maintain the interaction between these two residues, indicating that proper bond length is of critical importance to maintaining this interaction. Previous studies showed that double mutation of Trp 1020 to Gly and Glu 1024 to Ala (W1020G/E1024A) rendered Jak2 dominant negative (106). Here, we tested whether individual mutation at either Trp 1020 or Glu 1024 alone would not only render Jak2 catalytically inactive, but also dominant negative. We showed that substitution mutations at either position 1020 (W1020G) or position 1024 (E1024A) rendered Jak2 dominant negative. Interestingly, these individual mutants were not able to act as potent a dominant negative as the double Jak2-W1020G/E1024A mutant. We believe, therefore, that the strong dominant negative phenotype displayed by the double mutant is due to an additive dominant negative effect of the two individual mutations. This is a discovery of

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74 two new Jak2 dominant negative molecules, and these mutants may be useful research tools for studying the function of Jak2. In fact, these two dominant negative mutants may offer an advantage over the double W1020G/E1024A dominant negative mutant. Since their dominant negative character is not as strong as that of the double mutant, their expression in cells could be used as a knockdown approach to studying Jak2 function. This could be particularly useful for in vivo models, where Jak2 activation is critical to life. We next used COS-7 cells to examine the consequences of the Jak2-R1113E mutation in a ligand-dependent signaling system. These cells express very low levels of Jak2 and no AT 1 receptor. When Jak-STAT signaling is reconstituted in these cells they are a reliable, simple-to-use system for determining the functional consequences of Jak2 mutations. Using these cells, we showed that Jak2 containing an R1113E mutation is unable to become activated in response to angiotensin II. Furthermore, we showed that expression of Jak2-R1113E cDNA in cells prevented angiotensin II-dependent Jak2/AT 1 receptor co-association, Jak2/STAT1 co-association, STAT1 tyrosine phosphorylation, and STAT-mediated gene transcription. Together, these data show the critical importance of Arg 1113 to Jak2 function. Further supporting the indispensability of Arg 1113, is the conserved nature of this amino acid amongst Jak family members and throughout Jak2 from various species. We believe that the identification of this and other critical amino acid interactions within the Jak2 kinase domain could provide targets for drug design aimed at disabling Jak2 kinase function.

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CHAPTER 7 IDENTIFICATION OF A NOVEL Jak2 INHIBITOR Introduction The Jak2 protein is important in both physiology and pathophysiology, as it plays prominent roles in embryonic development, cell signaling, and in cancer and heart disease (61, 92, 102, 103,). Two impediments to better understanding Jak2 function are 1) the lack of an adult knockout animal model and 2) the lack of a Jak2-specific pharmacological inhibitor (102, 103, 130). Jak2 knockout mice die embyronically, around E10.5 because of a lack of erythropoesis (102, 103). This work showed the critical role that Jak2 plays in embryonic development and cytokine signal transduction, but also raised a barrier to research on elucidating the mechanisms of Jak2 cellular function. Without an adult Jak2 knockout animal available, studying the function of Jak2 in adult physiology and pathophysiology has been complicated. Furthermore, there is no Jak2-specific pharmacological inhibitor. AG490 is a commercially available Jak2 inhibitor, and while it has been instrumental in elucidating some functions of Jak2 and in identifying Jak2 as a therapeutic target, it suffers from a general lack of specificity. In fact, in Chapter 5, we showed that Jak2 nonspecifically inhibits angiotensin II-mediated ERK2 activation. Because of these problems, there are caveats in all research relying solely on AG490 to study Jak2 kinase function. Clearly, identification of a novel Jak2 inhibitor could aid research efforts. For these reasons, we sought to identify a potential novel Jak2 inhibitor. We first used homology modeling of the Jak2 kinase domain to identify exposed pockets on the 75

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76 surface of the protein. We then used a high-throughput program called DOCK, to predict the ability of 6,451 small molecules to interact with a solvent accessible pocket that is adjacent to the activation loop of Jak2, namely, Pocket 36. The compounds were scored in silico on their potential ability to interact with Pocket 36. We ordered the top seven scoring compounds, and tested their ability to inhibit Jak2 tyrosine kinase function. One of these, Compound 7, was found to be a potent inhibitor of Jak2. Results Homology Modeling and Target Pocket Identification We used the homology model of the Jak2 kinase domain described in Chapter 6 to identify pockets within the Jak2 kinase domain that could interact with potential small-molecule inhibitors of Jak2. The program SPHGEN identified 49 pockets within the Jak2 kinase domain. The pockets were designated based on their chemical and shape characteristics. We chose the pocket designated as Pocket as a target based on its proximity to the Jak2 activation loop and its large size, which makes it accessible to small-molecules (Fig. 7-1). Database Screening to Identify Potential Small-Molecule Inhibitors of Jak2 Using Pocket 36 as the target, we used the program DOCK to screen a National Cancer Institute database of known chemical structures for their ability to interact with Pocket 36. We used the program to screen 6,415 compounds of the over 140,000 compounds in the database. The program attempted, in silico, to fit each compound into Pocket 36 in 100 different orientations for each compound tested. The compounds were scored on their ability to fit into Pocket 36 and on their ability to interact chemically with Pocket 36. We ordered the seven top-scoring compounds for further testing. These

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77 Pocket 36 Pocket 36 Figure 7-1. SPHGEN identified 49 exposed pockets on the surface of the Jak2 protein Because of its large size, Pocket 36 was chosen as a target pocket for in silico compound screening. compounds were provided to us, free of charge, by the National Cancer Institute, through their Developmental Therapeutics Program. These non-proprietary compounds are offered to the extramural research community for the development of treatments for cancer, AIDS, and opportunistic infections afflicting patients with cancer or AIDS. These were designated Compounds 1-7 (Table 7-1). Table 7-1. Top 7 scoring compounds 558.0 Cyclohexane-1,2,3,4,5,6-hexabromoC6H6Br6 7908 125.0 Superacil C5H7N3O 7893 164.0 4,6-Dichloro-5-aminopyrimidine C4H3Cl2N3 7851 734.0 Acid Black S C36H25N5O6S2 2Na 7830 423.0 Chlorphenol Red C19H12Cl2O5S 7828 625.0 Acid Green 25 C28H22N2O8S2 2Na 7795 71.0 2-propenamide C3H5NO 7785 Mol. Weight Name Formula Cmpd#NSC # 1524367 558.0 Cyclohexane-1,2,3,4,5,6-hexabromoC6H6Br6 7908 125.0 Superacil C5H7N3O 7893 164.0 4,6-Dichloro-5-aminopyrimidine C4H3Cl2N3 7851 734.0 Acid Black S C36H25N5O6S2 2Na 7830 423.0 Chlorphenol Red C19H12Cl2O5S 7828 625.0 Acid Green 25 C28H22N2O8S2 2Na 7795 71.0 2-propenamide C3H5NO 7785 Mol. Weight Name Formula Cmpd#NSC # 1524367

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78 Compound 7 Inhibits Jak2 Autophosphorylation To test the ability of each of the seven compounds to inhibit Jak2 tyrosine kinase activity, we used the vaccinia virus transfection/infection protocol. Briefly, BSC-40 cells, a vaccinia virus permissive cell line, are transfected with an expression vector encoding the wild type murine Jak2 cDNA under the control of the T7 promoter. The cells are then infected with a vaccinia virus that produces T7 RNA polymerase. This results in high level Jak2 expression and subsequent tyrosine autophosphorylation independent of exogenous ligand addition. After the initial 1 h vaccinia virus infection, the cells were switched to serum containing media and each compound was added at a final concentration of 100 M and incubated overnight. Sixteen h later after the addition of the inhibitors, cellular lysates were prepared and immunoprecipitated with anti-Jak2 antibody and then immunoblotted with anti-phosphotyrosine antibody to detect tyrosine phosphorylated Jak2 (Fig. 7-2A, Top). The results showed that Compound 7 was the only compound to inhibit Jak2 tyrosine autophosphorylation. The membrane was then stripped and re-probed with anti-Jak2 antibody to demonstrate equal protein expression amongst all samples (Fig. 7-2A, Bottom). The identity and structure of compound 7 is shown (Fig. 7-2B). Cyclohexane-1,2,3,4,5,6-hexabromois a single aromatic ring structure with a halide on each carbon. It has a molecular weight of 125 daltons. Compound 7 Inhibits Jak2 Autophosphorylation in a Time-Dependent Manner We next wanted to determine whether Compound 7 could inhibit Jak2 tyrosine autophosphorylation in a time-dependent manner. For this, BSC-40 cells were transfected/infected as described. Before cell lysis, 100 M of Compound 7 was applied to the cells for 0, 1, 4, or 16 h. Cellular lysates were then prepared, immunoprecipitated

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79 WT Jak2WT Jak2+Cmpd 1WT Jak2+Cmpd 2WT Jak2+Cmpd 3WT Jak2+Cmpd 4WT Jak2+Cmpd 5WT Jak2+Cmpd 6WT Jak2+Cmpd 7IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111B BrBrBrBrBrBrCyclohexane-1,2,3,4,5,6-hexabromo-A WT Jak2WT Jak2+Cmpd 1WT Jak2+Cmpd 2WT Jak2+Cmpd 3WT Jak2+Cmpd 4WT Jak2+Cmpd 5WT Jak2+Cmpd 6WT Jak2+Cmpd 7IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111B BrBrBrBrBrBrCyclohexane-1,2,3,4,5,6-hexabromo-A Figure 7-2. Compound 7 inhibits Jak2 autophosphorylation A) The 7 compounds that received the highest score from the DOCK program for their ability to interact with Pocket 36 within the Jak2 kinase domain were tested for their ability to inhibit Jak2 autophosphorylation. BSC-40 cells were transfected with 5 g of Jak2 cDNA, and then infected with 1 MOI of vaccinia virus for 16 h to drive high-level expression of Jak2 and subsequent Jak2 autophosphorylation. During viral infection, the 7 compounds were incubated with the cells at a concentration of 100 M each. Cell lysates were immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-phosphotyrosine antibody to detect Jak2 tyrosine phosphorylation (Top). The membrane was stripped and re-probed with anti-Jak2 antibody to demonstrate equal Jak2 expression amongst all samples (Bottom). B) Shown is the structure and identity of Compound 7. Shown is one of three independent experiments. with anti-Jak2 antibody, and immunoblotted with anti-phosphotyrosine antibody to measure tyrosine phosphorylated Jak2 levels (Fig. 7-3, Top). The results showed that 1

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80 or 4 h treatment with Compound 7 was sufficient to block ~75% of the tyrosine autophosphorylation of Jak2. However, 16 h treatment with 50 M Compound 7 resulted in a virtual elimination of all Jak2 tyrosine autophosphorylation. The membrane was stripped and re-probed with anti-Jak2 antibody to demonstrate equal protein expression amongst all samples (Fig. 7-3, Bottom). Collectively, the data show that incubation of Compound 7 does inhibit Jak2 tyrosine autophosphorylation in a time-dependent manner; treatment of cells with 50 M Compound 7 for 1 or 4 h was sufficient to partially inhibit Jak2 tyrosine kinase autophosphorylation, while treatment of cells for 16 h resulted in near total elimination of Jak2 tyrosine autophosphorylation. WT-Jak2WT-Jak2+50 M Cmpd 7 (16 h)WT-Jak2+50 M Cmpd 7 (4 h)WT-Jak2+50 M Cmpd 7 (1 h)IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111 WT-Jak2WT-Jak2+50 M Cmpd 7 (16 h)WT-Jak2+50 M Cmpd 7 (4 h)WT-Jak2+50 M Cmpd 7 (1 h)IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111 Figure 7-3. Maximal Jak2 inhibition requires 16 h of incubation with Compound 7 Compound 7 was incubated with the cells for 0, 1, 4, or 16 h at a concentration of 50 M during infection. Cell lysates were immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-phosphotyrosine antibody to detect Jak2 tyrosine phosphorylation (Top). The membrane was stripped and re-probed with anti-Jak2 antibody to demonstrate equal Jak2 expression amongst all samples (Bottom). Shown is one of two representative experiments.

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81 Compound 7 Inhibits Jak2 Autophosphorylation in a Dose-Dependent Manner We next tested the ability of Compound 7 to inhibit Jak2 autophosphorylation in a dose-dependent manner. For this, we again used the BSC-40 cell transfection/infection protocol. The cells were treated for 16 h with Compound 7 at doses of 0, 1, 10, 50, 100, 250, or 500 M. The next morning, soluble protein lysates were immunoprecipitated with anti-Jak2 antibody and then immunoblotted with anti-phosphotyrosine antibody to measure the tyrosine phosphorylation levels of Jak2 (Fig. 7-4, Top). The results showed that Compound 7 inhibited Jak2 tyrosine autophosphorylation in a dose-dependent manner with maximal inhibition occurring at 50 M. The membrane was then stripped and re-probed with anti-Jak2 antibody to demonstrate equal protein expression amongst samples (Fig. 7-4, Bottom). WT-Jak2WT-Jak2+ 1M Cmpd7WT-Jak2+DMSOWT-Jak2+10 M Cmpd7WT-Jak2+50 M Cmpd 7WT-Jak2+100M Cmpd 7WT-Jak2+250 M Cmpd 7WT-Jak2+500 M Cmpd 7IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111 WT-Jak2WT-Jak2+ 1M Cmpd7WT-Jak2+DMSOWT-Jak2+10 M Cmpd7WT-Jak2+50 M Cmpd 7WT-Jak2+100M Cmpd 7WT-Jak2+250 M Cmpd 7WT-Jak2+500 M Cmpd 7IP=Jak2 pAbIB=Tyr(P) mAb 111 IP=Jak2 pAbIB=Jak2 pAb 111 Figure 7-4. Compound 7 inhibits Jak2 in a dose-dependent manner BSC-40 cells were again transfected/infected as described above. Compound 7 was incubated with the cells during infection at a dose of 0, 1, 10, 100, 250, or 500 M for 16 h. Cell lysates were immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-phosphotyrosine antibody to detect Jak2 tyrosine phosphorylation (Top). The membrane was stripped and re-probed with anti-Jak2 antibody to demonstrate equal Jak2 expression amongst all samples (Bottom). Shown is one of two representative experiments.

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82 Collectively, the data in Fig. 4 show that Compound 7 does in fact inhibit Jak2 autophosphorylation in a dose-dependent manner. The amount of material required to inhibit 50% of the Jak2 tyrosine autophosphorylation levels in this assay (IC 50 ) was in the low micromolar range. Additionally, 50 M Compound 7 was sufficient for maximal Jak2 inhibition. Compound 7 is Non-Cytotoxic at Concentrations that Maximally Inhibit Jak2 Tyrosine Autophosphorylation To determine whether Compound 7 was cytotoxic to the cultured cells, we treated BSC-40 cells with Compound 7 at doses of 0, 100, or 500 M for 16 h. The live cells were then stained with propidium iodide to determine whether Compound 7 was cytotoxic. Propidium iodide selectively stains necrotic cells and fluoresces red, but is excluded by the plasma membranes of healthy, intact cells. The results showed that cells treated with 100 M Compound 7 showed very little propidium iodide staining, akin to that of untreated cells (Fig. 7-5). In contrast, BSC-40 cells treated with 500 M Compound 7 did show increased propidium iodide staining, indicating that at a dose of 500 M Compound 7 is cytotoxic. Since the IC 50 of Compound 7 is estimated to be in the low micromolar range, and 50 M Compound 7 maximally inhibits Jak2 tyrosine kinase autophosphorylation, we conclude that the mechanism by which Compound 7 inhibits Jak2 tyrosine kinase autophosphorylation, at these concentrations, is independent of cellular cytotoxicity. Discussion Since its discovery in 1992, significant progress has been made in understanding the biochemical and cellular functions of Jak2 tyrosine kinase. Studies have shown essential roles for Jak2 in embryonic development, cell signaling, and the

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83 Bright Field Propidium Iodide UntreatedCmpd 7100 MCmpd7500 M UntreatedCmpd 7100 MCmpd7500 M Figure 7-5. Compound 7 is not cytotoxic at a dose of 100 M BSC-40 cells were grown on microscope slides and treated with 0, 100, or 500 M Compound 7 for 16 h. The live cells were then stained with 1 g/mL propidium iodide to determine whether Compound 7 was cytotoxic. The cells were visualized using confocal microscopy. Shown is one of two representative experiments. pathophysiology of heart disease and cancer (61, 92, 102, 103). Research on this protein, though, has been complicated by the lack of a Jak2-specific pharmacological inhibitor. AG490, a commercially available Jak2 inhibitor, also inhibits several other related tyrosine kinase signaling pathways (98-101). This work is significant for three fundamental reasons. First, we used homology modeling of the Jak2 kinase domain and high-throughput compound docking in silico to identify potential Jak2 inhibitors. We found that cyclohexane-1,2,3,4,5,6-hexabromo-, designated as Compound 7, potently inhibited Jak2 tyrosine autophosphorylation in cultured BSC-40 cells. Compound 7 inhibited Jak2 autophosphorylation in both a dose

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84 and time-dependent manner. Based on these autophosphorylation assays, it appears that a 16 h treatment with 1 M Compound 7 is sufficient to reduce Jak2 tyrosine autophosphorylation levels by about 50%, while 50 M Compound 7 eliminates virtually all detectable Jak2 tyrosine autophosphorylation. Furthermore, even at doses as high as 100 M, Compound 7 is not cytotoxic to cultured cells. As such, it inhibits Jak2 tyrosine autophosphorylation at concentrations that are well below its cytopathic threshold. Second, the results shown here using the DOCK program, demonstrate proof-of-principle in using in silico-based strategies for identifying biological interactions. Here, screening just 6,451 compounds for their ability to interact with one target pocket on the Jak2 kinase domain, we successfully used the DOCK program to identify a novel Jak2 inhibitor. We will therefore use the DOCK program for screening additional compounds for their ability to bind multiple targets within the Jak2 kinase domain. In fact, the library that we have available for screening contains over 140,000 compounds of known chemical structure. Furthermore, we identified 49 exposed pockets on the Jak2 kinase domain. By screening the entire library of compounds using multiple target pockets, we expect to identify several additional small molecule inhibitors of Jak2. Third, AG490 falls within the general class of tyrosine kinase inhibitors known as tyrphostins. The molecular structure of AG490 is known; it contains two aromatic ring structures linked by a spacer containing four carbons and an amide group. Compound 7 is noticeably different from AG490 in that it contains only a single aromatic ring without any spacers. As such, our work here suggests that Compound 7, with its single aromatic ring, could serve as a potential lead compound for future synthesis reactions with the hopes of identifying a specific Jak2 inhibitor.

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85 A final possibility for identifying additional small molecule inhibitors of Jak2 using database screening is to model the site where AG490 binds to the Jak2 kinase domain. We could then use this binding site as a target for database screening. This has the advantage of using an area that is known to bind a small molecule inhibitor of Jak2 as a target. This may allow us to identify Jak2 inhibitors that have the same site of action as AG490, but are more specific for Jak2 than AG490. Collectively, the work shown here has identified cyclohexane-1,2,3,4,5,6-hexabromoas a small molecule inhibitor of Jak2 tyrosine kinase. Because of the universal importance of Jak2 in mediating both the physiological and pathophysiological actions within animals, this compound, and potential derivatives of it, may have important therapeutic value.

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CHAPTER 8 CONCLUSIONS AND PERSPECTIVES The Jak2 tyrosine kinase protein was discovered in 1991. It was quickly identified as a key mediator of cytokine signaling. Since then, roles for Jak2 in mediating signaling through GPCRs and during cellular stress, including oxidative stress, have been identified. Despite this, study of Jak2 has been complicated for two reasons: 1) lack of an adult knockout animal and 2) lack of a specific Jak2 inhibitor. In these studies we used several strategies to circumvent these problems, and we significantly improved our understanding of Jak2 structure, function, and pharmacology. We used Jak2 -/cells to identify a novel role for Jak2 in angiotensin II-dependent inactivation of ERK2. We used cells expressing a Jak2 dominant negative mutant to identify Jak2 as an essential mediator of oxidative stress-induced apoptosis in vascular smooth muscle cells. Finally, we used homology modeling of the Jak2 kinase domain to identify an amino acid interaction within Jak2 that is critical for Jak2 function, and to identify a novel small molecule inhibitor of Jak2. Role of Jak2 in Angiotensin II-Dependent ERK2 Signaling Previous studies suggested that Jak2 was required for angiotensin II-dependent activation of ERK2. These studies, though, relied solely on the Jak2 inhibitor AG490 to determine this role for Jak2. While AG490 potently inhibits Jak2, it nonspecifically inhibits several other signaling pathways. For this reason, we used Jak2 -/cells to specifically study the role that Jak2 plays in angiotensin II-dependent ERK2 signaling. 86

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87 In Chapter 4, we showed that Jak2 is essential for inactivation of ERK2 after angiotensin II treatment. Moreover, we showed that the previously published results demonstrating that Jak2 is required for angiotensin II-dependent activation of ERK2, may be an artifact caused by using AG490 to study Jak2 function. These studies may open a new area of research that will further explore crosstalk between the Jak2 signaling pathway and the ERK signaling pathway. Future studies should further elucidate the role that Jak2 plays in the regulation of angiontensin II signaling. Ultimately, it will be interesting to determine the physiological importance of this novel role for Jak2 in vivo, where angiotensin II plays critical roles during cardiovascular disease. Role of Jak2 during Oxidative Stress It was discovered in 1998, that Jak2 is strongly activated in vascular smooth muscle cells by oxidative stress in the form of hydrogen peroxide. Since then, there has been little research into the physiological role that Jak2 plays during oxidative stress. In Chapter 5, we used expression of a Jak2 dominant negative mutant to show, for the first time, a physiological endpoint of Jak2 activation by hydrogen peroxide in vascular smooth muscle cells. We found that hydrogen peroxide resulted in apoptosis of control vascular smooth muscle cells, but failed to induce apoptosis in cells expressing a dominant negative Jak2, indicating that Jak2 activation is essential for oxidative stress-induced apoptosis of vascular smooth muscle cells. These results could have profound consequences on diseases where oxidative stress-induced apoptosis contributes to pathology. Atherosclerosis is one such disease. During atherosclerosis, circulating macrophages release high amounts of hydrogen peroxide on vascular smooth muscle cells within the fibrous cap of the atherosclerotic

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88 plaque. This can lead to apoptosis of the cells, and subsequent weakening of the fibrous cap. When the cap weakens, plaque rupture often occurs, which can result in thrombus formation, and subsequent heart attack or stroke. First, the role that Jak2 plays in oxidative stress in vivo must be determined. Jak2 Structure-Function Point mutations at both Trp 1020 and Glu 1024 render Jak2 dominant negative. As discussed above, we used this dominant negative mutant to determine the role of Jak2 during oxidative stress. We also used this dominant negative mutant to better understand the structure of Jak2. Previously, we showed that mutation of either Trp 1020 or Glu 1024 individually rendered Jak2 catalytically inactive. In Chapter 6, we showed that these individual point mutations also render Jak2 dominant negative. Moreover, we determined the reason that Glu 1024 is critical for Jak2 function. This amino acid forms two distinct hydrogen bonds Arg 1113. Critical amino acid interactions within the Jak2 kinase domain could be targets for drug design aimed at disabling Jak2 kinase function. Novel Jak2 inhibitors would be useful research tools and could possibly hold therapeutic potential. For this reason, the structure of the Jak2 protein should continue to be explored. Hopefully, in the future, the crystal structure of Jak2 will be determined. This will provide further insight into the Jak2 structure-function relationship, and could lead to design of Jak2 inhibitors. Identification of a Novel Jak2 Inhibitor A better understanding of Jak2 structure could lead to design of novel Jak2 inhibitors. In addition to pursuing a better understanding of Jak2 structure, we took a direct approach to identifying novel Jak2 inhibitors. We used homology modeling of the Jak2 kinase domain to identify target pockets for in silico compound docking. Using the

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89 DOCK program, we identified cyclohexane-1,2,3,4,5,6-hexabromo-, designated as Compound 7, as a novel Jak2 inhibitor. Compound 7 may prove to be a useful research tool for studying Jak2 function. With modification, Compound 7 could also be improved as a Jak2 inhibitor. Importantly, the identification of Compound 7 provides proof-of-principle that high throughput compound docking using a homology model of the Jak2 kinase domain can lead to the identification of novel Jak2 inhibitors. For this reason, additional compounds should be screened for their ability to interact with binding targets within the Jak2 kinase domain.

