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Phosphorylation Mediated Regulation of 14-3-3 Protein Dimerization in Arabidopsis Thaliana and the Effect of Dimerizatio...

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Title: Phosphorylation Mediated Regulation of 14-3-3 Protein Dimerization in Arabidopsis Thaliana and the Effect of Dimerization in 14-3-3/target Interactions
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Gokirmak, Tufan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: arabidopsis, difopein, dimerization, gbf3, interaction, localization, monomerization, phosphopeptide, phosphorylation, r18, regulation, target, toxicity
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: 14-3-3s are a family of regulatory proteins that are uniquely eukaryotic, evolutionarily conserved across all eukaryotes, and deeply involved in protein-protein interactions that mediate diverse biological processes. The 14-3-3 proteins commonly bind to target proteins containing well defined phosphothreonine (pThr) or phosphoserine (pSer) motifs. Though evolutionarily conserved, most eukaryotes have a range of 14-3-3 genes and proteins that provide functional divergence to the family. The families of 14-3-3 proteins are present in all cell types as homo- and hetero-dimers. The14-3-3/target interactions can be regulated at two different levels: First, the phosphorylation status of the target and second, the phosphorylation status of 14-3-3 itself, which has been shown to regulate the dimer/monomer status of the 14-3-3s. The present study demonstrated that in Arabidopsis there is phosphorylation dependent regulation of 14-3-3 dimerization that drastically affects the range of dimerization among 14-3-3s and severely affects target protein interactions. This phosphoylation dependent regulation of 14-3-3 dimerization has been conserved throughout the other eukaryotic organism. The data in this dissertation demonstrated that phosphorylation of 14-3-3 omega at Ser-62 has the potential regulatory role in both dimeriation and target interactions. In addition, phosphorylation at this conserved serine residue has the capacity to influence homo- and heterodimerization of 14-3-3 proteins.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Tufan Gokirmak.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Ferl, Robert J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-06-30

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Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042062:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042062/00001

Material Information

Title: Phosphorylation Mediated Regulation of 14-3-3 Protein Dimerization in Arabidopsis Thaliana and the Effect of Dimerization in 14-3-3/target Interactions
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Gokirmak, Tufan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: arabidopsis, difopein, dimerization, gbf3, interaction, localization, monomerization, phosphopeptide, phosphorylation, r18, regulation, target, toxicity
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 14-3-3s are a family of regulatory proteins that are uniquely eukaryotic, evolutionarily conserved across all eukaryotes, and deeply involved in protein-protein interactions that mediate diverse biological processes. The 14-3-3 proteins commonly bind to target proteins containing well defined phosphothreonine (pThr) or phosphoserine (pSer) motifs. Though evolutionarily conserved, most eukaryotes have a range of 14-3-3 genes and proteins that provide functional divergence to the family. The families of 14-3-3 proteins are present in all cell types as homo- and hetero-dimers. The14-3-3/target interactions can be regulated at two different levels: First, the phosphorylation status of the target and second, the phosphorylation status of 14-3-3 itself, which has been shown to regulate the dimer/monomer status of the 14-3-3s. The present study demonstrated that in Arabidopsis there is phosphorylation dependent regulation of 14-3-3 dimerization that drastically affects the range of dimerization among 14-3-3s and severely affects target protein interactions. This phosphoylation dependent regulation of 14-3-3 dimerization has been conserved throughout the other eukaryotic organism. The data in this dissertation demonstrated that phosphorylation of 14-3-3 omega at Ser-62 has the potential regulatory role in both dimeriation and target interactions. In addition, phosphorylation at this conserved serine residue has the capacity to influence homo- and heterodimerization of 14-3-3 proteins.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Tufan Gokirmak.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Ferl, Robert J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042062:00001


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1 PHOSPHORYLATION MEDIATED REGULATION OF 14 3 3 PROTEIN DIMERIZATION IN Arabidopsis thaliana AND THE EF FECT OF DIMERIZATION IN 143 3/TARGET INTERACTIONS By TUFAN GOKIRMAK 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 2010

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2 2010 Tufan Gokirmak

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3 To Mustafa Kemal Ataturk, the founder of the Modern Turkish Republic I am not leaving a spiritual legacy of dogmas, unchangeable petrified directives. My spiritual legacy is science and reason M. Kemal Ataturk

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4 ACKNOWLEDGMENTS I would like to give m y sincere appreciation to my major advisor Dr. Robert Ferl