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BIOGRAPHICAL SKETCH Eric Sandberg was born on May 12, 1978 in Ft. Lauderdale, FL. He grew up in South Florida and attended Chaminade-Madonna College Preparatory, where he was first inspired to pursue a career in science by his high school Chemistry teacher, Marcia Colon. He earned his Bachelor of Science degree in microbiology and cell science at the University of Florida, in May 2000. During his undergraduate years, Eric conducted research in plant molecular biology, under the supervision of Dr. William B. Gurley and his graduate student, Shai Lawit. Eric began pursuing his Ph.D. in biomedical science at the University of Florida College of Medicine in August 2000, under the mentorship of Dr. Peter P. Sayeski. Eric plans to pursue postdoctoral work in cancer biology. 106


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Jak2 TYROSINE KINASE: NEW INSIGHTS REGARDING STRUCTURE,
FUNCTION, AND PHARMACOLOGY














By

ERIC M. SANDBERG


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


2004
































Copyright 2004

by

Eric M. Sandberg


































This dissertation is dedicated to my parents, for their constant love and support.
















ACKNOWLEDGMENTS

I would like to thank, first and foremost, my adviser, Dr. Peter Sayeski. Dr.

Sayeski has an enthusiasm for science that is inspiring. His flexibility allowed me to

successfully pursue my own research interests, while his seemingly inborn mentoring

skills never let me flounder. I feel lucky to have been Dr. Sayeski's first graduate

student. He took the time to teach me research techniques, and also how to write

scientific papers, grantsmanship, and professionalism in science. He has been a mentor

and a friend.

Second, I want to thank my graduate committee. Drs. Mohan Raizada, Colin

Sumners, and Peggy Wallace have been very supportive; their technical input has been

extrememtly useful, and their encouragement has been invaluable.

Third, I want to thank all of the members of the Sayeski Lab, and everyone else

who trained and/or helped me along the way. Danielle Vonderlinden and Melissa Johns

kept the lab running smoothly, and their technical assistance significantly accelerated our

time to publication. Tiffany Wallace, Mike Godeny, Issam McDoom, and Dr. Xianyue

Ma have been both collaborators and friends. I also want to thank Drs. Michael Katovich

and Hideko Kasahara for serving on my examination committee, and Tim Vaught, for his

generous assistance with optical microscopy. Additionally, I want to thank Dr. David

Ostrov, who was an essential collaborator throughout my graduate studies. Without him,

our structure-function and inhibitor studies would not have been possible.









Finally, I want to thank my family and friends who supported me throughout

graduate school. In particular, my parents instilled in me a passion for learning and

science from an early age. With a mom who is a nurse, and a dad who drove his 1972

Datsun until it had precisely 176,000 miles on the odometer (so he could brag that he had

driven his car for 1 light second), it is no surprise that I pursued medical science as a

career. I also want to thank Mia DeBarros for her constant love, support, and technical

insight.




















TABLE OF CONTENTS

Page


ACKNOWLEDGMENT S .............. .................... iv


LI ST OF FIGURE S .............. .................... ix


AB STRAC T ................ .............. xi


CHAPTER


1 INTRODUCTION TO THE Jak-STAT PATHWAY ................. ........................1


Hi story of the Jak- ST AT Pathway ................. .......... ......... ...........
Initiation of Jak-STAT Signaling ................ ...................... ..................4
Physiology and Pharmacology of Jak2 ................. ........ ........ .......... ............
Target Genes and Regulation of Signal Transduction Pathways ..........................6
Regulation of Jak2 Signaling..........___..........__ ........._._ ...._.__.....7
Jak2 in Cancer ................. ...............10...
Jak2 in Cardiovascular Disease .....___.....__.___ .......____ ............1
Jak2 Pharmacology ........._........ .......___ _......_ ............ 1
Structure-Function Relationship of Jak2 ............__......__ ...._ .............16


2 EXPERIMENTAL METHODS .............. ...............23....


Cell Culture.................... ...................2
Vaccinia Virus Transfection/Infection .............. ...............23....
Immunoprecipitation/Western Blotting ................. ...............23.................
Immunocytochemistry .............. ...............24....
In Vitro Kinase As says .............. ...............24....
Site-Directed Mutagenesis............... ..............2
Luciferase As says .............. ...............25....
Molecular Modeling .............. ...............25....
Analysis of DNA Laddering ............ ...... .___ ...............26...
Hoechst 33342 Staining ........._.._ ..... .___ ...............26...
Propidium Iodide Staining ........._..... .... ...._ ._ ...............26....
Analysi s of Mitochondrial Membrane Integrity ................. ............................26














3 MEANS OF CIRCUMVENTING PROBLEMS WITH AG490 .............. ................27

Introducti on .........._.... ... .... ._._. ..... ..__. .............2
Primary Rat Aortic Smooth Muscle Cells .............. ...............27....
y2A Cell Line ................. ...............27........... ....

4 Jak2 REGULATES ANGIOTENSIN II-DEPEDENDENT ERK2 SIGNALINGG....28

Introducti on ................. ...............28.................
R e sults................ .. ........ .. .. ............... .. .... .... ...............2
ERK2 Activity is Sustained in y2A/AT1 Cells Compared to y2A/AT1+Jak2 Cells
after Angiotensin II Treatment..............._.._ .......... ......_ ............_. .......... 2
AG490 Suppresses Angiotensin II-Dependent ERK2 Activation Independent of
Jak2 Inhibition .............. ........ .. ...... .... .............3
There is a Marked Difference in the Angiotensin II-Induced Nuclear
Accumulation Pattern of Activated ERK2 between y2A/AT1 and
y2A /AT 1+Jak 2 Cells ............ ...... .................................3
ERK2-Dependent Gene Transcription in the y2A/AT1 and y2A/AT1+Jak2 Cells34
Jak2 is Essential for Angiotensin II-Induced MKP-1 Expression and Co-
association of MKP-1 with ERK2 ........._.._......... .. .. .._.. .. ....._.._...........3
MPK-1 is Required for Angiotensin II-Dependent Inactivation of ERK2..........3 8
Discussion ........._._. ._......_.. ...............42.....


5 Jak2 PROMOTES OXIDATIVE STRESS-INDUCED APOPTOSIS IN
VASCULAR SMOOTH MUSCLE CELLS .............. ...............46....

Introducti on ................. ...............46.................
R e sults................ .. .... ........ ......... .... .. .......... .........4
Jak2 Activation by Hydrogen Peroxide is Suppressed in RASM-DN Cells.......47
Jak2 Activation is Required for Oxidative Stress-Induced Apoptosis ................47
Quantification of Jak2-Mediated Apoptosis............... ...............5
Jak2 Activation by Oxidative Stress Mediates Bax Expression..........................51
Jak2 Activation by Oxidative Stress Promotes Mitochondrial Dysfunction....... 53
Jak2 is Required for Caspase-9 Cleavage During Oxidative Stress....................55
Discussion ................. ...............56.................


6 Jak2 RESIDUES GLU 1024 AND ARG 1113 FORM HYDROGEN BONDS, AND
ARE ES SENTIAL FOR Jak-STAT SIGNAL TRANSDUCTION ................... .........61

Introducti on ................. ...............61.................
R e sults.......... .................. .. ........... .... .. ..... ....... ....... ..........6
Molecular Modeling Identified a Putative Interaction between Jak2 Residues
Glu 1024 and Arg 1113.............. .. ................. .....................6
Mutation of Jak2 Residue Glu 1024 or Arg 1 113 Abolishes Jak2 Kinase Activity63
Individual Mutations of W1020G or E1024A Render Jak2 Dominant Negative65











Jak2-R1113E is Unable to Become Tyrosine Phosphorylated by Angiotensin II66
Jak2-R1113E is Unable to Tyrosine Phosphorylate STATs in Response to
Angiotensin II .............. ........... .. ... ... ............6
Jak2-R1113E is unable to mediate angiotensin II-dependent gene expression...70
Arg 1113 is conserved in different Jak kinase family members and among
species expressing Jak2............... ...............71..
Discussion ................. ...............72.................


7 IDENTIFICATION OF A NOVEL Jak2 INHIBITOR ......____ ....... ....._.........75

Introducti on ............ ..... ._ ...............75....
R e sults................ ........ ._ .. .......__ _............. ..........7
Homology Modeling and Target Pocket Identification................. ............7
Database Screening to Identify Potential Small-Molecule Inhibitors of Jak2 ....76
Compound 7 Inhibits Jak2 Autophosphorylation............... ... ...........7
Compound 7 Inhibits Jak2 Autophosphorylation in a Time-Dependent Manner78
Compound 7 Inhibits Jak2 Autophosphorylation in a Dose-Dependent Manner81
Compound 7 is Non-Cytotoxic at Concentrations that Maximally Inhibit Jak2
Tyrosine Autophosphorylation .............. ...............82....
Discussion ............ ..... ._ ...............82....


8 CONCLUSIONS AND PERSPECTIVES .............. ...............86....


Role of Jak2 in Angiotensin II-Dependent ERK2 Signaling ........._.._.. ........._.._.....86
Role of Jak2 during Oxidative Stress ................. .............._ ......._ ....... ..87
Jak2 Structure-Function .............. ........_ ...............88....
Identification of a Novel Jak2 Inhibitor. ....__ ......_____ .......__ ...........8



LIST OF REFERENCES ............ ..... ..__ ...............90...

BIOGRAPHICAL SKETCH ............_...... .__ ...............106...

















LIST OF FIGURES


Figure pg

1-1 Differences in the mechanism of Jak-STAT signaling through cytokine
receptors and GPCRs .............. ...............4.....

1-2 Summary of maj or Jak2 domains and amino acids critical to Jak2 tyrosine
kinase activity ........._... ...... ___ ...............17.....

4-1 ERK2 activity is sustained in y2A/AT1 cells compared to y2A/AT1+Jak2 cells
after angiotensin II treatment .............. ...............30....

4-2 In vitro kinase assay confirms that ERK2 activity is sustained in y2A/AT1 cells
compared to y2A/AT 1+Jak2 ........._._. ...._... ...............3 1...

4-3 AG490 suppresses angiotensin II-dependent ERK2 activation independent of
Jak2 inhibition... ........... ...............33......

4-4 Difference in the angiotensin II-dependent nuclear accumulation of
phospho-ERK2 between y2A/AT1 and y2A/AT1+Jak2 cells............... ..................3

4-5 No difference in angiotensin II-dependent ERK mediated gene transcription
between y2A/AT1 and y2A/AT1+Jak2 cells............... ...............36.

4-6 Jak2 is essential for angiotensin II-induced MKP-1 expression .............. ................39

4-7 Angiotensin II-dependent inactivation of ERK2 requires Jak2 and MKP-1 ............41

4-8 Proposed model of the mechanism by which Jak2 mediates ERK2 inactivation
after angiotensin II treatment .............. ...............44....

5-1 Hydrogen peroxide-induced Jak2 activity is suppressed in RASM-DN cells .........48

5-2 Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular smooth
m uscle cells .............. ...............49....

5-3 Hoechst staining to detect nuclear condensation............... ..............5

5-4 Quantification of apoptosis in RASM-Control and RASM-DN cells......................52

5-5 Jak2 mediates hydrogen peroxide-induced up regulation of Bax expression ..........54










5-6 Jak2 is essential for hydrogen peroxide-induced mitochondrial membrane
dysfunction ................. ...............55...... ......

5-7 Pixel intensity of each of 4 photographs from each condition in Fig. 5-6 was
determined, and intensity of green and red staining was determined for each ........56

5-8 Jak2 is required for oxidative stress-induced caspase-9 cleavage.............._._..........57

6-1 Molecular modeling of the Jak2 kinase domain suggested a putative interaction
between Glu 1024 and Arg 1113............... ...............62..

6-2 Mutation of Glu 1024 or Arg 1 113 abolishes the ability of Jak2 to
autophosphorylate .............. ...............64....

6-3 The Jak2-W1020G and E1024R mutations render Jak2 dominant negative ...........67

6-4 Jak2-R1113E mutant cannot become tyrosine phosphorylated in response to
angiotensin II............... ...............68...

6-5 Angiotensin II-dependent Jak2/AT1 receptor co-association does not occur in
cells expressing Jak2-R1113E ............... ...............68...___ ....

6-6 Jak2-R1113E is unable to activate STAT1 in response to angiotensin II................ 70

6-7 Jak2-R1113E is unable to activate STAT-mediated gene transcription in
response to angiotensin II............... ...............71...

6-8 Arg 1113 is conserved in Jak2 among species and in different Jak family
members ... ......_.__...... .__ ...............72....

7-1 SPHGEN identified 49 exposed pockets on the surface of the Jak2 protein ...........77

7-2 Compound 7 inhibits Jak2 autophosphorylation ......____ ... ......_ ..............79

7-3 Maximal Jak2 inhibition requires 16 h of incubation with Compound 7................80

7-4 Compound 7 inhibits Jak2 in a dose-dependent manner............. ..__.........__ ....81

7-5 Compound 7 is not cytotoxic at a dose of 100 CIM .............. ....................8
















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 TYROSINE KINASE: NEW INSIGHTS REGARDING STRUCTURE,
FUNCTION, AND PHARMACOLOGY

By

Eric M. Sandberg

December 2004

Chair: Peter P. Sayeski
Major Department: Physiology and Functional Genomics

The kinase Jak2 is a member of the Janus family of non-receptor tyrosine kinases.

One maj or impediment to understanding the role that Jak2 plays in physiology and

pathophysiology is the lack of a specific Jak2 inhibitor. We used several strategies to

circumvent this problem. First, using Jak2 -/- cells, we examined the role of Jak2 in

regulating angiotensin II-dependent ERK2 activity. We found that, contrary to

previously published work, Jak2 is required for inactivation of ERK2 after angiotensin II

treatment. In response to angiotensin II, Jak2 induces expression of MAP kinase

phosphatase-1 (MKP-1), a protein that dephosphorylates and inactivates ERK2.

Second, using stable expression of a Jak2 dominant negative mutant that

specifically suppresses endogenous Jak2 kinase activity, we found that Jak2 mediates

oxidative stress-induced apoptosis of vascular smooth muscle cells. In response to

hydrogen peroxide treatment, Jak2 induces expression of the pro-apoptotic Bax protein.










This causes a loss of mitochondrial transmembrane potential, cleavage of Caspase-9, and

subsequent apoptosis.

Third, we attempted to identify novel Jak2 inhibitors. For this, we used homology

modeling to analyze the structure of the Jak2 kinase domain, and we identified a

previously unknown amino acid interaction that is required for activation of Jak2. This

interaction, consisting of two distinct hydrogen bonds between Jak2 residues Glu 1024

and Arg 1113, may be a suitable target for drug design aimed at disabling Jak2 function.

Additionally, we used high-throughput compound docking in silico to identify a novel

Jak2 inhibitor. This compound, cyclohexane-1 ,2,3,4,5,6-hexabromo- (designated

Compound 7) potently inhibits Jak2 autophosphorylation in a time- and dose-dependent

manner.

In conclusion, using Jak2 -/- cells, stable expression of a Jak2 dominant negative

mutant, and structure-function studies, we successfully circumvented the problems that

lack of a Jak2-specific inhibitor pose. In doing so, we identified novel roles for Jak2

kinase function in regulation of angiotensin II-dependent ERK2 signaling and in

oxidative stress-induced apoptosis of vascular smooth muscle cells. Additionally, we

improved our understanding of Jak2 structure by identifying a previously unknown

amino acid interaction within the Jak2 kinase domain that is required for Jak2 kinase

function, and identified a novel Jak2 inhibitor.















CHAPTER 1
INTTRODUCTION TO THE Jak-STAT PATHWAY

History of the Jak-STAT Pathway

In 1990, the first member of the Jak family of cytoplasmic tyrosine kinases was

cloned and sequenced (1). The gene, termed Tyk2, was unique compared to previously

identified tyrosine kinases in that it had a kinase-like, or pseudokinase domain,

immediately N-terminal to a highly conserved protein tyrosine kinase domain. The

tandem manner by which these two domains adj oined one another was reminiscent of

Janus, Roman God of two opposing faces. As such, Tyk2 was classified as the first

member belonging to the JanJJJJJJJJ~~~~~~~~~us associated kinase family of protein tyrosine kinases (or

more simply) the Jaks. Other groups independently cloned the cDNAs encoding Jakl,

Jak2, and Jak3 (2-6). Because some of the genes were cloned from hematopoietic

tissues, it was hypothesized that the Jak kinases played a critical role in

cytokine-mediated signal transduction. This hypothesis was largely correct.

In 1992, Wilks and colleagues (3) were the first to clone and publish the Jak2

cDNA sequence. The gene encoded a protein of about 130 kDa in mass. Like the two

previously cloned Jaks (Tyk2 and Jakl) the predicted amino acid sequence of Jak2

contained the kinase and pseudokinase domains adj oining one another on the carboxyl

half of the protein. These regions are termed the Jak homology 1 (JH1) and Jak

homology 2 (JH2) domains, respectively. Wilks and colleagues also identified five other

domains that encompassed the amino half of the molecule. These were designated as the

JH3, JH4, JH5, JH6, and JH7 domains. The C-terminal half of the JH4 domain and the









entire JH3 domain were thought to encode a primitive SH2 domain spanning amino acids

412-480. This was significant because Jak family members lack any canonical SH2 or

SH3 domains. Aside from this putative SH2 domain, the remaining domains did not

possess the characteristics of any other known proteins. Wilks and colleagues found that,

like Tyk2 and Jakl, Jak2 was expressed in almost every tissue examined. In contrast,

Jak3 is expressed predominantly in hematopoietic cells (5, 7). Interestingly, subsequent

work showed that Jak2 homologs exist in animals as diverse as zebrafish (Danio rerio)

and fruitflies (Drosophila melan2oga~ster) (8, 9).

Collectively, these studies showed that a new family of cytoplasmic protein

tyrosine kinases existed in animals. These family members shared properties that were

unique only unto them. Of these genes, Jak2 is expressed in numerous tissues and in

evolutionarily diverse species.

The importance of Jak2 in cellular signaling was realized when it was discovered

that Jak2 appeared to be a critical mediator of cytokine-dependent signal transduction

(1-5.Subsequent work quickly identified a correlation between activation of Jak2 in

the cytoplasm and increased gene transcription in the nucleus. This observation

suggested that a specific class of cytokine-responsive transcription factors was mediating

this transcriptional effect. This hypothesis was proven correct when concurrent studies

identified a new class of cytokine-responsive transcription factors, termed the Signal

Transducers and Activators of Transcription (STAT) proteins (16, 17). These proteins

(when tyrosine phosphorylated by Jak2 in the cytoplasm) translocate to the nucleus and

mediate gene transcription. Thus, within 2 years, the broad framework of the Jak-STAT

signaling paradigm was elucidated.










More recently, it was discovered that in addition to mediating cytokine signal

transduction, Jak2 also mediates signaling through G protein coupled receptors (GPCRs)

(18). In fact, the number of cytokines and GPCR agonists that activate Jak-STAT

signaling has grown steadily since the discovery of the pathway. The cytokines currently

known to activate Jak2 include IL-2, IL-3, IL-5, IL-6, IL-11, IL-12, granulocyte

macrophage colony-stimulating factor, ciliary neurotrophic factor, leukemia inhibitory

factor, oncostatin M, granulocyte colony-stimulating factor, interferon-y, growth

hormone, prolactin, erythropoietin, thrombopoietin, and leptin (10-15, 19-27). The

GPCR agonists that activate Jak2 include angiotensin II, bradykinin, endothelin, platelet

activating factor, co-melanocyte stimulating hormone, isoproterenol, and phenylephrine

(28-32). The mechanism of Jak2 activation by these two receptor subtypes differs, but

the downstream effects of Jak2 activation are similar. In both cases, Jak2 acts a critical

link in coupling ligand binding at the cell surface with gene transcription in the nucleus.

Key differences in Jak-STAT signaling through cytokine receptors and GPCRs are shown

in Fig. 1-1 and discussed in the next section.

In addition to cytokines and GPCR agonists, Jak2 can also be activated in response

to a number of cellular stressors. These include mechanical cell stretch, ischemia-

reperfusion, and hydrogen peroxide (33-36). The upstream activators of Jak2 and the

downstream effects of Jak2 activation by these stimuli are not well characterized. In

Chapter 5, we elucidate the role that Jak2 plays in hydrogen peroxide-induced signaling

in vascular smooth muscle cells.











~Ligand
Ligand





Jak Jak2 Jak2 Jak2
PI P
STAT~ STA






Ligand


Ligand















Figure 1-1. Differences in the mechanism of Jak-STAT signaling through cytokine
receptors (Top) and GPCRs (Bottom)

Initiation of Jak-STAT Signaling

Jak2 signaling through cytokine receptors is initiated by the binding of an

extracellular cytokine to its cognate monomeric receptor on the cell surface, resulting in

receptor dimerization. This oligomerization of two distinct receptors results in the









activation of Jak2 molecules bound non-covalently to the receptors before ligand binding.

An activated Jak2 then phosphorylates specific tyrosine residues on the cytoplasmic tails

of the receptors creating docking sites for SH2 domain-containing proteins, such as the

STATs. Once bound to the receptors, STATs are themselves phosphorylated by Jak2 on

tyrosine residues. The tyrosine phosphorylated STATs then dissociate from the receptor

to form active homo- and hetero-dimer protein complexes. The STAT complexes then

translocate into the nucleus where they bind specific DNA sequences in gene promoter

elements and modulate gene transcription. Interestingly, the increased tyrosine

phosphorylation, nuclear translocation, and DNA binding activity of the STATs occurs in

the presence of cycloheximide, suggesting that this signaling pathway uses a post-

translational modification of existing proteins and does not require de-novo protein

synthesis (37). Thus, Jak2 is capable of transducing a signal from the cell surface to the

nucleus through a tyrosine phosphorylation signal transduction cascade.

While the downstream consequences of Jak2 signaling through GPCRs are similar

to Jak2 signaling through cytokine receptors, there are key differences in how Jak2

signaling is initiated in these two pathways. Similar to Jak2 signaling through cytokine

receptors, Jak2 signaling through GPCRs is initiated by binding of an extracellular ligand

to its cognate receptor on the cell surface. However, Jak2 is not ubiquitously bound to

GPCRs. Instead, it becomes activated in the cytoplasm after ligand binding (18). The

exact mechanism by which this occurs has not been elucidated. In the case of angiotensin

II (a Jak2-activating GPCR ligand), Jak2 is recruited to the angiotensin II type I receptor

(AT1R) after its activation. Then Jak2 acts as a molecular bridge, linking STAT proteins









to the AT1R. Jak2 phosphorylates the STAT proteins, and the signal cascade proceeds

similar to cytokine-mediated Jak2 signaling (3 8).

Physiology and Pharmacology of Jak2

Jak2 signaling plays a critical role in normal physiological processes, including

development, regulation of other signaling pathways, and cellular stress responses. In

addition, Jak2 signaling has been implicated in the pathophysiology of cancer and heart

disease. The role that Jak2 plays in physiology and pathophysiology will be discussed in

this section.

Target Genes and Regulation of Signal Transduction Pathways

Since its discovery, Jak2 activation has been linked to mediation of gene

expression. It accomplishes this through activation of the STAT family of transcription

factors. Despite this, the identities of downstream target genes of the Jak-STAT pathway

are largely unknown. Additionally, Jak2 has been shown to directly regulate other signal

transduction pathways. In fact, data in the results section show that Jak2 can indirectly

regulate angiotensin II-induced extracellular signal regulated kinase 2 (ERK2) signaling

by mediating expression of an ERK2 regulatory protein.

Jak2 can influence other angiotensin-dependent signaling events as well. For

example, when Jak2 is activated by angiotensin II, Jak2 recruits the Src family tyrosine

kinase Fyn to the Jak2-based signaling complex (39). Jak2 then activates Fyn, by binding

the SH2 domain of Fyn with very high affinity (Kd = 2.36 nM). This strong interaction

results in a conformational change within Fyn that allows the Fyn kinase domain to

become accessible to substrate. Thus, Jak2 serves as a potent activator of Fyn kinase.

Additionally, Jak2 has been shown to regulate ERK2 activity in response to

angiotensin II (40). Jak2 was found to be essential for activation of ERK2. Despite this,









our study (Chapter 4) suggests that these findings may be an artifact of using a

nonspecific inhibitor of Jak2 kinase function in the studies.

Other molecules that are recruited into Jak2-based signaling complexes include c-

Src, Grb2, PI3 kinase, PP2A, Yes, Raf-1, Shc, Syp, and FAK (41-47). Furthermore, a

review of the literature found that more than 50 different cellular proteins associate with

Jak2 in some manner. While the precise relationship of each of these proteins to Jak2 is

not known, it would seem that each interaction is occurring for a specific cellular and

biochemical reason. As such, these studies demonstrate a role for Jak2 as a cellular

headquarters for the recruitment, modification, and modulation of numerous signaling

pathways. While Jak2 clearly can regulate other intracellular signal transduction

pathways, the Jak2 pathway itself is tightly regulated within the cell.

Regulation of Jak2 Signaling

For Jak2 to initially become activated, a single tyrosine within the Jak2 activation

loop must be phosphorylated. Site-directed mutagenesis studies showed that

phosphorylation of Tyr 1007 is required for ligand-induced Jak2 activation and

subsequent phosphorylation of several STATs (48). Activation of Jak2 may depend on

its interaction with ancillary molecules such as SH2B-P and SHP-2. The mere expression

of SH2B-P in the same cell as Jak2 increases Jak2 tyrosine phosphorylation levels and its

catalytic activity (49). It is known that SH2B-P directly binds Jak2, however exactly how

SH2B-P biochemically modifies Jak2 to promote its activation has not been determined.

The tyrosine phosphatase SHP-2 is a more controversial Jak2-regulatory protein; there

are conflicting data regarding whether this protein acts as an activator or an inhibitor of

Jak2 function. The discrepancies are due to several factors, including the specific ligand









and cell types used in each experiment. For example, when growth hormone binds to its

receptor on transfected COS-7 cells, SHP-2 augments the level by which Jak2 increases

c-fos expression (50). Jak2 shows higher tyrosine phosphorylation levels in these cells

and it is thus believed that SHP-2 augments Jak2 function by elevating Jak2 tyrosine

kinase activity. In contrast, when fibroblasts are treated with interferons, SHP-2 inhibits

Jak2 function (51).

Equally important to the activators of Jak2 are the inhibitors, that serve to

terminate Jak2-dependent signaling. These inhibitors work at different levels of the

signal transduction cascade to attenuate Jak2 signaling. In addition to providing a certain

level of redundancy in this inhibitory process, the inhibitors also appear to act in a

temporal or sequential manner.

Since the Jak2 activation state is dependent on its tyrosine phosphorylation levels,

one obvious mechanism of inactivation is tyrosine dephosphorylation of Jak2. This is

accomplished by protein tyrosine phosphatases, including SHP-1. The binding of ligands

such as growth hormone, erythropoietin, IL-2, and IL-4 to their cognate receptors

promotes the binding of SHP-1 to the Jak2-based receptor-signaling complex. The SH2

domain of SHP-1 binds a phosphotyrosine residue on Jak2, thus inhibiting Jak2 activity

(52). Not surprisingly, loss of SHP-1 expression in cells lead to a variety of transformed

phenotypes, owing to the growth-promoting actions of Jak2 (53, 54).

Suppressors of Cytokine Signaling (SOCS) also act as potent inhibitors of Jak

kinase function. They act in a classical negative feedback mechanism. Cytokine

inducible SH2 domain-containing protein (CIS) was the first SOCS family member to be

cloned. It was initially identified as a gene that was rapidly induced by IL-3 (55). Based









on sequence homology, subsequent groups cloned and characterized seven additional

SOCS family members, termed SOCS1-SOCS7. Accumulation of SOCS mRNA is

rapidly induced by a variety of cytokines and growth factors including IL-2, IL-4, IL-6,

leukemia inhibitory factor, granulocyte colony stimulating factor, interferon-y, growth

hormone, prolactin, erythropoietin, and leptin. There are multiple mechanisms by which

SOCS proteins suppress Jak2 function. For instance, SOCS1 binds Jak2 via a direct

interaction between the SH2 domain of SOCS1 and phosphotyrosine residue 1007 on

Jak2. Structure-function studies have shown that this interaction is required for

suppression of Jak2 kinase activity, as deletion of the SOCS1 SH2 domain results in the

inability of SOCS1 to reduce Jak2 kinase function (56). However, the exact biochemical

modifications) that occur on Jak2 after co-association with SOCS1 is not known.

A final group of Jak-STAT inhibitory proteins are termed the Protein Inhibitors of

Activated STATs (PIAS). The four members identified so far are PIAS-1, PIAS-3,

PIAS-X, and PIAS-Y (57, 58). They differ from protein tyrosine phosphatases and

SOCS in that they bind STATs and not Jaks. While they share homology among

themselves, they have no previously characterized protein domains. These proteins are

constitutively expressed in numerous tissues, and do not appear to be highly specific for

which STATs they bind. Unfortunately, the biochemical and cellular mechanisms by

which PIAS proteins suppress STAT function are not well understood.

Specific activators and inhibitors of Jak2 signaling have been identified. While

the activators allow maximal Jak2 activation and kinase function, the inhibitors work in

concert to suppress Jak2 signaling at different levels of the Jak-STAT pathway. These

regulatory proteins control the magnitude and duration of Jak-STAT signaling. Although









Jak2 activity is tightly controlled within cells, aberrant regulation of this pathway

contributes to the pathological progression of certain diseases.

Jak2 in Cancer

In 1995, studies of the Drosophila Jak2 homolog, hopscotch, were the first to

implicate Jak2 in tumorigenesis (59). Specifically, a Glu 695 to Lys mutation in the

HopT42 prOtein increased the intrinsic tyrosine kinase activity of Jak2, and led to

malignant neoplasia of Drosophila blood cells. When the same mutation was introduced

into the JH2 domain of mammalian Jak2, the mutant protein similarly had significantly

increased kinase activity, and hyperactivated the mammalian Jak-STAT signaling

pathway (60). Concurrent work by Meydan and colleagues (61) was the first to implicate

Jak2 in human cancer, as they reported an inhibition of acute lymphoblastic leukemia via

treatment with the Jak2 pharmacological inhibitor AG490. Collectively, these works

suggested a direct link between activated Jak2 and neoplastic cell growth.

Tel-Jak2 fusion proteins result from a translocation event between the kinase

domain of Jak2 and the HLH domain of Tel. The first reports describing Tel-Jak2 fusion

proteins came from a child with early B-precursor acute lymphoid leukemia, and an adult

with atypical chronic myeloid leukemia (62, 63). The tumors of these 2 patients are

differ because of distinct translocations within the Jak2 and Tel genes, which then give

rise to distinct chimeras. Nonetheless, it appears that all Tel-Jak2 fusions confer

constitutive Jak-STAT activity. They have been shown to increase NF-icB signaling and

induce growth factor-independent proliferation in Ba/F3 hematopoietic cells (64, 65).

More importantly, the creation of Tel-Jak2 transgenic mice revealed a causal relationship









between the Tel-Jak2 gene product and leukemogenesis, as over expression of this fusion

protein stimulated development of T-cell leukemia in these animals (66).

While many of the mechanisms involving Jak2 activation during cancer are

incompletely understood, striking data have surfaced implicating Jak2 activation in

human hepatocellular carcinoma (HCC) cells. Methylation of CpG islands within the

SOCS-1 gene, which results in reduced SOCS-1 expression, was directly responsible for

constitutive Jak2 activity. Furthermore, restoration of SOCS-1 expression and/or

treatment with AG490 reduced Jak2 tyrosine kinase activity and the growth rate of these

cells (67). Therefore, increased activation of Jak2 was triggered by inactivation of its

inhibitor, and this finding identified a new area of therapeutic research.

Increased Jak2 tyrosine kinase activity is associated with many other types of

cancers as well. Specifically, Jak2 has been shown to activate ErbB-2, whose oncogenic

activity is associated with various human breast cancers (68-70). Additionally, BRCAl

over expression enhanced the ability of Jak2 to activate STAT3 in human prostate cancer

cells (71).

Jak2 in Cardiovascular Disease

Jak2 plays a pivotal role in various cardiovascular signaling systems. Therefore, it

is not surprising that activation of this protein has been implicated in the molecular events

of certain cardiovascular disease states including cardiac hypertrophy, ischemia-

reperfusion injury, and heart failure. A review of the literature presents strong evidence

that Jak2 plays a role in the cellular signaling processes associated with these and other

pathological disease states.

Cardiac hypertrophy (or the increase of cardiac muscle mass) is a natural defense

for coping with cardiovascular diseases, and is a maj or cause of mortality in the United









States (72-74). Cardiac hypertrophy occurs in response to an increased workload on the

heart and/or the secretion of certain humoral factors (75-85). Interestingly, cardiac

hypertrophy-inducing stimuli also activate Jak2. For example, acute pressure overload in

rats increases Jak2 tyrosine phosphorylation levels by causing autocrine/paracrine

secretion of angiotensin II (86, 87). Jak2 is also activated by cardiotrophin-1, another

potent activator of cardiomyocyte hypertrophy (88, 89). Cardiotrophin-1, a member of

the interleukin-6 related cytokine family, induces cardiomyocyte hypertrophy by

increasing expression of angiotensinogen mRNA through the activation of STAT3.

Activated STAT3 forms dimer complexes that translocate into the nucleus and bind

ST-domains within the angiotensinogen gene promoter (90). Jak2 was implicated in this

pathological process, as AG490 treatment suppressed the cardiotrophin-induced STAT3

binding to the angiotensinogen promoter and subsequent gene activation (90). Thus, Jak2

appears to have a key role in the signaling system leading to cardiotrophin-1 induced

cardiac hypertrophy via increased expression of the angiotensinogen gene.

Heart failure is another disease that has been linked to Jak2, but in a different

fashion. In short, heart failure is defined as inadequate cardiac output. The progression

of heart failure is dependent on a balance between cardiomyocyte hypertrophy and

apoptosis (91). Podewski et al. (92) examined signaling events associated with one

disease that leads to heart failure, dilated cardiomyopathy (DCM). They showed that

Jak2 tyrosine phosphorylation levels were decreased in patients with DCM, while levels

of the Jak2 inhibitor, SOCS1, were increased. Based on this and subsequent work, they

proposed a model in which decreased Jak2 tyrosine phosphorylation levels (attributable

to an increase in SOCS1 activity) result in decreased phosphorylated STAT3. The result









of this is that STAT3 fails to increase expression of cardioprotective genes that would

save the heart from failure. What makes this intriguing is that during DCM, reduced Jak2

activity is pathological; while during cardiac hypertrophy, increased Jak2 activity is

pathological. Clearly, future work will need to better define the signaling pathways

leading to cardiac hypertrophy and heart failure. Presently, these and other results show

that either too much, or too little Jak2 activity, can have negative consequences.

Jak2 activation is also associated with cardiac injury during ischemia-reperfusion

(34). Ischemia-reperfusion is a pathological condition characterized by impeded blood

flow to an area of tissue followed by the reestablishment of circulation to that same area.

It has been shown that treatment with AG490 leads to a reduction in cardiac infarct size

and a reduction in apoptotic cell death of cardiomyocytes after ischemia-reperfusion in an

isolated perfused rat heart (34). Furthermore, ischemia-reperfusion leads to STAT~a and

STAT6 binding to the angiotensinogen gene promoter. Treatment with either AG490 or

the AT1 receptor antagonist losartan resulted in the loss of STAT/St-domain complex

formation, and a subsequent reduction in angiotensinogen mRNA levels. Thus, a positive

feedback model in which Jak2 activates Stat~a and Stat6 which bind to the

angiotensinogen gene promoter, resulting in an increase in angiotensinogen mRNA, and

subsequent angiotensin II production and activation of the AT1 receptor.

Recent evidence has linked Jak2 to vascular injury. Jak2 and STAT3 protein

expression levels are increased after balloon injury of rat carotid arteries, and increased

activity of the Jak-STAT pathway is involved in the ensuing vascular smooth muscle cell

proliferation and neointima formation seen in this model of vascular injury (93). In

addition, oxidative stress in the form of hydrogen peroxide potently activates Jak2 in









vascular smooth muscle cells, suggesting a possible role for Jak2 during oxidative stress

(35). Evidence supporting a pro-apoptotic role for Jak2 during oxidative stress in

vascular smooth muscle cells is given in Chapter 5.

In conclusion, reports have shown a clear relationship between Jak2 and

neoplastic transformation, and between Jak2 and cardiovascular disease. Researchers are

now trying to determine the cellular and biochemical mechanisms by which aberrant Jak2

activity leads to cancerous cell growth; and to determine the precise role that Jak2 plays

in cardiovascular disease progression. These results show that in the near future, Jak2

may be an attractive target for pharmacological inhibition during cancer and heart

disease.

Jak2 Pharmacology

Based on the role that Jak2 plays in cancer and cardiovascular disease,

pharmacological inhibition of Jak2 may soon hold therapeutic promise. This section

reviews research that highlights Jak2 as a therapeutic target.

Many studies have used the commercially available Jak2 inhibitor, tyrphostin

AG490, to demonstrate the benefits of inhibiting this signaling pathway during certain

disease states. For instance, AG490 suppressed growth of human hepatocellular

carcinoma cells (67). Jak2 is constitutively activated in these cells because of

methylation and transcriptional silencing of the SOCS-1 gene, a negative regulator of

Jak2 signaling. AG490 prevented constitutive Jak2 activation, and induced apoptosis in

these cells (67). Similarly, AG490 sensitized metastatic breast cancer cells to

chemotherapy-induced apoptosis (68), induced apoptosis in myeloblastic cells (94), and

blocked growth of acute lymphoblastic leukemia cells in vitro and in vivo by inducing

apoptosis (61). AG490 also prevented Jak2-mediated constitutive tyrosine









phosphorylation of ErbB-2 and DNA synthesis in breast cancer cells (68), and abrogated

growth of human B-precursor leukemic cells (95).

In addition to the potentially therapeutic effects of inhibiting Jak2 in various types

of cancer, Jak2 inhibition via AG490 was shown to be therapeutic in several

cardiovascular disease models. For instance, AG490 reduced neointima formation in the

carotid artery of rats after balloon injury (92). In cultured cardiomyocytes, AG490

attenuated leukemia inhibitory factor-induced hypertrophy and myofilament

reorganization (96). Additionally, AG490 inhibited several signaling pathways rapidly

induced after myocardial infarction that are thought to contribute to diastolic dysfunction

and arrythmogenicity in the post-myocardial infarcted heart (97). Finally, AG490-treated

hearts showed a reduction in myocardial infarct size and in the number of

cardiomyocytes undergoing apoptosis after ischemia-reperfusion (34). It is apparent

from these studies that inhibition of Jak2 in various types of cancer, heart, and vascular

disease holds therapeutic promise.

While AG490 has been used extensively to study the Jak-STAT pathway in health

and disease, and has been instrumental in identifying Jak2 as a therapeutic target, it

suffers from a lack of specificity. For instance, AG490 inhibits activation of cyclin

dependent kinases and causes growth arrest of cells in G1 phase (98). It inhibits calf

serum inducible cell growth and DNA synthesis (98), and is a partial blocker of c-Src

activity (99). Most critically, AG490 inhibits epidermal growth factor receptor

autophosphorylation more potently than it inhibits Jak2 activity (100, 101). Moreover,

no assay is available for quantifying tissue AG490 concentrations, and no data exist









describing the in vivo degradation rate of AG490 (93). Thus, the issue of specificity of

AG490 for Jak2 is a maj or concern.

Lack of a specific Jak2 inhibitor has made study of the Jak-STAT pathway

difficult. Additionally, Jak2 knockout mice die embryonically, further complicating

research efforts (102, 103). Therefore, the identification or development of new, Jak2-

specific pharmacological inhibitors would provide useful research tools and potentially

therapeutic drugs. As such, investigation into the structural characteristics of the Jak2

protein as they relate to kinase function may be an important step towards achieving this

goal .

Structure-Function Relationship of Jak2

When the Jak family of protein kinases was first discovered, their unique structural

characteristics were quickly noted. The proteins consist of seven novel protein domains

now termed Jak homology (JH) domains. Most of these JH domains have a distinct role

in controlling Jak2 function. Figure 1-2 is cartoon summarizing the major domains of

Jak2. This section reviews the structural characteristics of the Jak2 protein, and recent

research that advanced our understanding of the structure-function relationship of Jak2.

The carboxyl terminus of Jak2 contains the JH1 and JH2 domains. The JH1

domain is the highly conserved kinase domain, which contains the ATP-binding region

and the activation loop. Most of the structure-function data available for Jak2 concerns

this domain. Studies have shown the requirement for the JH1 domain for Jak2

tyrosine kinase function (104). In addition, a number of individual amino acids within

the JH1 domain have bee identified that are critical to this function. Two adjacent

tyrosines, Tyr 1007 and Tyr 1008, were shown to be phosphorylation sites within the












Unique N Terminus Conserved C Terminus


FERM SH2 Pseudokinase Knase


JH7 JH6 JH5 JH4 JH3 JH2 JH1



1 130 283 307 450 543 827 1129


Amino Acid Function
2-240 Critical for Jak2/GHR co-association
23 1-235 Critical for Jak2/AT 1R co- association
Lys 882 Critical for Jak2 autophosphorylation
994-1024 Activation loop
Try 1007 Phosphorylated upon Jak2 activation
Tyr 1008 Phosphorylated upon Jak2 activation
Trp 1020 Critical for Jak2 autophosphorylation
Glu 1024 Critical for Jak2 autophosphorylation
Glu 1046 Stabilizes activation loop via H bond with Trp 1020

Figure 1-2. Summary of maj or Jak2 domains and amino acids critical to Jak2 tyrosine
kinase activity

Jak2 activation loop. The tyrosine at position 1007 is required for Jak2 tyrosine kinase

activity, while the tyrosine at position 1008 seems to be dispensable for the tyrosine

kinase function of Jak2 (48). Additionally, Tyr 1007 interacts with a Jak binding protein

(JAB), also known as suppressor of cytokine signaling 1 (SOCS1), a protein that

negatively regulates the kinase activity of Jak2 (56). Also, mutation of the invariant Lys

882 in the JH1 domain rendered Jak2 catalytically inactive (104, 105).

In 1994, a Jak2 containing mutations at both Trp 1020 and Glu 1024

(W1020G/E1024A) was shown to not only be catalytically inactive, but also dominant

negative (106). We recently showed that mutation of either of these amino acids









individually rendered Jak2 catalytically inactive (107). Moreover, we elucidated the

requirement for Trp 1020 for Jak2 kinase activity, by demonstrating that Trp 1020 forms

a hydrogen bond with Glu 1046, an amino acid that previously had not been shown to be

required for Jak2 function. The importance of this interaction is that it appears to

stabilize the three dimensional structure of the Jak2 activation loop. More recently, we

elucidated the requirement for Glu 1024. These data are presented in Chapter 7.

The JH2 domain of Jak2 is the pseudokinase domain. While it shares conserved

motifs with other protein kinases, this region is catalytically inactive. In fact, the active

site and activation loop are modified compared to traditional protein tyrosine kinase

activation loops (108). These modifications are, however, conserved amongst Jak family

members, suggesting a regulatory role for this pseudokinase domain (109).

Other lines of evidence point to a critical role for the JH2 domain. In Drosophila,

a mutation in the JH2 domain of the Jak homolog Hop, rendered the protein hyperactive

and caused hematopoietic hyperplasia (59). This was the first evidence of a negative

regulatory role for the JH2 domain. Subsequent work by Saharinen et al. showed that the

JH2 domain of mammalian Jak2 interacts with the JH1 kinase domain and suppresses the

activity of Jak2 at the basal level (109). Following this work, another group constructed

a homology model of the JH1 and JH2 domains. Using this model, they predicted two

maj or interactions between the JH1 and JH2 domains of Jak2. The first interaction is

between the two oc helices of the two domains. The second interaction occurs between a

loop that lies between two P stands of the JH2 domain and the activation loop of the JH1

domain (110). Incidentally, another group, Chen et al., showed a similar role for the JH2

domain in Jak3, suggesting functional conservation amongst Jak family members (111).









In addition, Saharinen et al. showed that the JH2 domain is also required for the ability of

Jak2 to become maximally activated in response to cytokine stimulation (112).

This same group further clarified the regulatory role of the JH2 domain by

identifying three specific regions within this domain that are involved in the

auto-inhibition of Jak2, which are termed IR1, IR2, and IR3 (113). IR3, which spans

amino acid residues 758 to 807, directly inhibited the JH1 kinase domain, while IR1

spanning residues 619 to 670 and IR2 spanning residues 725 to 757, enhanced

IR3-mediated inhibition of the JH1 domain. In this work, the authors present an

explanatory model for the function of JH2 in regulating JH1. In it, they suggest that

under basal conditions, JH2 is bound to JH1. Upon cytokine binding and receptor

aggregation, the inhibitory JH1-JH2 interaction is displaced, possibly through homotypic

interaction of the JH1 domains of the two Jak2 molecules. Why the JH2 domain is

required for maximal cytokine-induced Jak2 activation still remains to be determined.

The amino terminal region of Jak2 comprises the JH3 through JH7 domains. This

region is largely responsible for receptor interactions and may be responsible for

interactions between Jak2 and regulatory molecules. Interestingly, although the Jak

family members have traditionally been considered absent of a canonical SH2 domain, it

was noted that in Tyk2, the second half of the JH4 domain plus the whole of the JH3

domain weakly resembles an SH2 domain (114). Upon cloning of murine Jak2, it was

similarly noted that the sequence GLYVLRWS bore weak homology to the core

sequence element of SH2 domains (FLVRES) (3). Using multiple sequence alignments

and secondary and tertiary structure predictions, Kampa et al. presented for the first time

a three dimensional view of a putative Jak family SH2 domain spanning amino acids 412-









480 of Jak3. Furthermore, this group demonstrated binding of phosphorylated proteins to

the putative SH2 domain of Jak3, thus proving an SH2-like function for this domain (1 15,

116).

Despite these results, Giordanetto and Kroemer recently published a predicted

Jak2 structure comprising JH domains 1 through 7, whereby they contended that the

putative SH2 domain of Jak2 might not be fully functional (117). Comparing the SH2

domain of p561ck with the putative Jak2 SH2 domain, they demonstrated several key

differences at amino acids involved in phosphotyrosine binding between the two that may

preclude the Jak2 SH2 domain from being fully functional. They stated that despite

conservation between the two SH2 domains of one arginine known to interact with

phosphate groups (Arg 426 of Jak2, Arg 134 of p561ck), another highly conserved

arginine known to interact with phosphate groups is replaced by a methionine at the

corresponding position in Jak2 (Met 406 of Jak2, Arg 154 of p561ck). This, they point

out, would abolish interaction with a phosphate group. Furthermore, a lysine within the

putative Jak2 SH2 domain corresponds to a highly conserved serine residue in other SH2

domains (Lys 430 of Jak2, Ser 158 of p561ck). The authors contend that the longer side

chain of lysine would present difficulties in allowing it to interact with a phosphotyrosine

residue. Finally, they show that there are key differences in the phosphotyrosine binding

pockets of the two SH2 domains. Specifically, Glu 157 of p561ck is replaced by Pro 429

in Jak2. In the predicted model, this proline appears to restrict access to the

phosphotyrosine binding pocket, because of the rigid side chain of proline. Whether an

SH2 domain exists within Jak2 and whether it is fully functional may be clarified in the

future by determining the crystal structure of Jak2.









Adj acent to the putative SH2 domain of Jak2 lays the FERM domain, which spans

from the middle of the JH4 domain through the JH7 domain (1 18). This domain is

present in a number of other proteins including the band 4. 1 protein from erythrocytes,

some protein tyrosine phosphatases, and the focal adhesion kinases (119-121). Often,

this domain is involved in stable association with membrane bound proteins (122). In

fact, the amino terminal region of Jak2, especially the JH6 and JH7 domains (comprising

part of the FERM domain), has been shown to be crucial for Jak2/receptor interactions.

For example, deletion of amino acids 2-239, which deletes the JH7 domain and

part of the JH6 domain, abrogated Jak2 association with the growth hormone receptor

(123). It was then shown, though, that neither the JH6 and JH7 domains alone, nor the

entire Jak2 FERM domain (comprising the JH6 and JH7 domains with the JH5 domain

and half of the JH4 domain) were sufficient for Jak2 association with the growth

hormone receptor. This suggested that multiple JH domains must interact to allow Jak2

association with cytokine receptors (124). Additionally, amino acids 1-294 were found to

be essential for Jak2 binding to the granulocyte-macrophage colony stimulating factor

(GM-CSF)-Pe receptor (125). More recently, our lab found that the YRFRR motif within

the JH6 domain of Jak2, spanning amino acids 23 1-23 5, was essential for both Jak2

association with the angiotensin II type 1 receptor (AT1 receptor) and STAT1

translocation to the nucleus in response to angiotensin II treatment (126). Similar

requirements for the amino terminal region for receptor association have been shown for

the other Jak family members (127, 128). The Jak2 FERM domain interacts with

cytokine receptors via a proline rich box1 motif on the cytoplasmic chain of the cytokine

receptor (129, 124). A recent paper describing a homology model of Jak2 comprising the









JH1-7 domains highlighted hydrophobic amino acids within the FERM domain predicted

to be essential for Jak2 association with the box1 motif of cytokine receptors (117). The

analysis predicted that Met 181, Phe 236, Phe 240, and Iso 223 within the Jak2 FERM

domain would mediate interaction with receptors. These results elucidated the

requirement of the FERM domain for receptor interaction, but mutational studies on the

predicted amino acids are required for confirmation of these predictions.

Collectively, structure-function studies involving the Jak family of non-receptor

tyrosine kinases identified seven conserved Jak homology domains in each protein.

Studies have described essential functions for each of these domains, including kinase

activation for the JH1 domain, autoregulation for the JH2 domain, and interactions with

substrates, regulatory proteins, and membrane-bound receptors for the JH3-7 domains.

Future structure-function studies will be critical in advancing our understanding of Jak2,

and may lead to new ways to control its function, including Jak2-specific

pharmacological inhibition.















CHAPTER 2
EXPERIMENTAL METHODS

Cell Culture

All cells were cultured at 37oC in a 5% CO2 humidified atmosphere and were

maintained in DMEM containing 10% fetal bovine serum. Ample stocks of these cells

are stored in liquid nitrogen.

Vaccinia Virus Transfection/Infection

BSC-40 cells were transfected in serum free media with Jak2 cDNA cloned into the

pRC-CMV plasmid. After 4 h of transfection, cells were infected with 1.0 MOI of

vaccinia virus vTF7-3. After 1 h of incubation with the virus, complete media was put on

the cells to stop transfection, and the cells were allowed to become infected with virus for

16 h. The virus expresses a T7 RNA polymerase, which drives Jak2 expression from a

T7 promoter upstream of the Jak2 cDNA in the pRC-CMV plasmid, allowing

overexpression of the Jak2 protein.

Immunoprecipitation/Western Blotting

To prepare lysates, cells were washed with two volumes of ice-cold PBS containing

1 mM Na3VO4 and lysed in 1.0 mL of ice-cold RIPA buffer containing protease

inhibitors. The samples were sonicated and incubated on ice for 30 min. Samples were

spun at 16,000 x g for 5 min at 40C, and supernatants were normalized for protein content

using the Bio-Rad De assay. Normalized lysates (approx. 400 Clg/mL) were

immunoprecipitated for 2-4 h at 40C with 2 Clg of antibody and 20 Cll of Protein A/G Plus

agarose beads (Santa Cruz Biotechnology). After centrifugation, protein complexes were









washed 3 times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaC1, and 0.1% Triton

X-100) and resuspended in SDS sample buffer. Bound proteins were boiled, separated

by SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking

membrane for 1 h in 5% dry milk/TBST (100 mM Tris, pH 7.5, 0.9% NaCl and 0.05%

Tween 20) at room temperature, nitrocellulose membranes were probed with primary

antibody for 1-2 h at room temperature in 5% milk/TBST. Blots were washed with

TBST and proteins were visualized using enhanced chemiluminescence (ECL) following

the manufacturers instructions (Amersham).

Immunocytochemistry

Cells were grown on microscope slides. After treatment, cells were washed twice

with K+ free PBS and fixed for 60 min at room temperature with 4% paraformaldehyde.

After fixation, cells were washed 4 times with K' free PBS, permeabilized for 10 min at

room temperature with 0.2% Triton X-100 in K+ free PB S (vol/vol), washed an additional

4 times, and then blocked with 5 mg/mL BSA in K' free PB S for 4 h at room

temperature. After blocking, cells were incubated with primary antibody overnight at

4oC in K' free PBS containing 5 mg/mL BSA. The next day, cells were washed 5 times

and incubated with secondary antibody conjugated to Texas Red for 4 h at room

temperature. The cells were then dehydrated through increasing concentrations of

ethanol, dipped into xylene, and mounted. The next day, the cells were visualized with a

fluorescent microscope.

Inz Vitro Kinase Assays

ERK2 immunoprecipitates were washed twice with wash buffer, followed by two

washes in kinase reaction buffer (25 mM HEPES pH 7.4 and 20 mM MgCl2). The

precipitates were resuspended in 50 Cll of the same kinase buffer containing 50 CIM ATP,










2 CICi 32P y-ATP, and 5 Clg myelin basic protein (MBP). The samples were incubated for

15 min at 30oC. Reactions were terminated by adding sample buffer. The samples were

separated by SDS-PAGE, transferred onto nitrocellulose membranes, and subj ected to

autoradi ography.

Site-Directed Mutagenesis

Mutations of Jak2 amino acids were generated using the QuikChange site-directed

mutagenesis system (Stratagene). All mutations were confirmed by DNA sequence

analy si s.

Luciferase Assays

100 mm dishes of 70% confluent COS-7 cells were transfected with 10 Clg

c-fos/1uciferase in 12 Cll Lipofectin for 5 h. The cells were then trypsinized and seeded

into 6-well plates at 4.5 x 105 cells per well, and allowed to attach overnight in serum

containing medium. The next morning, the cells were washed and placed into serum free

DMEM. The next morning, the cells were stimulated with angiotensin II, and luciferase

activity was measured from detergent extracts in the presence of ATP and luciferin using

the Reporter Lysis Buffer System (Promega) and a luminometer (Moonlight 3010).

Molecular Modeling

A structural homology model of the Jak2 kinase domain ranging from amino acid

814 to the stop codon (position 1129) was generated using the program Swiss Model.

The model was based on the known crystal structure of the kinase domain of the

fibroblast growth factor tyrosine kinase receptor. The program HBPLUS Hydrogen Bond

Calculator, version 3.15, was used to determine hydrogen bond interactions and bond

lengths.









Analysis of DNA Laddering

Genomic DNA was isolated using the Easy-DNA kit from Invitrogen. 20 Clg of

DNA was separated on a 1.8% agarose gel and stained with ethidium bromide.

Laddering was analyzed under ultraviolet light using the GelDoc system.

Hoechst 33342 Staining

Cells were grown on microscope slides, serum starved for 48 h, and treated. Cells

were washed twice with PBS and incubated for 30 min at room temperature with 50

Clg/mL Hoechst 33342 nuclear stain. Cells were then washed, fixed, and mounted as

described in the immunocytochemistry section. The cells were visualized using a

fluorescent microscope with appropriate filters. Apoptotic cells were counted as those

showing condensed and/or fragmented nuclei.

Propidium Iodide Staining

Cells were grown on microscope slides and stained with 1 Clg/mL propidium iodide

for 10 min at 37oC. Live cells were examined using confocal microscopy. Same field

images were captured under phase contrast and fluorescent conditions.

Analysis of Mitochondrial Membrane Integrity

Mitochondrial membrane integrity was analyzed using the MitoCapture Apoptosis

Detection Kit from BioVision. Cells were treated with 1 mM hydrogen peroxide for 2 h

and stained according to the manufacturer' s protocol. Live cells were then visualized

using appropriate filters on a confocal microscope. Predominant green staining occurs in

cells with a disrupted mitochondrial membrane potential, while predominant red staining

occurs in cells with an intact mitochondrial membrane potential.















CHAPTER 3
MEANS OF CIRCUMVENTING PROBLEMS WITH AG490

Introduction

As discussed in Chapter 1, one of the central problems facing researchers studying

Jak2 tyrosine kinase function is the lack of a specific Jak2 inhibitor. While AG490 is a

useful research tool, results acquired using AG490 must be corroborated using other

methodology. Here we will briefly discuss in vitro strategies that we use to circumvent

the problems with using AG490.

Primary Rat Aortic Smooth Muscle Cells

To study the role that Jak2 tyrosine kinase plays in VSMC physiology, we use

primary rat aortic smooth muscle cells (RASM cells) stably transfected with a Jak2

dominant negative mutant (RASM-DN). As controls, we use the same cells expressing a

neomycin resistant cassette (RASM-Control). Creation of these cells has been described

previously (39). In the RASM-DN cells, Jak2 tyrosine kinase function is suppressed by

the Jak2 dominant negative mutant, allowing us to reliably study the function of Jak2.

y2A Cell Line

To study the role that Jak2 plays in intracellular Ang II-dependent signaling, we

used y2A cells, which are human fibrosarcoma cells that were gamma-irradiated and

screened to identify Jak2-deficient cells. The cells were then stably transfected with the

angiotensin II type I receptor (y2A/AT1). Controls are y2A/AT1 cells stably transfected

with Jak2 cDNA to reconstitute Jak2 signaling (y2A/AT1+Jak2) (130).















CHAPTER 4
Jak2 REGULATES ANGIOTENSIN II-DEPEDENDENT ERK2 SIGNALING

Introduction

Extracellular signal regulated kinase 2 (ERK2) is a member of the mitogen-

activated protein (MAP) kinase family of serine/threonine protein kinases. These

proteins become activated by phosphorylation on tyrosine and threonine residues in

response to a variety of ligands binding their cognate receptors at the cell surface (131-

134). Angiotensin II is one such ligand; it exerts many of its mitogenic effects by

binding to the angiotensin II type 1 (AT1) receptor and activating ERKl/2. This occurs

via rapid angiotensin II-dependent phosphorylation of the dual specificity kinase MEK,

which in turn phosphorylates ERKl/2 on both tyrosine and threonine residues (135).

ERK activity is tightly regulated. The duration of ERK activation is regulated by

the intracellular signals that phosphorylate and dephosphorylate it (136). While ERKs

are activated by ligand binding at the cell surface, they are inactivated by several

dual-specificity phosphatases (137). One of these, MAP kinase phosphatase 1 (MKP-1),

associates with and is phosphorylated by activated ERK2, and thus protected from

proteasomal degradation. The phosphorylated MKP-1 then dephosphorylates ERK2,

thereby inactivating it (138).

Evidence shows that Jak2 forms a membrane complex with the intermediate

signaling molecules Ras and Rafl, and may therefore play a role in the regulation of ERK

activity (44, 139-140). In fact, previous work suggested that inhibition of Jak2 using the

pharmacological compound AG490, blocks the angiotensin II-dependent activation of









ERK2 (18). One problem, though, with using AG490 to study the function of Jak2 is its

lack of specificity for Jak2. To examine the role that Jak2 plays in the regulation of

Ang II-induced ERK2 signaling, we used the y2A cells described in Chapter 3. Briefly,

these are Jak2 -/- cells that stably express the AT1 receptor (y2A/AT1). Controls cells are

the y2A cells stably transfected with both the AT1 receptor and Jak2 (y2A/AT1+Jak2), to

reconstitute Ang II-dependent Jak2 signaling.

Results

ERK2 Activity is Sustained in y2A/AT1 Cells Compared to y2A/AT1+Jak2 Cells after
Angiotensin II Treatment

We sought to determine the role that Jak2 plays in angiotensin II-dependent ERK2

activity using the y2A-derived cells. y2A/AT1 and y2A/AT1+Jak2 cells were stimulated

with 100 nM angiotensin II for 0, 6, 12, 18, 24, and 30 min. The cells were lysed and

protein was extracted. The protein extracts were immunoblotted with an

anti-ACTIVE-ERK2 antibody that detects phosphorylated ERK2 protein. In the cells

lacking Jak2, angiotensin II stimulation resulted in a rapid and sustained increase in

ERK2 activation that persisted for 30 min (Fig. 4-1A, Top). However, in the

y2A/AT1+Jak2 cells, angiotensin II caused an increase in ERK2 activity that peaked at 6-

12 min and returned to basal levels 18-24 min after angiotensin II stimulation. The

membrane was then stripped and reprobed with an anti-ERK2 antibody to show constant

ERK2 expression at all time points (Fig. 4-1A, Bottom).

The 3 different membranes representing Fig. 4-1A were scanned for densimetric

analysis, and ERK2 phosphorylation was plotted as a function of angiotensin II treatment

(Fig. 4-1B). The graph shows that in the cells expressing Jak2, ERK2 phosphorylation

transiently increased, peaking 6 min after angiotensin II treatment. However, in cells

















y2A/AT,

Ang II (min) 0 6 12 18 24 30


y2A/AT,+Jak2

0 6 12 18 24 30


ACTIVE-
ERK2


- *


36-


-- ,, Total
36- ERK2


U
a,
iij 6
E

5
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9!
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3
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2
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cn
o
r
a
hi
Y O
[r
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SAT1
o AT1 +Jak2


0 5 10 15

Ang II (min)


20 25 30 35


Figure 4-1.


ERK2 activity is sustained in y2A/AT1 cells compared to y2A/AT1+Jak2

cells after angiotensin II treatment A) y2A/AT1 and y2A/AT1+Jak2 cells

were treated with 100 nM angiotensin II for the indicated times. Whole

cell lysates were prepared and Western blotted with anti-phospho-ERK2

antibody to detect activated ERK2 (Top). The membrane was then

stripped and reprobed with anti-ERK2 antibody to confirm total ERK2

protein levels (Bottom). B) The three membranes representing Fig. 5-1A
were subjected to densimetric analysis. Anti-phospho-ERK2 signal was

plotted as a function of both angiotensin II treatment and Jak2 expression.
Values are expressed as the mean +/- SD. p<0.01, **p<0.05 (Student's t

test). Adapted from Sandberg et al. Journal of Biological Chemistry.

(2004) 279, pg 1960, Fig. 3A and 3B with permission from publisher.


lacking Jak2, ERK2 phosphorylation was significantly elevated 30 min after angiotensin


II stimulation. Thus, the data in Fig. 4-1 suggest that loss of Jak2 expression via a null


mutation results in sustained ERK2 phosphorylation in response to angiotensin II. This is









contrary to previously published data, which suggested that inhibiting Jak2 kinase

function using AG490 results in diminished angiotensin II-dependent ERK2

phosphorylation (18).

To verify our results using an alternate protocol, in vitro kinase assays were

performed using myelin basic protein (MBP) as a substrate for ERK2 phosphorylation.

In y2A/AT1 cells, phosphorylation of MBP remained elevated 20 min after angiotensin II

treatment, suggesting that ERK2 was catalytically active at this time point (Fig. 4-2).

y2A/AT, y2A/AT +Jak
Ang II (min) 0 5 10 15 20 0 5 10 15 20

13-51 a 32P M/BP


Figure 4-2. In vitro kinase assay confirms that ERK2 activity is sustained in y2A/AT1
cells compared to y2A/AT1+Jak2 y2A/ATi cells and y2A/AT1+Jak2 cells
were treated with 100 nM angiotensin II for the indicated times. Lysates
were prepared and immunoprecipitated with anti-ERK2-mAb and then
resuspended in kinase reaction buffer. Phosphorylation of myelin basic
protein was detected by autoradiography. Shown is one of three
independent results. Adapted from Sandberg et al. Journal of Biological
Chemistry. (2004) 279, pg 1960, Fig. 3C with permission from publisher.

However, in y2A/AT1+Jak2 cells, angiotensin II stimulated ERK2 activity peaked at 5-10

min. After 15-20 min, the 32P-labeled MBP signal was similar to that of basal conditions.

Collectively, the results in Fig. 4-1 and 4-2 clearly show that loss of Jak2

expression via a Jak2 null mutation results in enhanced ERK2 activation in response to

angiotensin II when compared to Jak2-expressing control cells.

AG490 Suppresses Angiotensin II-Dependent ERK2 Activation Independent of Jak2
Inhibition

We next wanted to determine whether treating the Jak2 expressing cells with

AG490 could recapitulate the observations seen in Fig. 4-1. In other words, could we









reproduce the enhanced ERK2 activation phenomenon seen in the Jak2 deficient cells, by

treating the Jak2 expressing cells with AG490? For this, y2A/AT1+Jak2 cells were

treated with either AG490 or the inert control compound, AG9. The cells were then

treated with angiotensin II and phospho-ERK2 levels were directly measured via Western

blot analysis. First, y2A/AT1 cells were treated with angiotensin II to reproduce the

enhanced ERK2 activation seen 30 min after angiotensin II treatment (Fig. 4-3A, lanes 1-

3). In y2A/AT1+Jak2 cells pretreated with the AG9 control compound, Ang II-induced

ERK2 phosphorylation levels were high at 5 min, but returned to basal levels by 30 min,

similar to that shown in Fig. 4-1A (Fig. 4-3A, lanes 4-6). However, in y2A/AT1+Jak2

cells pretreated with AG490, the ability of angiotensin II to induce ERK2

phosphorylation appeared to be lost (Fig 4-3A, lanes 7-9). As such, this result indicates

that pharmacological suppression of Jak2 via AG490 does not recapitulate the effect of

the Jak2 null mutation and suggests that AG490 is blocking angiotensin II-dependent

ERK2 activation via a mechanism that is independent of Jak2.

Recent work suggests that one mechanism by which G protein coupled receptors

activate ERKl/2 is via transactivation of the epidermal growth factor receptor (EGFR)

(14-143). This is important because, as indicated in Chapter 1, AG490 inhibits epidermal

growth factor receptor autophosphorylation more potently that it inhibits Jak2 kinase

activity (100, 101). To determine whether this mechanism of action is responsible for the

angiotensin II-dependent activation of ERK2 in the y2A-derived cells, y2A/AT1+Jak2

cells were treated with the EGFR tyrosine kinase inhibitor, AGl478, before measuring

angiotensin II-dependent ERK2 activation. AGl478 treatment, at a dose that has















y2A/AT,+Jak2


previously been shown to fully suppress EGFR kinase activity (144, 145), failed to

inhibit the angiotensin II-mediated activation of ERK2 in y2A/AT1+Jak2 cells, when


y2A/ATz


AG-9
0 5 30 0 5 30


AG-490
0 5 30


Ang II (min)


ACTIVE-
ERK2


36 -


y2A/AT +Jak2


Control
0 5 30


AG1478
0 5 30


Ang II (min)


--- ~ -- -- 4- Active ERK2


36 -


Figure 4-3.


AG490 suppresses angiotensin II-dependent ERK2 activation independent
of Jak2 inhibition A) Cells were pretreated for 16 hrs with either 100 CIM
AG9 or 100 CIM AG490 and then stimulated with 100 nM angiotensin II
for the indicated times. Whole cell lysates were prepared and then
Western blotted with anti-phospho-ERK2 antibody to detect activated
ERK2 protein. B) Cells were pretreated for 1 hr with 10 CIM AGl478 and
then stimulated with 100 nM angiotensin II for the indicated times. Whole
cell lysates were prepared and then Western blotted with anti-phospho-
ERK2 antibody to detect activated ERK2 protein. Shown is one of three
representative results for each. Adapted from Sandberg et al. Journal of
Biological Chemistry. (2004) 279, pg 1961, Fig. 4A and 4B with
permission from publisher.


compared to similarly treated control cells (Fig. 4-3B). This result suggests that in the

y2A-derived cells, angiotensin II mediates activation of ERK2 via a mechanism that is

independent of EGFR tyrosine kinase activity.

In summary, the data in Fig. 4-3A show that AG490 blocks the angiotensin II-

dependent activation of ERK2 via a mechanism that is independent of Jak2. The data in

Fig. 4-3B show that this nonspecific effect of AG490 is not because of reduced EGFR

tyrosine kinase activity, as direct suppression of EGFR tyrosine kinase activity with









AGl478 does not inhibit angiotensin II-dependent activation of ERK2. Collectively, the

data suggest that AG490 is inhibiting ERK2 activation via nonspecific suppression of a

tyrosine kinase that is not the EGFR.

There is a Marked Difference in the Angiotensin II-Induced Nuclear Accumulation
Pattern of Activated ERK2 between y2A/AT1 and y2A/AT1+Jak2 Cells

ERK2, when activated by angiotensin II, accumulates in the nucleus of cells and

modulates the expression of a variety of genes by activating nuclear transcription factors

such as AP-1 (136). We next wanted to determine if a difference in nuclear accumulation

of activated ERK2 existed between y2A/AT1 and y2A/AT1+Jak2 cells. For this, both cell

types were immunostained with an anti-ACTIVE-ERK2 antibody. The cells were then

visualized using a fluorescent microscope to measure angiotensin II-dependent ERK2

nuclear accumulation, as a function of Jak2 expression (Fig. 4-4). There was a marked

difference in the nuclear accumulation pattern of phospho-ERK2 between the two cell

types. Specifically, in the cells lacking Jak2, angiotensin II treatment facilitated a rapid

nuclear accumulation of activated ERK2. This staining persisted for at least 30 min, but

also appeared to be perinuclear in nature at this time point (arrows). For the Jak2

expressing cells however, angiotensin II treatment promoted a transient nuclear

accumulation of activated ERK2 that was visible at 5 min, but virtually gone at 30 min.

Collectively, these data suggest that the sustained levels of phospho-ERK2 seen in

Fig. 4-1 correlate with increased phospho-ERK2 immunoreactivity in the nucleus of the

cell .

ERK2-Dependent Gene Transcription in the y2A/AT1 and y2A/AT1+Jak2 Cells

To determine if differences in ERK2-mediated gene expression existed between the

y2A/AT1 and y2A/AT1+Jak2 cells, we measured angiotensin II-dependent, ERK2










y2A/AT


y2A/AT +Jak2


Figure 4-4.


Difference in the angiotensin II-dependent nuclear accumulation of
phospho-ERK2 between y2A/AT1 and y2A/AT1+Jak2 cells y2A/AT1 cells
and y2A/AT1+Jak2 cells were treated with 100 nM angiotensin II for
either 0, 5 or 30 min. The cells were incubated with anti-phospho-ERK2-
pAb and immunostained with goat-anti-rabbit antibody conjugated to
Texas Red. The cells were visualized using a fluorescent microscope to
detect nuclear accumulation of activated ERK2 protein. Arrows indicate
apparent perinuclear staining. Shown is one of three independent results.
Adapted from Sandberg et al. Journal of Biological Chemistry. (2004)
279, pg 1962, Fig. 5 with permission from publisher.


mediated gene transcription in the two cell types. This was accomplished using a

synthetic promoter containing seven copies of the AP-1 binding element upstream of a

luciferase reporter. After transfection with this construct, the cells were treated with 100

nM angiotensin II for the indicated times and then lysed. Angiotensin II treatment

elicited a marked increase in luciferase activity in both cell types (Fig. 4-5). Although

the y2A/AT1 cells repeatedly generated higher fold changes in luciferase activity










compared to the Jak2-expressing control cells, the difference failed to reach statistical

significance at any time point.

Jak2 is Essential for Angiotensin II-Induced MKP-1 Expression and Co-association
of MKP-1 with ERK2

The data in Fig. 4-1 and 4-2 show that in the cells lacking Jak2, ERK2 has

sustained levels of activation when compared to the Jak2-expressing cells. We

hypothesized that the signal transduction pathways leading to the induction of ERK2


SAT1
-- AT1 + Jak2













20 25 30


O 5 10 15
100 nM Ang II (hrs)


Figure 4-5.


No difference in angiotensin II-dependent ERK mediated gene
transcription between y2A/AT1 and y2A/AT1+Jak2 cells y2A/AT1 and
y2A/AT1+Jak2 cells were transfected with 10 Clg of a luciferase reporter
whose expression is driven by a synthetic promoter containing seven
copies of the AP-1 binding element to measure ERK-mediated gene
transcription. The cells were treated with 100 nM angiotensin II for the
indicated times. Lysates were prepared and luciferase activity was
measured. Shown is one of three independent results. Adapted from
Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 1963, Fig.
6 with permission from publisher.










specific phosphatases were different in the two cell types. Specifically, we hypothesized

that in the cells lacking Jak2, there would be little to no angiotensin II-mediated induction

of the phosphatases that inactivate ERK2. We therefore tested the ability of angiotensin

II to induce gene expression of two such phosphatases, PP2A and MKP-1, in both cell

types. Quiescent cells were treated with 100 nM angiotensin II for 0, 15, 30, or 60 min,

and protein lysates were prepared. The whole cell lysates were first immunoblotted with

an anti-PP2A antibody (Fig. 4-6A). The results show that there was no marked

difference in PP2A protein expression between the y2A/AT1 and y2A/AT1+Jak2 cells in

response to angiotensin II. Based on these results, we concluded that angiotensin II was

not modulating gene expression of PP2A.

We next immunoblotted similarly prepared protein extracts with an anti-MKP-1

antibody, and found that in y2A/AT1 cells, angiotensin II induced very little MKP-1

expression (Fig 4-6B). In the y2A/AT1+Jak2 cells however, angiotensin II induced

marked MKP-1 expression, demonstrating that maximal angiotensin II-induced MKP-1

protein expression requires Jak2.

MKP-1 associates with ERK2 in response to angiotensin II treatment, and is

activated by ERK2. MKP-1, in turn dephosphorylates ERK2 (138). We next

investigated if Jak2 is required for co-association of MKP-1 with ERK2. y2A/AT1 and

y2A/AT1+Jak2 cells were treated with 100 nM angiotensin II for 0, 15, and 30 min. The

cells were lysed and cellular protein was extracted. The protein extracts were

immunoprecipitated with anti-ERK2 antibody and then immunoblotted with anti-MKP-1

antibody. The results show that no increase in co-association of ERK2 and MKP-1 was










observed in y2A/AT1 cells, whereas in y2A/AT1+Jak2 cells, angiotensin II induced a

substantial increase in co-association of ERK2 and MKP-1 (Fig. 4-6C).

The data in Fig. 4-6B suggest that Jak2 is playing a key role in the angiotensin II-

dependent increased expression of MKP-1. Previous work has shown that ERK2 itself

can also be a critical mediator of MKP-1 gene expression (137, 138). To determine the

relative contribution of ERK2 and Jak2 in mediating angiotensin II-dependent MKP-1

gene expression, both cell types were treated with angiotensin II in the presence or

absence of the MEK specific inhibitor, PD98059 (Fig. 4-6D). For the cells lacking Jak2,

ligand treatment only modestly induced MKP-1 expression (lanes 1-3), and this was

completely blocked with PD98059 (lanes 4-6). However, for the cells expressing Jak2,

ligand treatment again induced marked MKP-1 expression (lanes 7-9), and this was

partially blocked with PD98059 (lanes 10-12). Thus, the data indicate that there is both a

Jak2-dependent component and an ERK2-dependent component to the angiotensin II-

mediated induction of MKP-1, as maximal MKP-1 expression is only attained when cells

have both functional Jak2 and ERK2.

Collectively, the data in Fig. 4-6 suggest that Jak2 is not only required for induction

of MKP-1 expression in response to angiotensin II, but also for co-association of MKP-1

and ERK2. Additionally, the data indicate that that both Jak2 and ERK2 are required for

maximal angiotensin II-mediated MKP-1 protein expression.

MPK-1 is Required for Angiotensin II-Dependent Inactivation of ERK2

The data in Fig. 4-6 show that Jak2 is required for angiotensin II-dependent

induction of MKP-1, but not PP2A. However, the data clearly show that both proteins

are present in the cell. To determine the extent to which these two phosphatases regulate















L~ -PP2A


y2A/AT, y2A/AT,+Jak
0 15 30 60 0 15 30 60


Ang II (min)


24-


y2A/AT, y2A/AT,+Jak
0 30 60 0 30 60


Ang II (min)


- -- +-~ MKP-1


111-


C

Ang II (min)






D



Ang II (min)


+- MKP-1


y2A/AT, y2A/AT,+Jak
0 15 30 0 15 3

Ali~ib w g

IP :aERK2-mAb
IB :aMKP l-pAb


y2A/AT,
Control PD98059
0 30 60 0 30 60


y2A/AT,+Jak
Control PD98059
0 30 60 0 30 60


.+ MKP-1


- -


Figure 4-6.


Jak2 is essential for angiotensin II-induced MKP-1 expression y2A/AT1
and y2A/AT1+Jak2 cells were treated with angiotensin II for the indicated
times and cell lysates were prepared. A) Whole cell lysates were Western
blotted with anti-PP2A-mAb to detect PP2A expression. B) Whole cell
lysates were Western blotted with anti-MKP-1-mAb to detect MKP-1
expression. C) Lysates were immunoprecipitated with anti-ERK2-mAb
and Western blotted with anti-MKP-1-mAb to detect co-association of
MKP-1 with ERK2. D) Cells were pre-treated for 60 min with either
DMSO or 50 C1M PD98059 and then stimulated with 100 nM angiotensin
II for the indicated times. Whole cell lysates were prepared and then
Western blotted with anti-MKP-1-mAb to detect MKP-1 protein. Shown
is one of four (A) or three (B, C, D) independent results for each. Adapted
from Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg
1964, Fig. 7 with permission from publisher.









the dephosphorylation of ERK2, angiotensin II-mediated ERK2 phosphorylation was

measured in the presence, or absence, of PP2A and MKP-1 specific inhibitors.

To inhibit PP2A, which is a serine/threonine specific phosphatase, y2A/AT1 and

y2A/AT1+Jak2 cells were pretreated with the PP2A specific inhibitor, okadaic acid (146).

The cells were then treated with angiotensin II and ERK2 phosphorylation was measured

via Western blot analysis (Fig. 4-7 A). In the y2A/AT1 cells, before ligand treatment,

ERK2 was already phosphorylated, possibly because of the presence of a phosphatase

inhibitor. Addition of angiotensin II did not significantly increase the signal at either the

5 or 30 min time points, thereby suggesting that the signal was at, or near, maximal

phosphorylation levels before ligand treatment. Additionally, the data suggest that

angiotensin II treatment did not induce the necessary cellular factors, such as Jak2, that

are required for dephosphorylating ERK2, since ERK2 remained phosphorylated 30 min

after ligand treatment. For the y2A/AT1+Jak2 cells, ERK2 was also basally

phosphorylated before ligand treatment. In contrast however, 30 min of angiotensin II

treatment promoted its relative dephosphorylation. This observation suggests two

important things. First, it indicates that the y2A/AT1+Jak2 cells contain the necessary

components) to promote the dephosphorylation of ERK2 (ie. Jak2). Second, the data

suggest that this ligand dependent dephosphorylation does not require PP2A since the

dephosphorylation occurs in the presence of okadaic acid.

To inhibit MKP-1, which is a threonine/tyrosine dual specificity phosphatase,

y2A/AT1 and y2A/AT1+Jak2 cells were pretreated with the MKP-1 inhibitor, vanadate

(147). The cells were then treated with angiotensin II and ERK2 phosphorylation was

again measured via Western blot analysis with anti-ACTIVE-ERK2 antibody (Fig. 4-7B).










In the y2A/AT1 cells, before ligand treatment, ERK2 showed some basal phosphorylation.

Addition of angiotensin II modestly increased the signal at both the 5 and 30 min time

points. These cells were once again unable to dephosphorylate ERK2 after 30 min of

ligand treatment. Similarly, in the y2A/AT1+Jak2 cells, ERK2 was basally

phosphorylated before ligand treatment and 5 min of angiotensin II treatment

A Okadaic Acid
y2A/AT, y2A/AT +Jak2
Ang II (min) 0 5 30 0 5 30

- + Active ERK2
36 -



B Vanadate

y2A/AT, y2A/AT +Jak2
Ang II (min) 0 5 30 0 5 30
~~ Active ERK2
36 -


Figure 4-7. Angiotensin II-dependent inactivation of ERK2 requires Jak2 and MKP-1
A) y2A/AT1 and y2A/AT1+Jak2 cells were pretreated for 1 hr with 500 nM
okadaic acid and then stimulated with 100 nM angiotensin II for the
indicated times. Whole cell lysates were prepared and then Western
blotted with anti-phospho-ERK2 antibody to detect activated ERK2
protein. B) y2A/AT1 and y2A/AT1+Jak2 cells were pretreated for 1 hr
with 100 CIM sodium ortho-vanadate and then stimulated with 100 nM
angiotensin II for the indicated times. Whole cell lysates were prepared
and then Western blotted with anti-phospho-ERK2 antibody to detect
activated ERK2 protein. Shown is one of three independent results for
each. Adapted from Sandberg et al. Journal of Biological Chemistry.
(2004) 279, pg 1965, Fig. 8 with permission from publisher.

modestly increased its signal. However, unlike the previous experiments in the Jak2

expressing cells, ERK2 phosphorylation levels remained elevated at the 30 min time

point. These data thereby suggest that MKP-1 is critical for mediating the angiotensin II-

dependent dephosphorylation of ERK2, as vanadate treatment of these cells blocks the










angiotensin II-dependent dephosphorylation of ERK2. Collectively, the data in Fig. 4-7

suggest that PP2A and MKP-1 play distinct roles in the dephosphorylation of ERK2;

PP2A appears to be largely responsible for the basal phosphorylation state of ERK2

while MKP-1 appears to regulate angiotensin II-dependent dephosphorylation.

Moreover, this ligand dependent dephosphorylation of ERK2 by MKP-1 requires Jak2.

Discussion

Here, we report several new observations. First, lack of Jak2 signaling in a cell

increases the duration of ERK2 activity after angiotensin II stimulation. This was shown

by both Western blot analysis and in vitro kinase assays. This observation is contrary to

what has been reported previously in studies using AG490 to study the role that Jak2

plays in ERK activation. One possible reason for this discrepancy is that AG490 has

been shown to inhibit other tyrosine kinases nonspecifically (98-101). A second

observation that we report is that angiotensin II induces rapid induction of the MKP-1

gene. We report that angiotensin II not only causes activation of ERK2, but also

simultaneously induces upregulation of MKP-1, a phosphatase that inactivates ERK2.

This is an interesting example of the tight regulation of ERK signaling within a cell.

Furthermore, we report that angiotensin II-induced upregulation of MKP-1 gene

expression is dependent on Jak2 expression. We believe that this is the reason that in

y2A/AT1 cells lacking Jak2, ERK2 activity is sustained after angiotensin II stimulation

compared to y2A/AT1+Jak2 cells. Without Jak2 present, angiotensin II is simply unable

to upregulate MKP-1 expression, and thus the cell is slower to inactivate ERK2.

Interestingly, in the y2A/AT1 cells lacking Jak2, after 30 min of angiotensin II

treatment the active ERK2 showed predominantly perinuclear localization. There was










not as much nuclear localization of active ERK2 after 30 min of treatment compared to 5

min of treatment. Therefore, despite the sustained ERK2 activation in the y2A/AT1 cells

lacking Jak2, there is not a sustained ERK2 nuclear localization. This is probably the

reason that no significant difference in gene transcription between the two cell types was

observed. Furthermore, it suggests that ERK2 nuclear localization is not dependent only

upon ERK2 activation. Instead, ERK2 nuclear localization appears to be controlled in a

temporal manner, so that even though a cell may have sustained ERK2 activity, it may

not show sustained ERK2 nuclear localization and ERK2-mediated gene transcription.

This presents an interesting possibility. Perhaps in cells with sustained ERK2 activity,

the cytosolic actions, rather than the nuclear actions, of ERK2 are sustained. This could

be particularly relevant in cancer cells showing constitutive ERK activity. Further testing

is needed to determine this.

Fig. 4-8 shows a model depicting what we believe is occurring in normal cells.

Upon angiotensin II stimulation, ERK2 becomes phosphorylated on threonine and

tyrosine residues. Simultaneously, Jak2 becomes tyrosine phosphorylated and associates

with the AT1 receptor. Jak2 induces expression of IVKP-1, presumably through

activation of one or more STAT proteins. ERK2 also acts on the IVKP-1 promoter to

increase its expression. The IVKP-1 protein that is generated then associates with and

inactivates ERK2.

Previous work has shown that the duration of ERK activation is critical for

determining cell fate (148-150). In some cell signaling systems for instance, transient

activation of ERK2 is a common feature of cell proliferation. Sustained activation on the

other hand, is associated with very different cellular events such as apoptosis or









senescence. Previously published work has shown that Jak2 can promote both cellular

proliferation and apoptosis (151, 152). How Jak2 elicits such different cellular responses

is presumably dependent on the specific cell type and ligand used in each experiment.

However, the exact cellular and biochemical mechanisms) by which Jak2 accomplishes

this is not fully known. Our work here shows that Jak2 plays an important role in

determining whether ERK2 activation is transient or sustained. As such, we may have

AT, Receptor













(+)







Promoter MKP-1
Nucleus


Figure 4-8. Proposed model of the mechanism by which Jak2 mediates ERK2
inactivation after angiotensin II treatment Angiotensin II binding to its
type 1 receptor activates ERK2, while simultaneously activating Jak2.
Jak2, probably through the action of STAT proteins, increases expression
of MKP-1. ERK2 also increases MKP-1 expression. The expressed
MKP-1 protein then associates with and dephosphorylates ERK2, thus
inactivating the signal. Adapted from Sandberg et al. Journal of
Biological Chemistry. (2004) 279, pg 1966, Fig. 9 with permission from
publisher.











determined that one mechanism by which Jak2 influences cell fate is by altering the

duration of ERK2 activation via induction of IVKP-1. Clearly, further studies are needed

to fully address this issue.















CHAPTER 5
Jak2 PROMOTES OXIDATIVE STRESS-INDUCED APOPTOSIS IN VASCULAR
SMOOTH MUSCLE CELLS

Introduction

In 1998, it was demonstrated that Jak2 is activated in response to oxidative stress

(36). While the signaling cascade responsible for oxidative stress-induced Jak2 activity

was at least partially elucidated in vascular smooth muscle cells, no physiological role

has been ascribed to this pathway. Runge's group (35) showed that Jak2 activation by

oxidative stress caused up regulation of heat-shock protein 70, a protein that can protect

cells from oxidative stress. These data were generated using AG490 to inhibit Jak2

function. Based on these data, they suggested that Jak2 might help vascular smooth

muscle cells adapt to oxidative stress (35).

Oxidative stress in vascular smooth muscle cells can cause proliferation,

contraction, or apoptosis (153-155). How the same stimulus can result in such opposing

endpoints is unknown, but is probably dependent on oxidant dose. Furthermore, the

signaling proteins that mediate these different responses are unknown. Oxidative stress-

induced apoptosis of vascular smooth muscle cells contributes to the progression of a

number of vascular pathologies, including atherosclerosis and restenosis (156, 157).

Identifying the mediators of oxidant-induced apoptosis may therefore uncover novel

therapeutic targets. Interestingly, both pro- and anti-apoptotic roles have been ascribed to

Jak2 tyrosine kinase activity in a variety of signaling systems (34, 97, 158). We therefore










sought to determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis in

vascular smooth muscle cells.

Results

Jak2 Activation by Hydrogen Peroxide is Suppressed in RASM-DN Cells

To study the role that Jak2 activation plays in oxidative stress-induced apoptosis,

we used rat aortic smooth muscle cells stably expressing a Jak2 dominant negative

protein (RASM-DN). We used the same cells, expressing only a neomycin resistant

cassette, as controls (RASM-Control). To show that Jak2 activation by hydrogen

peroxide is inhibited in RASM-DN cells, we treated RASM-Control and RASM-DN cells

with 0.2, 0.5, or 1.0 mM hydrogen peroxide for 0, 5, or 10 min (Fig. 5-1, Top). Cellular

lysates were immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted

with anti-Jak2 antibody, to detect tyrosine phosphorylated Jak2. Aliquots from the

cellular lysates were Western blotted with anti-Jak2 antibody to demonstrate equal

protein loading amongst all samples (Fig. 5-1, Bottom).

The results show that Jak2 is strongly activated by hydrogen peroxide in RASM-

Control cells, but Jak2 activation is greatly reduced in RASM-DN cells. This shows that

RASM-Control and RASM-DN cells are good models for studying the physiological role

of Jak2 during oxidative stress in vascular smooth muscle cells.

Jak2 Activation is Required for Oxidative Stress-Induced Apoptosis

To determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis,

RASM-Control and RASM-DN cells were treated with 1 mM hydrogen peroxide for 24

h. We assessed apoptosis by analyzing genomic DNA isolated from each cellular

condition (Fig. 5-2A). Genomic DNA isolated from RASM-Control cells that were

treated with hydrogen peroxide was fragmented into approximately 200 base pair bands,









RASM-Control RASM-DN

0.2 mM 0.5 mM 1.0 mM 0.2 mM 0.5 mM 1.0 mM
H202 (min) 0 5 10 5 10 5 10 0 5 10 5 10 5 10


111- ** +-I~P Jak2(Pl lt'kP)







Figure 5-1. Hydrogen peroxide-induced Jak2 activity is suppressed in RASM-DN
cells RASM-Control and RASM-DN cells were treated with 0.2 mM, 0.5
mM, or 1.0 mM hydrogen peroxide for 0, 5, or 10 min. Cellular lysates
were immunoprecipitated with anti-phosphotyrosine-mAb antibody and
immunoblotted with anti-Jak2-pAb antibody to detect tyrosine
phosphorylated Jak2 (Top). An aliquot from each lysate was Western
blotted with anti-Jak2-pAb to demonstrate equal Jak2 expression amongst
all samples (Bottom). Shown is one of three representative experiments.
Adapted from Sandberg et al. Journal of Biological Chemistry. (2004)
279, pg 34548, Fig. 1 with permission from publisher.

characteristic of apoptosis. In contrast, genomic DNA from RASM-DN cells treated with

hydrogen peroxide showed no evidence of DNA fragmentation. Furthermore, AG490

prevented genomic DNA fragmentation in RASM-Control cells treated with hydrogen

peroxide. Next, to show that RASM-DN cells could undergo apoptosis in response to

another pro-apoptotic stimulus, genomic DNA was isolated from RASM-DN cells treated

for 4 h with 5 CIM staurosporine, a potent activator of the intrinsic apoptosis pathway

(Fig. 5-2B). The banding pattern characteristic of fragmented DNA was clearly seen in

these cells. Finally, to determine the effect of different doses of hydrogen peroxide on

vascular smooth muscle cell apoptosis, we treated RASM-Control and RASM-DN cells

with 0.2, 0.5, or 1.0 mM hydrogen peroxide for 24 h and examined genomic DNA for

fragmentation (Fig. 5-2C). The results show that, as expected no DNA fragmentation









occurs in RASM-DN cells. In RASM-Control cells, 1.0 mM hydrogen peroxide was

required to induce DNA fragmentation. Collectively, these results show that Jak2

activation is required for oxidative stress-induced apoptosis in vascular smooth muscle

cells.


RASM-DN RASM-Control


RASMDN

c~C~'e
~P c~,~3
~~Sdv ,,10
~," ,;cp


RASMDN RASMControl

5~.be~ .~L1:~ -1~5~?1~5~-~~9~~ -~PyS:~i~h
tI~0,(24h) Ij~v~" o d~ 29 ~"' o o~ 2;


Figure 5-2.


Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular
smooth muscle cells A) RASM-DN and RASM-Control cells were either
left untreated, or treated with 1 mM hydrogen peroxide for 24 h, or with 1
mM hydrogen peroxide for 24 h after 16 h of pretreatment with 100 C1M
AG490. Genomic DNA was isolated and separated on a 1.8% agarose gel.
The gel was stained with EtBr and visualized under U.V. light to detect
genomic DNA laddering. B) RASM-DN cells were either left untreated or
treated with 5 C1M staurosporine for 4 h. Genomic DNA was isolated and
separated on a 1.8% agarose gel. The gel was stained and visualized to
detect genomic DNA laddering. C) RASM-Control and RASM-DN cells
were treated with 0.2 mM, 0.5 mM, or 1.0 mM hydrogen peroxide for 24
h. Genomic DNA was isolated and separated on a 1.8% agarose gel. The
gel was stained with EtBr and visualized under U.V. light to detect
genomic DNA laddering. Shown is one of four (A and B) or three (C)
representative experiments for each. Adapted from Sandberg et al. Journal
of Biological Chemistry. (2004) 279, pg 34548, Fig. 2 with permission
from publisher.









Quantification of Jak2-Mediated Apoptosis

We next quantified the amount of apoptosis occurring in RASM-Control and

RASM-DN cells treated with hydrogen peroxide. The cells were grown on microscope

slides and treated with 1 mM hydrogen peroxide for either 0 or 24 h. The cells were then

stained with the nucleus-specific Hoechst 33342 dye. Representative photomicrographs

of RASM-Control and RASM-DN cells either left untreated, or treated with hydrogen

peroxide, or hydrogen peroxide after pretreatment with the Jak2 inhibitor AG490, are

shown (Fig. 5-3). RASM-Control cells treated with hydrogen peroxide had shrunken

nuclei that fluoresced more intensely than untreated cells, which is indicative of apoptotic

cells. In contrast, RASM-Control cells treated with AG490 before hydrogen peroxide

treatment appeared similar to untreated cells. Similarly, RASM-DN cells either treated

with hydrogen peroxide alone, or treated with both AG490 and hydrogen peroxide

showed nuclear staining akin to untreated cells.

Four replicate experiments were performed for each condition, and a minimum of

100 cells were counted from each replicate. Cells clearly showing condensed and/or

fragmented nuclei were counted as apoptotic. Data are presented as percentage of cells

undergoing apoptosis (Fig. 5-4). These data show that untreated RASM-Control and

RASM-DN cells showed very low levels of apoptosis (2.4+/-0.54% and 2.9+/-0.85%

respectively). RASM-Control cells treated with hydrogen peroxide showed 54.1+/-

3.71% of total cells undergoing apoptosis, while only 12.3+/-1.93% of RASM-DN cells

treated with hydrogen peroxide were apoptotic. Finally, 5.7+/-0.81% of RASM-Control

cells and 4.4+/-0.98% of RASM-DN cells that were pretreated with AG490 before

hydrogen peroxide treatment were apoptotic.









Untre ated H 02 AG490+H O,



RASM-Control I




RASM-DN I



Figure 5-3. Hoechst staining to detect nuclear condensation RASM-Control and
RASM-DN cells were grown on microscope slides and treated with 1 mM
hydrogen peroxide for 0 or 24 h, or with 1 mM hydrogen peroxide for 24 h
after 16 h of pretreatment with 100 CIM AG490. The cells were then
stained with 50 Clg/mL Hoechst 33342 nuclear stain, fixed, mounted, and
visualized using a florescent microscope to detect nuclear condensation.
Shown is one of four representative photographs of each condition.
Adapted from Sandberg et al. Journal of Biological Chemistry. (2004)
279, pg 34549, Fig. 3A with permission from publisher.

Jak2 Activation by Oxidative Stress Mediates Bax Expression

We hypothesized that Jak2 was promoting apoptosis by regulating expression of a

pro-apoptotic protein. Since Bax is a pro-apoptotic protein required for oxidative stress-

induced apoptosis, we used Western blotting to determine whether Jak2 was required for

oxidative stress-mediated up regulation of Bax expression. For this, RASM-Control and

RASM-DN cells were treated with 1 mM hydrogen peroxide for 0, V/2, 1, 2, or 3 h (Fig. 5-

5A). Cellular lysates were Western blotted with anti-Bax antibody (Fig. 5-5A, Top). The

data show that in RASM-Control cells, hydrogen peroxide induced rapid and transient

induction of Bax expression, which peaked at 1-2 h. In contrast, hydrogen peroxide did

not induce Bax expression in RASM-DN cells.











70



50 -





20 I

10 -

0
Untreated LO, AG490 Untreated HO, AG490
+H, O, +H, O,
RASM-Control RASM-DN

Figure 5-4. Quantification of apoptosis in RASM-Control and RASM-DN cells. A
minimum of 100 cells from each of 4 replicates for each of the 6 treatment
conditions represented in Fig 5-3 was counted. Apoptotic cells were
counted as those showing condensed and/or fragmented nuclei. Data are
presented as percentage of cells undergoing apoptosis +/- S.D. *, p<0.001.
Statistical analysis was performed using Student's t test. printed with
Adapted from Sandberg et al. Journal of Biological Chemistry. (2004)
279, pg 34549, Fig. 3B with permission from publisher.

We next stripped and re-probed the membrane with anti-BCL-2 antibody to

determine whether Jak2 influences expression of theanti-apoptotic BCL-2 protein (Fig. 5-

5A, Middle). We found that BCL-2 expression increased slightly in both cell types in

response to hydrogen peroxide. Because there was no difference in the two cell types, we

concluded that Jak2 does not effect expression of BCL-2. To demonstrate equal protein

loading amongst all samples, the membrane was stripped and re-probed with anti-STAT1

antibody (Fig. 5-5A, Bottom). These data show that Jak2 activation is required for

induction of the pro-apoptotic Bax protein in response to oxidative stress. To confirm

this result, RASM-Control cells were retreated with either the Jak2 inhibitor AG490 or

its inactive analog AG9, and then treated with hydrogen peroxide for 0, 1/2, 1, 2, or 3 h










(Fig. 5-5B). The data show that AG490 attenuates hydrogen peroxide-mediated Bax

induction in RASM-Control cells (Fig. 5-5B, Top). The membrane was stripped and

reprobed with anti-STAT1 antibody to demonstrate equal loading amongst all samples

(Fig. 5-5B, Bottom). Collectively, the data in Fig. 5-5 present strong evidence that Jak2

activation promotes oxidative stress-induced apoptosis by mediating an increase in Bax

protein expression levels in vascular smooth muscle cells.

Jak2 Activation by Oxidative Stress Promotes Mitochondrial Dysfunction

During oxidative stress, the pro-apoptotic Bax protein localizes to the outer

mitochondrial membrane and increases mitochondrial membrane permeability (159, 160).

This is an essential step in the intrinsic apoptosis pathway (161, 162). Since we showed

in Fig. 5-5 that Jak2 tyrosine kinase mediates induction of Bax expression, we

hypothesized that activation of Jak2 by hydrogen peroxide may also contribute to

mitochondrial dysfunction. To test this, we used the MitoCapture reagent to stain live

cells treated with hydrogen peroxide and visualized the cells using confocal microscopy

(Fig. 5-6). In healthy, non-apoptotic cells, the dye accumulates predominantly in the

mitochondria where it forms aggregates, and fluoresces red. In contrast, in apoptotic

cells, because of the change in mitochondrial trasnsmembrane potential, the dye remains

predominantly in the cytosol as a monomer, and fluoresces green. The data show that in

untreated RASM-Control and RASM-DN cells, the MitoCapture dye fluoresces

predominantly red, indicating that these cells are non-apoptotic. After 1 mM hydrogen

peroxide treatment for 2 h, the RASM-Control cells show a decrease in red staining and a

dramatic increase in green staining, indicative of mitochondrial dysfunction in these cells.

In contrast, the RASM-DN cells treated with hydrogen peroxide actually show increased
























--i ~~5~__


RASM-Control


RASM-DN


1 mM HO (h)


0 1/2 1 2 3 0


1/2 1 2 3


- -


IB: aBax-pAb



IB: aBCL2-mAb



IB: a STAT1l-pAb


AG9


AG490


1 mM H,O, (h) 0 1/2 1 2 3 0 1/2 1 2 3


IB: aBax-pAb

IB: aSTAT1-pAb


Figure 5-5.


Jak2 mediates hydrogen peroxide-induced up regulation of Bax expression
A) RASM-Control and RASM-DN cells were treated with 1 mM
hydrogen peroxide for 0, '/, 1, 2, or 3 h. Cellular lysates were Western
blotted with anti-Bax-pAb antibody (Top). The membrane was stripped
and reprobed with anti-BCL2-mAb (Middle). Finally, the membrane was
stripped and re-probed with anti-STAT1-pAb to demonstrate equal
loading amongst all samples (Bottom). B) RASM-Control cells were
pretreated with 100 CIM AG9 or 100 CIM AG490 for 16 h, followed by
treatment with 1 mM hydrogen peroxide for 0, '/, 1, 2, or 3 h. Cellular
lysates were Western blotted with anti-Bax-pAb (Top). The membrane
was stripped and re-probed with anti-STAT1-pAb to demonstrate equal
loading amongst all samples (Bottom). Shown is one of three
representative experiments for each. Adapted from Sandberg et al. Journal
of Biological Chemistry. (2004) 279, pg 34550, Fig. 4 with permission
from publisher.


red staining, and only marginally increased green staining compared to control cells.

This indicates that hydrogen peroxide-induced Jak2 activation promotes mitochondrial

dysfunction in vascular smooth muscle cells. To quantify these results, we used









morphometric scanning software to determine the ratio of green staining intensity to red

staining intensity (Green:Red) (Fig. 5-7). A higher Green:Red staining intensity is

indicative of a greater loss of mitochondrial transmembrane potential. The results

indicate that the Green:Red staining intensity was significantly higher in RASM-Control

Untreated 2 hr H202




Control





DN




Figure 5-6. Jak2 is essential for hydrogen peroxide-induced mitochondrial membrane
dysfunction RASM-Control and RASM-DN cells were grown on
microscope slides and treated with 1 mM hydrogen peroxide for 0 or 2 h.
Cells were stained with the MitoCapture reagent and visualized using a
confocal microscope. Predominant red staining is indicative of healthy
cells, while predominant green staining is indicative of apoptotic cells.
Shown is one of three representative experiments. Adapted from
Sandberg et al. Journal of Biological Chemistry. (2004) 279, pg 34551,
Fig. 5A with permission from publisher.

cells treated with hydrogen peroxide than in similarly treated RASM-DN cells, showing

that relative to RASM-Control cells, RASM-DN cells maintained their mitochondrial

transmembrane potential.

Jak2 is Required for Caspase-9 Cleavage During Oxidative Stress

Caspase-9 is the primary initiator caspase of the intrinsic apoptosis pathway, and is

involved in hydrogen peroxide-induced apoptosis. Caspase-9 is cleaved to its active form

following disruption of mitochondrial integrity (163). We therefore examined caspase-9










cleavage after hydrogen peroxide treatment in RASM-Control and RASM-DN cells (Fig.

5-8). The two cell types were treated with 1 mM hydrogen peroxide for 0, V/2, 1, 2, or 3 h,

and cellular lysates were Western blotted with anti-CLEAVED-Caspase-9 antibody (Fig.

5-8, Top). The data show that in RASM-Control cells, hydrogen peroxide treatment

causes accumulation of cleaved caspase-9. In contrast, little caspase-9 cleavage occurred


0.9
0.8
S0.7
e 0.6


S0.4
2' 0.3

S0.1
0.

Conr~ol Control DN DN+H,O,
+H,O,


Figure 5-7. Pixel intensity of each of 4 photographs from each condition in Fig. 5-6
was determined, and intensity of green and red staining was determined
for each. Data are presented as average ratio of green to red pixel
intensity +/- S.D. for each condition. *, p<0.01. Statistical analysis was
performed using Student's t test Adapted from Sandberg et al. Journal of
Biological Chemistry. (2004) 279, pg 34451, Fig. 5B with permission
from publisher.

in RASM-DN cells. The membrane was stripped and re-probed with anti-STAT1

antibody to show equal protein loading amongst all samples (Fig. 5-8, Bottom). Jak2

tyrosine kinase activity is therefore required for cleavage and activation of caspase-9 in

vascular smooth muscle cells during oxidative stress.

Discussion

Although Jak2 is traditionally considered a mediator of cytokine signaling, other

ligands and stimuli can activate this signaling pathway (18, 36). Oxidative stress is one









such stimulus; Jak2 is potently activated by hydrogen peroxide in a number of cell types,

yet no physiological endpoint has been attributed to hydrogen peroxide-induced Jak2

activity (35, 36). Additionally, hydrogen peroxide induces apoptosis in vascular smooth

muscle cells, yet few intracellular mediators of hydrogen peroxide-induced apoptosis

have been identified (164).

RASM-Control RASM-DN
1 mM H202(h) 0 1/2 1 2 3 0 1/2 1 2 3

CLE VD-as de- IB: uCLEAVED-Caspase-9



80- IB: uSTAT1-pAb


Figure 5-8. Jak2 is required for oxidative stress-induced caspase-9 cleavage RASM-
Control and RASM-DN cells were treated with 1 mM hydrogen peroxide
for 0, V/2, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-
CLEAVED-Caspase-9-pAb to detect Caspase-9 activation (Top). The
membrane was stripped and re-probed with anti-STAT1-pAb to
demonstrate equal loading amongst all samples (Bottom). Shown is one
of three representative experiments. Adapted from Sandberg et al. Journal
of Biological Chemistry. (2004) 279, pg 34551, Fig. SC with permission
from publisher.

Here, we report that Jak2 activation by oxidative stress in the form of hydrogen

peroxide mediates apoptosis of vascular smooth muscle cells. We demonstrated

fragmentation of genomic DNA, a characteristic of apoptosis, in control rat aortic smooth

muscle cells treated with hydrogen peroxide (RASM-Control), but that fragmentation

was non-existent in the same cells expressing a Jak2 dominant negative protein (RASM-

DN), or in RASM-Control cells treated with the Jak2 inhibitor AG490. We observed a

significant decrease in the percentage of cells undergoing apoptosis in response to

hydrogen peroxide in the RASM-DN cells compared to RASM-Control cells. Moreover,

pretreatment with AG490 reduced the percentage of cells undergoing hydrogen peroxide-









induced apoptosis nearly to the level of untreated cells, in both cell types, again

suggesting a critical role for Jak2 in apoptosis. Finally, we provided evidence of the

mechanism by which this occurs. In RASM-Control cells, expression of the pro-

apoptotic Bax protein was rapidly induced. There was no such Bax induction in the

RASM-DN cells. Furthermore, in RASM-Control cells, AG490 attenuated hydrogen

peroxide-mediated Bax induction. This is the first evidence that Jak2 mediates Bax

protein expression in response to hydrogen peroxide.

During apoptosis, Bax is largely responsible for loss of mitochondrial

transmembrane potential and subsequent increase in mitochondrial membrane

permeability. This mitochondrial dysfunction allows translocation of macromolecules

such as cytochrome c from the inner mitochondrial membrane to the cytosol, leading to

cleavage and activation of caspase-9. We found that mitochondrial membrane integrity

was compromised and caspase-9 was cleaved in RASM-Control cells, but not in RASM-

DN cells, indicating an essential role for Jak2 in these events.

This report has therefore identified apoptosis as a physiological endpoint of Jak2

activation by hydrogen peroxide. Moreover, this work shows that Jak2 is a novel

mediator of hydrogen peroxide-induced apoptosis in vascular smooth muscle cells.

These results could have profound consequences for the treatment of a number of

vascular diseases in which oxidative stress-mediated cell death plays a prominent role.

As such, this work identifies Jak2 as a potential therapeutic target in vascular diseases

associated with oxidative stress. Atherosclerosis is one such disease. During the

development of an atherosclerotic plaque, a fibrous cap forms over the plaque. Vascular

smooth muscle cells that have migrated from the medial layer of the blood vessel are









found within the fibrous cap. These cells are exposed to large amounts of oxidative stress

derived largely from circulating macrophages. This oxidative stress can cause apoptosis

of the vascular smooth muscle within the fibrous cap, leading to cap weakening, and

accelerating the time to plaque rupture. If Jak2 plays a role in oxidative stress-induced

apoptosis in vivo during atherosclerosis, inhibition of Jak2 could stabilize the plaque.

Since Jak2 knockout mice die embryonically, investigating the role that Jak2 plays

during atherosclerosis is difficult. One possibility is to use vascular smooth muscle cell

restricted expression of the Jak2 dominant negative protein to study the in vivo role of

Jak2. Mice expressing Jak2 only in vascular smooth muscle cells could then be crossed

with apolipoprotein deficient mice, which develop atherosclerosis when fed a high fat

diet, to examine the role of Jak2 during atherosclerosis.

Interestingly, Jak2 can play either pro- or anti-apoptotic roles depending on the

signaling system and apoptotic stimulus examined. Where, then, is the specificity of the

response controlled? One possibility is that the specific STAT proteins that are activated

by Jak2 determine whether Jak2 plays a pro- or anti-apoptotic role. For instance,

activation of STAT3 is usually associated with inhibition of apoptosis, while STAT 1

activation has been associated with induction of apoptosis. It is possible that STAT1 is

the predominant STAT activated during oxidative stress. Studies using dominant

negative mutants of the STAT1 and STAT3 proteins could be used to determine which

STAT protein is responsible for the pro-apoptotic role for Jak2.

Finally, whether Jak2 plays a role in oxidative stress-induced apoptosis in other cell

types should be examined. Many cell types are exposed to high oxidative stress,

especially during disease. These include endothelial cells and cardiomyocytes. It will be






60


interesting to determine if the pro-apoptotic role of Jak2 is restricted to vascular smooth

muscle cells, or if it is a ubiquitous role.















CHAPTER 6
Jak2 RESIDUES GLU 1024 AND ARG 1113 FORM HYDROGEN BONDS, AND ARE
ESSENTIAL FOR Jak-STAT SIGNAL TRANSDUCTION

Introduction

Structure-function studies have identified several specific amino acid residues

within Jak2 that are essential for its activation. For example, conversion of Lys 882 to

Glu (K882E) within subdomain II rendered Jak2 catalytically inactive (104, 105).

Similarly, conversion of Tyr 1007 to Phe (Y1007F) prevented ligand-mediated Jak2

activation (48). A double mutation of W1020G/E1024A within sub domain VIII not only

inactivated Jak2, but also rendered the molecule dominant negative (106). This double

mutant is of interest to our laboratory because we have shown that expression of it in

cells inhibits angiotensin II-mediated Jak2 activation, Jak2/AT1 receptor co-association,

STAT1 tyrosine phosphorylation, and ligand-dependent gene transcription (38, 39). We

recently showed that mutation of either Trp 1020 or Glu 1024 individually renders Jak2

catalytically inactive (107). We are interested in elucidating the requirement of these two

amino acids for Jak2 kinase function. Recently, we showed that Trp 1020 forms a

hydrogen bond with Glu 1046 that is critical to maintain the structural integrity of the

Jak2 activation loop (107). The role that Glu 1024 plays in Jak2 kinase function,

however, is not known.

Here, we investigated the requirement for Glu 1024 for Jak2 kinase function using

homology modeling of the Jak2 kinase domain and site-directed mutagenesis. Our data

indicate that Glu 1024 forms an interaction with an arginine at position 1113, via two









distinct hydrogen bonds, and is essential for angiotensin II-dependent activation of the

Jak-STAT signaling pathway. Conversion of Arg 1 113 to lysine, alanine, or glutamic

acid renders Jak2 catalytically inactive. Consequently, this is the first report describing

Arg 1113 as being essential for Jak2 kinase activity.

Results

Molecular Modeling Identified a Putative Interaction between Jak2 Residues Glu
1024 and Arg 1113

Previously, we showed that mutation of Glu 1024 to Ala rendered Jak2 catalytically

inactive (107). To understand how Glu 1024 contributes to Jak2 kinase function, we

generated a molecular model of the Jak2 kinase domain (Fig. 6-1).



















Figure 6-1. Molecular modeling of the Jak2 kinase domain suggested a putative
interaction between Glu 1024 and Arg 1113 The model was designed
using the program Swiss Model and was based on the known crystal
structure of the kinase domain of the fibroblast growth factor tyrosine
kinase receptor. Shown are bond distances in angstroms.

The model was based on the known crystal structure of the basic fibroblast growth factor

receptor. Our model indicated that Glu 1024 forms a critical interaction with Arg 1113,

an amino acid that thus far has not been shown to be essential for Jak2 function. The









model predicted that the activation loop of the Jak2 kinase domain is maintained in its

proper conformation via critical interactions between the oxygen groups on the side chain

of Glu 1024 and the terminal amino groups on the side chain of Arg 1 113. Using the

program HBPLUS Hydrogen Bond Calculator, we determined that these interactions

were hydrogen bonds, based on the bond lengths (2.91 angstroms and 2.51 angstroms,

respectively) between the atoms involved. In each bond, the amino group is the electron

donor, while the oxygen group is the electron acceptor. Furthermore, our model

suggested that permutation of Arg 1 113 would render Jak2 catalytically inactive.

Mutation of Jak2 Residue Glu 1024 or Arg 1113 Abolishes Jak2 Kinase Activity

We tested the ability of a Jak2 protein containing point mutations at either Glu

1024 or Arg 1 113 to autophosphorylate, to determine if indeed an interaction between

these two amino acids, as predicted by our model, exists. For this, we transfected BSC-

40 cells with 10 Clg of Jak2 cDNA containing E1024R, E1024D, R1 113E, R1 113A, or

R1113K mutations. BSC-40 cells transfected with 5 Clg of wild type Jak2 cDNA served

as a positive control, while cells transfected with empty plasmid (pRC) served as a

negative control. Cells were lysed and protein was extracted. Protein extracts were

immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-

phosphotyrosine antibody to detect tyrosine phosphorylated Jak2. Previously, we showed

that conversion of Glu 1024 to Ala (E1024A), a neutrally charged amino acid, rendered

Jak2 catalytically inactive (107). Here, we show that conversion of Glu 1024 to Asp

(E1024D), a negatively charged amino acid, or Arg (E1024R), a positively charged

amino acid, similarly abolishes Jak2 tyrosine kinase activity (Fig. 7.2, Top). Likewise,

conversion of Arg 1 113 to Glu (R1 113E), a negatively charged amino acid, Ala









(R1113A), a neutrally charged amino acid, or Lys (R1113K), a positively charged amino

acid, rendered Jak2 catalytically inactive (Fig. 2, Top). In addition, a double mutation

whereby Glu 1024 was converted to Arg, and Arg 1113 was converted to Glu, thus

effectively switching the positions of these two amino acids, also rendered Jak2

catalytically inactive (Fig. 6-2, Top). We confirmed expression of all transfected

constructs by Western blotting the same membrane with anti-Jak2 antibody (Fig. 6-2,

Bottom). These data show that both Glu 1024 and Arg 1113 are critical for the ability of

Jak2 to autophosphorylate, thus supporting the prediction that these two amino acids

form a critical interaction.








~,IP: u2ak2-p;

111~ -- IB uyr(P)-mgb



8 IP: aJak2-pAb
111 11 ... IB: aJak2-pAb




Figure 6-2. Mutation of Glu 1024 or Arg 1 113 abolishes the ability of Jak2 to
autophosphorylate. BSC-40 cells were transfected with 10 Clg of the
indicated plasmids and infected with vaccinia virus clone vTF7-3 to
induce high-level protein expression. Lysates were prepared,
immunoprecipitated with anti-Jak2-pAb, and Western blotted with anti-
phosphotyrosine-mAb to assess the ability of Jak2 containing mutations to
autophosphorylate (top). The nitrocellulose membranes used for Western
blotting were stripped and reprobed with anti-Jak2-pAb to assess Jak2
protein expression (bottom). Shown is one of three independent
experiments.









Individual Mutations of W1020G or E1024A Render Jak2 Dominant Negative

It is known that double mutation of W1020G/E1024A renders Jak2 dominant

negative (106). This means that when these two amino acids are both mutated, the

resulting mutant Jak2 can inhibit the ability of wild type Jak2 to autophosphorylate. We

recently showed that mutation of either Trp 1020 or Glu 1024 individually renders Jak2

catalytically inactive (107). We therefore sought to determine if mutation of either of

these amino acids individually would render Jak2 dominant negative, as well as

catalytically inactive. For this, we transfected BSC-40 cells with either 5 Clg of wild type

Jak2 cDNA alone, or with 5 Clg of Jak2 cDNA plus increasing amounts of mutant Jak2

cDNAs. If the mutation tested does render Jak2 dominant negative, then upon co-

transfection with increasing amounts of mutant cDNA, the ability of the wild type Jak2 to

autophosphorylate will be inhibited. Addition of wild type plasmid alone results in

increased Jak2 tyrosine phosphorylation levels (Fig. 6-3A, Top, lane 2 vs. lane 1).

Addition of increasing amounts of the previously characterized Jak2 dominant negative

mutant (W1020G/E1024A) inhibits wild type phosphorylation (lanes 3 and 4). Mutation

at either Trp 1020 (W1020G) or at Glu 1024 (E1024A) alone is also sufficient to render

Jak2 dominant negative in a dose-dependent manner (lanes 5-8). It is clear, though, that

the dominant negative that results from mutation of W1020G or E1024A alone is not

nearly as potent an inhibitor as the double W1020G/E1024A mutation. For instance,

when 5 Clg of wild type Jak2 was co-transfected with 5 Clg of the Jak2-W1020G or the

Jak2-E 1024A mutant, we observed a diminished, but existent degree of Jak2

autophosphorylation. In comparison, when 5 Clg of wild type Jak2 was co-transfected

with 5 Clg of Jak2 containing the W1020G/E1024A double mutation, the ability of wild









type Jak2 to autophosphorylate was almost completely lost. Therefore, individual

mutations at either Trp 1020 or Glu 1024 can render Jak2 both catalytically inactive and

dominant negative. The individual mutants, though, do not show as strong a dominant

negative character as the double Jak2-W1020G/E1024A mutant.

We next tested the ability of the R1 113E mutant to render Jak2 dominant

negative. Again, we transfected BSC-40 cells with either 5 Clg of wild type Jak2 cDNA

alone, or with increasing amounts of the R1 113E mutant plasmid. The results show that

even when 5 Clg of wild type Jak2 cDNA is co-transfected with 15 Clg of Jak2 containing

the R1113E mutation, wild type Jak2 is able to autophosphorylate (Fig. 6-3B).

Therefore, the data suggest that mutation of Arg 1 113, while disrupting Jak2 kinase

function, does not render a dominant negative phenotype.

Jak2-R1113E is Unable to Become Tyrosine Phosphorylated by Angiotensin II

The data in Figs. 6-2 and 6-3 were generated using ligand-independent

experimental conditions. To determine whether Arg 1113 is critical for proper Jak2

function in a ligand-dependent signaling system, we used COS-7 cells to study the role

that Arg 1113 plays in angiotensin II-induced activation of the Jak-STAT signaling

pathway. We first tested the ability of angiotensin II to activate Jak2 containing the

R1 113E point mutation. For this, COS-7 cells were transfected with 10 Clg of AT1

receptor, and either 10 Clg of wild type Jak2 cDNA or 12.5 Clg of Jak2-R1 113E cDNA.

The cells were treated with 100 nM angiotensin II for either 0, 5, or 10 min. Cells were

lysed and protein was extracted. Protein extracts were immunoprecipitated with anti-

phosphotyrosine antibody and immunoblotted with anti-Jak2 antibody to detect tyrosine

phosphorylated Jak2. The results show that in response to angiotensin II, wild type Jak2






67

becomes increasingly tyrosine phosphorylated over the time course, while Jak2-R1113E

is unable to become activated in response to ligand treatment (Fig. 6-4).


A



111-




111-


8


IP: uJak2-pAb
IB: uTyr(P)-mAb


CI


I) I)~'rttIP: uJak2-pAb
IB: uJak2-pAb
5 5 5 5pR -a2-T(g
0 5 15 5 15 5 15 pRC-Jak2-mutan (yg)


IP: uJak2-pAb
IB: uTyr(P)-mAb


111-


pRC-Jak2-WT (yg)
SpRC-Jak2-R1113E (pg)


Figure 6-3.


The Jak2-W1020G and E1024R mutations render Jak2 dominant negative
BSC-40 cells were transfected with the indicated plasmids and infected
with vaccinia virus clone vTF7-3. A) Lysates were prepared,
immunoprecipitated with anti-Jak2-pAb, and Western blotted with anti-
phosphotyrosine-mAb to assess the ability of Jak2-W 1020G or Jak2-
E1024A to act as a dominant negative (top). The membrane was stripped
and reprobed with anti-Jak2-pAb to assess Jak2 protein expression in each
sample (bottom). B) Using the same experimental paradigm, the ability of
Jak2-R1113E was tested for its ability to act as a dominant negative.
Shown is one of two independent experiments for each.










Jak2-WT


Jak2-R1113E


Ang II (min) 0


5 10 0 5 10


IP. aTyr(P)-mAb
IB aJak2-pAb


I... -~ -


Figure 6-4.


Jak2-R1113E mutant cannot become tyrosine phosphorylated in response
to angiotensin II COS-7 cells were transfected with 10 Clg of AT1 receptor
and either 10 Clg of wild type Jak2 or 12.5 Clg Jak2-R1113E cDNA. The
cells were treated with 100 nM angiotensin II for the indicated times and
lysates were prepared to assess Jak2 tyrosine phosphorylation. Lysates
were immunoprecipitated with anti-Tyr(P)-mAb and Western blotted with
anti-Jak2-pAb. Shown is one of three independent experiments.


Jak2-WT Jak2-R1113E
0 3 6 03 6


Ang II (min)


IP: uHA-mAb
IB: aJak2-pAb


Figure 6-5.


Angiotensin II-dependent Jak2/AT1 receptor co-association does not occur
in cells expressing Jak2-R1113E COS-7 cells were transfected with HA-
tagged AT1 receptor and either wild type Jak2 or Jak2-R1113E cDNA.
The cells were treated with 100 nM angiotensin II for the indicated times
and lysates were prepared to assess Jak2/AT1 receptor co-association.
Lysates were immunoprecipitated with anti-HA-mAb and Western blotted
with anti-Jak2-pAb. Shown is one of three independent experiments.


Following angiotensin II-induced Jak2 tyrosine phosphorylation, Jak2 associates

with the AT1 receptor (18). To determine if Jak2 containing the R1 113E mutation could

associate with the AT1 receptor, we transfected COS-7 cells similar to those described

above. The cells were then treated with 100 nM angiotensin II for either 0, 3, or 6 min,

lysed, and protein was extracted. Protein lysates were immunoprecipitated with anti-HA

antibody to immunoprecipitate the HA-tagged AT1 receptor, and immunoblotted with

anti-Jak2 antibody to detect Jak2/AT1 receptor co-association (Fig. 6-5). The results

show that in response to angiotensin II, wild type Jak2 rapidly associates with the AT1


rlll










receptor, while Jak2-R1113E is unable to do so, thus demonstrating that Arg 1113 is also

essential for Jak2/AT1 receptor co-association.

Jak2-R1113E is Unable to Tyrosine Phosphorylate STATs in Response to
Angiotensin II

Once Jak2 associates with the AT1 receptor in response to angiotensin II, STAT

proteins are recruited to the Jak2/AT1 receptor complex (38). Jak2 then tyrosine

phosphorylates these receptor-bound STATs (18). To examine angiotensin II-dependent

STAT activation in cells expressing Jak2-R1113E, we again transfected COS-7 cells

similar to those described above. The cells were then treated with 100 nM angiotensin II

for either 0, 5, or 10 min, lysed, and protein was extracted. The protein lysates were

immunoprecipitated with anti-STAT1 antibody and immunoblotted with anti-Jak2

antibody to detect STAT1/Jak2 co-association (Fig. 6-6, Top).

We show that in cells expressing wild type Jak2, angiotensin II induced

STAT1/Jak2 co-association, whereas in cells expressing Jak2-R1113E, no STAT1/Jak2

co-association was seen in response to angiotensin II. We stripped the same membrane

and reprobed it with anti-phosphotyrosine antibody to detect tyrosine phosphorylated

STAT1 protein (Fig. 6-6, Middle). The results show that in cells expressing wild type

Jak2, angiotensin II induces marked STAT1 tyrosine phosphorylation, whereas in cells

expressing Jak2-R1113E, STAT1 does not become tyrosine phosphorylated in response

to angiotensin II. Finally, we stripped the same membrane and reprobed it with anti-

STAT1 antibody to demonstrate equal sample loading (Fig. 6-6, Bottom). Collectively,

the data in Fig. 6-6 demonstrate that in cells expressing Jak2-R1113E, angiotensin II-

dependent activation of STAT 1 is disrupted, as measured by reduced

STAT1/Jak2 co-association and STAT1 tyrosine phosphorylation.










Jak2-WT Jak2-R1113E
Ang II (min) 0 5 10 0 5 10
IP: aSTAT1-pAb
111 4ggIB: a Jak2-pAb


IP: aSTAT1-pAb
79 IB: a Tyr(P)-mAb


PB : aSAA1 -ppAbb


7 9 '~

Figure 6-6. Jak2-R1113E is unable to activate STAT1 in response to angiotensin II
Transfected COS-7 cells were treated with angiotensin II for the indicated
times and lysates were prepared. Lysates were immunoprecipitated with
anti-STAT 1-pAb and Western blotted with anti-Jak2-pAb to assess
Jak2/STAT1 co-association (Top). The membrane was stripped and
reprobed with anti-Tyr(P)-mAb to assess STAT1 tyrosine phosphorylation
(Middle). The membrane was again stripped, and then reprobed with anti-
STAT1-pAb to demonstrate equal sample loading (Bottom). Shown is
one of three independent experiments.

Jak2-R1113E is unable to mediate angiotensin II-dependent gene expression

We tested the ability of cells expressing the Jak2-R1 113E mutant to activate

STAT-mediated gene transcription. For this, COS-7 cells were transfected with 10 Clg of

AT1 receptor, 10 Clg of a luciferase reporter plasmid encoding a Statl-binding sis-

inducible element, and either 10 Clg of wild type Jak2 cDNA or 12.5 Clg of Jak2-R1 113E

cDNA. The luciferase reporter plasmid contains a tandem repeat of a minimal DNA

enhancer element, the thymidine kinase TATA-containing promoter, and the firefly

luciferase cDNA. Each copy of the DNA enhancer contains a STAT1-inducible SIE

element, a serum response element, and an AP-1 binding site. Others and we previously

showed that this plasmid is a good indicator of Jak-STAT-mediated gene transcription

(108, 126). The cells were serum-starved for 24 h, and then treated with 100 nM

angiotensin II for 0, 4, 8, or 12 h. We show that in cells expressing wild type Jak2,







































O 2 4 6 8 10 12 14


angiotensin II treatment produced a rapid and transient increase in Jak-STAT mediated

gene expression that peaked at 4-8 h after angiotensin II treatment (Fig. 6-7). This

response was greatly attenuated in cells expressing the Jak2-R1113E plasmid. Thus,

these data show that Arg 1113 is also critical for angiotensin II-induced STAT-mediated

gene transcription.


Ang II (hrs)


Figure 6-7.


Jak2-R1113E is unable to activate STAT-mediated gene transcription in
response to angiotensin II. COS-7 cells were transfected with 10 Clg of
AT1 receptor, 10 Clg of a luciferase reporter plasmid encoding a STAT1-
binding sis-inducible element, and either 10 Clg of wild type Jak2 or 12.5
Clg of Jak2-R1 113E cDNA. The cells were treated with 100 nM
angiotensin II for the indicated times and detergent extracts were prepared.
Luciferase activity was measured using the Reporter Lysis Buffer System
(Promega). Shown is one of two independent experiments. Values are
expressed as the mean +/- SD. n=4 for each time point, p<0.05, **
p<0.005 (Student' st test).


Arg 1113 is conserved in different Jak kinase family members and among species
expressing Jak2

The data in Figs. 6-4 through 6-7 show that Arg 1113 is essential for ligand

dependent Jak2-mediated signaling. We therefore wanted to determine if this amino acid









was highly conserved throughout the evolutionary history of Jak2. Comparison of the

amino acid sequence of the different Janus kinase family members, and the amino acid

sequence of Jak2 from various species expressing the gene, indicates that Arg 1 113 is a

highly conserved residue (Fig. 6-8). The highly conserved nature of Arg 1 113 further

indicates a critical role for this amino acid in Jak2 function.

Mouse Jak2 1102 MT EC WN NN V S QR P SF R DL S FG W
Mouse Jak1 1102 M R CC W E F Q PS NIR TT F Q N LIE G F
Mouse Jak3 1102M Q LCW A PS P H DR P AF GT LS PQ L
Mouse Tyk2 1102 MQ N C WE TEAS FIR PT F Q N L V PIL

Mouse Jak2 M T EC WN N NV SQ R P SF R D LSF GW
Rat Jak2 MTE CWN NN V NQ R IPS FR D LS L R V
Pig Jak2 M TE CW NN NV N Q R P SF R D LAL R V
Human Jak2 MT E CWNNN V N RI PSF R D LAL R V
Zebra Fish Jak2 M QE CW DN D PS LI R IPNF KE LAL R V
Puffer Fish Jak2 M E Q CWDN DP Y L R IPS F K E LAL S I
Chicken Jak2 M LS C WAF AP S A R PT F TE LA AR V

Figure 6-8. Arg 1113 is conserved in Jak2 among species and in different Jak family
members Comparison of the amino acid sequence of the different murine
Janus kinase family members, and the amino acid sequence of Jak2 from
various species, indicates that Arg 1113 is a highly conserved residue
throughout evolution.

Discussion

We provided evidence that Glu 1024 forms two distinct hydrogen bonds with Arg

1113 that are critical for Jak2 tyrosine kinase activity. As such, these are the first data

describing Arg 1113 as being critical for Jak2 kinase function. Molecular modeling

studies identified a putative interaction between Glu 1024 and Arg 1113. Using the

program HBPLUS Hydrogen Bond Calculator, we determined that this interaction

consisted of two distinct hydrogen bonds between the oxygen groups on the side chain of









Glu 1024 and the terminal amino groups on the side chain of Arg 1 113. Using site-

directed mutagenesis, we showed that E1024R, E1024D, R1113K, R1113A, and R1113E

point mutations all rendered Jak2 catalytically inactive. Converting Glu 1024 to Arg

(E1024R), and Arg 1113 to either Ala (R1113A) or Glu (R1113E) changed the charge on

the amino acid at those positions and thus likely disrupted the ionic interaction between

amino acids 1024 and 1113. While the E1024D and R1113K substitutions maintained

the charge at those positions, both of these mutations shortened the side chains of the

amino acids at their respective positions. Aspartic acid has a side chain that is one carbon

shorter than that of glutamic acid; lysine has a side chain that is one amino group shorter

than that of arginine. Thus, despite the conservative nature of the R1 113K and E1 024D

mutations, the kinase function of Jak2 was lost. Consequently, we believe that the

shorter side chains were not sufficiently long to maintain the interaction between these

two residues, indicating that proper bond length is of critical importance to maintaining

this interaction.

Previous studies showed that double mutation of Trp 1020 to Gly and Glu 1024 to

Ala (W1020G/E1024A) rendered Jak2 dominant negative (106). Here, we tested whether

individual mutation at either Trp 1020 or Glu 1024 alone would not only render Jak2

catalytically inactive, but also dominant negative. We showed that substitution mutations

at either position 1020 (W1020G) or position 1024 (E1024A) rendered Jak2 dominant

negative. Interestingly, these individual mutants were not able to act as potent a

dominant negative as the double Jak2-W1020G/E1024A mutant. We believe, therefore,

that the strong dominant negative phenotype displayed by the double mutant is due to an

additive dominant negative effect of the two individual mutations. This is a discovery of









two new Jak2 dominant negative molecules, and these mutants may be useful research

tools for studying the function of Jak2. In fact, these two dominant negative mutants may

offer an advantage over the double W1020G/E1024A dominant negative mutant. Since

their dominant negative character is not as strong as that of the double mutant, their

expression in cells could be used as a knockdown approach to studying Jak2 function.

This could be particularly useful for in vivo models, where Jak2 activation is critical to

life.

We next used COS-7 cells to examine the consequences of the Jak2-R1 113E

mutation in a ligand-dependent signaling system. These cells express very low levels of

Jak2 and no AT1 receptor. When Jak-STAT signaling is reconstituted in these cells they

are a reliable, simple-to-use system for determining the functional consequences of Jak2

mutations. Using these cells, we showed that Jak2 containing an R1113E mutation is

unable to become activated in response to angiotensin II. Furthermore, we showed that

expression of Jak2-R1 113E cDNA in cells prevented angiotensin II-dependent Jak2/AT1

receptor co-association, Jak2/STAT1 co-association, STAT1 tyrosine phosphorylation,

and STAT-mediated gene transcription. Together, these data show the critical

importance of Arg 1 113 to Jak2 function. Further supporting the indispensability of Arg

1 113, is the conserved nature of this amino acid amongst Jak family members and

throughout Jak2 from various species. We believe that the identification of this and other

critical amino acid interactions within the Jak2 kinase domain could provide targets for

drug design aimed at disabling Jak2 kinase function.















CHAPTER 7
IDENTIFICATION OF A NOVEL Jak2 INHIBITOR

Introduction

The Jak2 protein is important in both physiology and pathophysiology, as it plays

prominent roles in embryonic development, cell signaling, and in cancer and heart

disease (61, 92, 102, 103,). Two impediments to better understanding Jak2 function are

1) the lack of an adult knockout animal model and 2) the lack of a Jak2-specific

pharmacological inhibitor (102, 103, 130).

Jak2 knockout mice die embyronically, around E10.5 because of a lack of

erythropoesis (102, 103). This work showed the critical role that Jak2 plays in embryonic

development and cytokine signal transduction, but also raised a barrier to research on

elucidating the mechanisms of Jak2 cellular function. Without an adult Jak2 knockout

animal available, studying the function of Jak2 in adult physiology and pathophysiology

has been complicated. Furthermore, there is no Jak2-specific pharmacological inhibitor.

AG490 is a commercially available Jak2 inhibitor, and while it has been instrumental in

elucidating some functions of Jak2 and in identifying Jak2 as a therapeutic target, it

suffers from a general lack of specificity. In fact, in Chapter 5, we showed that Jak2

nonspecifieally inhibits angiotensin II-mediated ERK2 activation. Because of these

problems, there are caveats in all research relying solely on AG490 to study Jak2 kinase

function. Clearly, identification of a novel Jak2 inhibitor could aid research efforts.

For these reasons, we sought to identify a potential novel Jak2 inhibitor. We first

used homology modeling of the Jak2 kinase domain to identify exposed pockets on the









surface of the protein. We then used a high-throughput program called DOCK, to predict

the ability of 6,451 small molecules to interact with a solvent accessible pocket that is

adj acent to the activation loop of Jak2, namely, Pocket 36. The compounds were scored

in silico on their potential ability to interact with Pocket 36. We ordered the top seven

scoring compounds, and tested their ability to inhibit Jak2 tyrosine kinase function. One

of these, Compound 7, was found to be a potent inhibitor of Jak2.

Results

Homology Modeling and Target Pocket Identification

We used the homology model of the Jak2 kinase domain described in Chapter 6 to

identify pockets within the Jak2 kinase domain that could interact with potential small-

molecule inhibitors of Jak2. The program SPHGEN identified 49 pockets within the Jak2

kinase domain. The pockets were designated based on their chemical and shape

characteristics. We chose the pocket designated as Pocket "36" as a target based on its

proximity to the Jak2 activation loop and its large size, which makes it accessible to

small-molecules (Fig. 7-1).

Database Screening to Identify Potential Small-Molecule Inhibitors of Jak2

Using Pocket 36 as the target, we used the program DOCK to screen a National

Cancer Institute database of known chemical structures for their ability to interact with

Pocket 36. We used the program to screen 6,415 compounds of the over 140,000

compounds in the database. The program attempted, in silico, to fit each compound into

Pocket 36 in 100 different orientations for each compound tested. The compounds were

scored on their ability to fit into Pocket 36 and on their ability to interact chemically with

Pocket 36. We ordered the seven top-scoring compounds for further testing. These




























Figure 7-1. SPHGEN identified 49 exposed pockets on the surface of the Jak2 protein
Because of its large size, Pocket 36 was chosen as a target pocket for in
silico compound screening.

compounds were provided to us, free of charge, by the National Cancer Institute, through

their Developmental Therapeutics Program. These non-proprietary compounds are

offered to the extramural research community for the development of treatments for

cancer, AIDS, and opportunistic infections afflicting patients with cancer or AIDS.

These were designated Compounds 1-7 (Table 7-1).

Table 7-1. Top 7 scoring compounds
Cmd SC#Formula Name Mol. Weight


1 7785 C3HsNO 2-pro pena mide 71 .0

27795 C28 H22 20,S 22Na Acid Green 25 625.0

3 7828 C19H12 20sS Chlorphe nolI Red 423.0

4 7830 C36H25 506S,22Na Acid Black S 734.0

7851 C4H3C 2, 3 4,6-Dichloro-5- 164.0
5 arrnopyrirrdine

6 7893 CH7N30 Superacil 125.0

77908 C6H6Br6 CyCIOhexane- 1,2,3,4,5,6- 558.0
hexa bro mo-










Compound 7 Inhibits Jak2 Autophosphorylation

To test the ability of each of the seven compounds to inhibit Jak2 tyrosine kinase

activity, we used the vaccinia virus transfection/infection protocol. Briefly, BSC-40

cells, a vaccinia virus permissive cell line, are transfected with an expression vector

encoding the wild type murine Jak2 cDNA under the control of the T7 promoter. The

cells are then infected with a vaccinia virus that produces T7 RNA polymerase. This

results in high level Jak2 expression and subsequent tyrosine autophosphorylation

independent of exogenous ligand addition. After the initial 1 h vaccinia virus infection,

the cells were switched to serum containing media and each compound was added at a

final concentration of 100 CIM and incubated overnight. Sixteen h later after the addition

of the inhibitors, cellular lysates were prepared and immunoprecipitated with anti-Jak2

antibody and then immunoblotted with anti-phosphotyrosine antibody to detect tyrosine

phosphorylated Jak2 (Fig. 7-2A, Top). The results showed that Compound 7 was the

only compound to inhibit Jak2 tyrosine autophosphorylation. The membrane was then

stripped and re-probed with anti-Jak2 antibody to demonstrate equal protein expression

amongst all samples (Fig. 7-2A, Bottom). The identity and structure of compound 7 is

shown (Fig. 7-2B). Cyclohexane-1,2,3 ,4,5,6-hexabromo- is a single aromatic ring

structure with a halide on each carbon. It has a molecular weight of 125 daltons.

Compound 7 Inhibits Jak2 Autophosphorylation in a Time-Dependent Manner

We next wanted to determine whether Compound 7 could inhibit Jak2 tyrosine

autophosphorylation in a time-dependent manner. For this, BSC-40 cells were

transfected/infected as described. Before cell lysis, 100 CIM of Compound 7 was applied

to the cells for 0, 1, 4, or 16 h. Cellular lysates were then prepared, immunoprecipitated


















EI lwl PBI)Iy~rl 'P="p Abb




B


Cyclohexane- 1,2,3,4,5,6-hexabromo-


Figure 7-2.


Compound 7 inhibits Jak2 autophosphorylation A) The 7 compounds that
received the highest score from the DOCK program for their ability to
interact with Pocket 36 within the Jak2 kinase domain were tested for their
ability to inhibit Jak2 autophosphorylation. BSC-40 cells were transfected
with 5 Clg of Jak2 cDNA, and then infected with 1 MOI of vaccinia virus
for 16 h to drive high-level expression of Jak2 and subsequent Jak2
autophosphorylation. During viral infection, the 7 compounds were
incubated with the cells at a concentration of 100 CIM each. Cell lysates
were immunoprecipitated with anti-Jak2 antibody and immunoblotted
with anti-phosphotyrosine antibody to detect Jak2 tyrosine
phosphorylation (Top). The membrane was stripped and re-probed with
anti-Jak2 antibody to demonstrate equal Jak2 expression amongst all
samples (Bottom). B) Shown is the structure and identity of Compound 7.
Shown is one of three independent experiments.


with anti-Jak2 antibody, and immunoblotted with anti-phosphotyrosine antibody to

measure tyrosine phosphorylated Jak2 levels (Fig. 7-3, Top). The results showed that 1









or 4 h treatment with Compound 7 was sufficient to block ~75% of the tyrosine

autophosphorylation of Jak2. However, 16 h treatment with 50 CIM Compound 7 resulted

in a virtual elimination of all Jak2 tyrosine autophosphorylation. The membrane was

stripped and re-probed with anti-Jak2 antibody to demonstrate equal protein expression

amongst all samples (Fig. 7-3, Bottom).

Collectively, the data show that incubation of Compound 7 does inhibit Jak2

tyrosine autophosphorylation in a time-dependent manner; treatment of cells with 50 C1M

Compound 7 for 1 or 4 h was sufficient to partially inhibit Jak2 tyrosine kinase

autophosphorylation, while treatment of cells for 16 h resulted in near total elimination of

Jak2 tyrosine autophosphorylation.








~ r g IPuak ~
111-~ IBuyrP msi b




,,, IP-uJak2 pAb


111- ~ l l IB=uJak2 pAb

Figure 7-3. Maximal Jak2 inhibition requires 16 h of incubation with Compound 7
Compound 7 was incubated with the cells for 0, 1, 4, or 16 h at a
concentration of 50 CIM during infection. Cell lysates were
immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-
phosphotyrosine antibody to detect Jak2 tyrosine phosphorylation (Top).
The membrane was stripped and re-probed with anti-Jak2 antibody to
demonstrate equal Jak2 expression amongst all samples (Bottom). Shown
is one of two representative experiments.










Compound 7 Inhibits Jak2 Autophosphorylation in a Dose-Dependent Manner

We next tested the ability of Compound 7 to inhibit Jak2 autophosphorylation in a

dose-dependent manner. For this, we again used the BSC-40 cell transfection/infection

protocol. The cells were treated for 16 h with Compound 7 at doses of 0, 1, 10, 50, 100,

250, or 500 CIM. The next morning, soluble protein lysates were immunoprecipitated

with anti-Jak2 antibody and then immunoblotted with anti-phosphotyrosine antibody to

measure the tyrosine phosphorylation levels of Jak2 (Fig. 7-4, Top). The results showed

that Compound 7 inhibited Jak2 tyrosine autophosphorylation in a dose-dependent

manner with maximal inhibition occurring at 50 CIM. The membrane was then stripped

and re-probed with anti-Jak2 antibody to demonstrate equal protein expression amongst

samples (Fig. 7-4, Bottom).


IP-a Jak2 p Ab
--IB=aTyr(P) mAb


,, lr ri IP-a Jak2 pbIaak pAb


Compound 7 inhibits Jak2 in a dose-dependent manner BSC-40 cells
were again transfected/infected as described above. Compound 7 was
incubated with the cells during infection at a dose of 0, 1, 10, 100, 250, or
500 C1M for 16 h. Cell lysates were immunoprecipitated with anti-Jak2
antibody and immunoblotted with anti-phosphotyrosine antibody to detect
Jak2 tyrosine phosphorylation (Top). The membrane was stripped and re-
probed with anti-Jak2 antibody to demonstrate equal Jak2 expression
amongst all samples (Bottom). Shown is one of two representative
experiments.


Figure 7-4.


..* eme -









Collectively, the data in Fig. 4 show that Compound 7 does in fact inhibit Jak2

autophosphorylation in a dose-dependent manner. The amount of material required to

inhibit 50% of the Jak2 tyrosine autophosphorylation levels in this assay (ICso) was in the

low micromolar range. Additionally, 50 C1M Compound 7 was sufficient for maximal

Jak2 inhibition.

Compound 7 is Non-Cytotoxic at Concentrations that Maximally Inhibit Jak2
Tyrosine Autophosphorylation

To determine whether Compound 7 was cytotoxic to the cultured cells, we treated

BSC-40 cells with Compound 7 at doses of 0, 100, or 500 C1M for 16 h. The live cells

were then stained with propidium iodide to determine whether Compound 7 was

cytotoxic. Propidium iodide selectively stains necrotic cells and fluoresces red, but is

excluded by the plasma membranes of healthy, intact cells. The results showed that

cells treated with 100 C1M Compound 7 showed very little propidium iodide staining, akin

to that of untreated cells (Fig. 7-5). In contrast, BSC-40 cells treated with 500 CIM

Compound 7 did show increased propidium iodide staining, indicating that at a dose of

500 C1M Compound 7 is cytotoxic. Since the ICso of Compound 7 is estimated to be in

the low micromolar range, and 50 CIM Compound 7 maximally inhibits Jak2 tyrosine

kinase autophosphorylation, we conclude that the mechanism by which Compound 7

inhibits Jak2 tyrosine kinase autophosphorylation, at these concentrations, is independent

of cellular cytotoxicity.

Discussion

Since its discovery in 1992, significant progress has been made in understanding

the biochemical and cellular functions of Jak2 tyrosine kinase. Studies have shown

essential roles for Jak2 in embryonic development, cell signaling, and the








Bright Field Propidium Iodide




Untreated ~rq






Cmpd 7
100 tLM






Cmpd 7
500 tLM




Figure 7-5. Compound 7 is not cytotoxic at a dose of 100 C1M BSC-40 cells were
grown on microscope slides and treated with 0, 100, or 500 C1M
Compound 7 for 16 h. The live cells were then stained with 1 Clg/mL
propidium iodide to determine whether Compound 7 was cytotoxic. The
cells were visualized using confocal microscopy. Shown is one of two
representative experiments.

pathophysiology of heart disease and cancer (61, 92, 102, 103). Research on this protein,

though, has been complicated by the lack of a Jak2-specific pharmacological inhibitor.

AG490, a commercially available Jak2 inhibitor, also inhibits several other related

tyrosine kinase signaling pathways (98-101).

This work is significant for three fundamental reasons. First, we used homology

modeling of the Jak2 kinase domain and high-throughput compound docking in silico to

identify potential Jak2 inhibitors. We found that cyclohexane-1 ,2,3,4,5,6-hexabromo-,

designated as Compound 7, potently inhibited Jak2 tyrosine autophosphorylation in

cultured BSC-40 cells. Compound 7 inhibited Jak2 autophosphorylation in both a dose-









and time-dependent manner. Based on these autophosphorylation assays, it appears that a

16 h treatment with 1 C1M Compound 7 is sufficient to reduce Jak2 tyrosine

autophosphorylation levels by about 50%, while 50 CIM Compound 7 eliminates virtually

all detectable Jak2 tyrosine autophosphorylation. Furthermore, even at doses as high as

100 C1M, Compound 7 is not cytotoxic to cultured cells. As such, it inhibits Jak2 tyrosine

autophosphorylation at concentrations that are well below its cytopathic threshold.

Second, the results shown here using the DOCK program, demonstrate proof-of-

principle in using in silico-based strategies for identifying biological interactions. Here,

screening just 6,451 compounds for their ability to interact with one target pocket on the

Jak2 kinase domain, we successfully used the DOCK program to identify a novel Jak2

inhibitor. We will therefore use the DOCK program for screening additional compounds

for their ability to bind multiple targets within the Jak2 kinase domain. In fact, the library

that we have available for screening contains over 140,000 compounds of known

chemical structure. Furthermore, we identified 49 exposed pockets on the Jak2 kinase

domain. By screening the entire library of compounds using multiple target pockets, we

expect to identify several additional small molecule inhibitors of Jak2.

Third, AG490 falls within the general class of tyrosine kinase inhibitors known as

tyrphostins. The molecular structure of AG490 is known; it contains two aromatic ring

structures linked by a spacer containing four carbons and an amide group. Compound 7

is noticeably different from AG490 in that it contains only a single aromatic ring without

any spacers. As such, our work here suggests that Compound 7, with its single aromatic

ring, could serve as a potential lead compound for future synthesis reactions with the

hopes of identifying a specific Jak2 inhibitor.









A final possibility for identifying additional small molecule inhibitors of Jak2 using

database screening is to model the site where AG490 binds to the Jak2 kinase domain.

We could then use this binding site as a target for database screening. This has the

advantage of using an area that is known to bind a small molecule inhibitor of Jak2 as a

target. This may allow us to identify Jak2 inhibitors that have the same site of action as

AG490, but are more specific for Jak2 than AG490.

Collectively, the work shown here has identified cyclohexane-1,2,3,4,5,6-

hexabromo- as a small molecule inhibitor of Jak2 tyrosine kinase. Because of the

universal importance of Jak2 in mediating both the physiological and pathophysiological

actions within animals, this compound, and potential derivatives of it, may have

important therapeutic value.















CHAPTER 8
CONCLUSIONS AND PERSPECTIVES

The Jak2 tyrosine kinase protein was discovered in 1991. It was quickly identified

as a key mediator of cytokine signaling. Since then, roles for Jak2 in mediating signaling

through GPCRs and during cellular stress, including oxidative stress, have been

identified. Despite this, study of Jak2 has been complicated for two reasons: 1) lack of an

adult knockout animal and 2) lack of a specific Jak2 inhibitor. In these studies we used

several strategies to circumvent these problems, and we significantly improved our

understanding of Jak2 structure, function, and pharmacology. We used Jak2 -/- cells to

identify a novel role for Jak2 in angiotensin II-dependent inactivation of ERK2. We used

cells expressing a Jak2 dominant negative mutant to identify Jak2 as an essential

mediator of oxidative stress-induced apoptosis in vascular smooth muscle cells. Finally,

we used homology modeling of the Jak2 kinase domain to identify an amino acid

interaction within Jak2 that is critical for Jak2 function, and to identify a novel small

molecule inhibitor of Jak2.

Role of Jak2 in Angiotensin II-Dependent ERK2 Signaling

Previous studies suggested that Jak2 was required for angiotensin II-dependent

activation of ERK2. These studies, though, relied solely on the Jak2 inhibitor AG490 to

determine this role for Jak2. While AG490 potently inhibits Jak2, it nonspecifically

inhibits several other signaling pathways. For this reason, we used Jak2 -/- cells to

specifically study the role that Jak2 plays in angiotensin II-dependent ERK2 signaling.









In Chapter 4, we showed that Jak2 is essential for inactivation of ERK2 after

angiotensin II treatment. Moreover, we showed that the previously published results

demonstrating that Jak2 is required for angiotensin II-dependent activation of ERK2, may

be an artifact caused by using AG490 to study Jak2 function.

These studies may open a new area of research that will further explore crosstalk

between the Jak2 signaling pathway and the ERK signaling pathway. Future studies

should further elucidate the role that Jak2 plays in the regulation of angiontensin II

signaling. Ultimately, it will be interesting to determine the physiological importance of

this novel role for Jak2 in vivo, where angiotensin II plays critical roles during

cardiovascular disease.

Role of Jak2 during Oxidative Stress

It was discovered in 1998, that Jak2 is strongly activated in vascular smooth muscle

cells by oxidative stress in the form of hydrogen peroxide. Since then, there has been

little research into the physiological role that Jak2 plays during oxidative stress. In

Chapter 5, we used expression of a Jak2 dominant negative mutant to show, for the first

time, a physiological endpoint of Jak2 activation by hydrogen peroxide in vascular

smooth muscle cells. We found that hydrogen peroxide resulted in apoptosis of control

vascular smooth muscle cells, but failed to induce apoptosis in cells expressing a

dominant negative Jak2, indicating that Jak2 activation is essential for oxidative stress-

induced apoptosis of vascular smooth muscle cells.

These results could have profound consequences on diseases where oxidative

stress-induced apoptosis contributes to pathology. Atherosclerosis is one such disease.

During atherosclerosis, circulating macrophages release high amounts of hydrogen

peroxide on vascular smooth muscle cells within the fibrous cap of the atherosclerotic










plaque. This can lead to apoptosis of the cells, and subsequent weakening of the fibrous

cap. When the cap weakens, plaque rupture often occurs, which can result in thrombus

formation, and subsequent heart attack or stroke. First, the role that Jak2 plays in

oxidative stress in vivo must be determined.

Jak2 Structure-Function

Point mutations at both Trp 1020 and Glu 1024 render Jak2 dominant negative. As

discussed above, we used this dominant negative mutant to determine the role of Jak2

during oxidative stress. We also used this dominant negative mutant to better understand

the structure of Jak2. Previously, we showed that mutation of either Trp 1020 or Glu

1024 individually rendered Jak2 catalytically inactive. In Chapter 6, we showed that

these individual point mutations also render Jak2 dominant negative. Moreover, we

determined the reason that Glu 1024 is critical for Jak2 function. This amino acid forms

two distinct hydrogen bonds Arg 1113.

Critical amino acid interactions within the Jak2 kinase domain could be targets for

drug design aimed at disabling Jak2 kinase function. Novel Jak2 inhibitors would be

useful research tools and could possibly hold therapeutic potential. For this reason, the

structure of the Jak2 protein should continue to be explored. Hopefully, in the future, the

crystal structure of Jak2 will be determined. This will provide further insight into the

Jak2 structure-function relationship, and could lead to design of Jak2 inhibitors.

Identification of a Novel Jak2 Inhibitor

A better understanding of Jak2 structure could lead to design of novel Jak2

inhibitors. In addition to pursuing a better understanding of Jak2 structure, we took a

direct approach to identifying novel Jak2 inhibitors. We used homology modeling of the

Jak2 kinase domain to identify target pockets for in silico compound docking. Using the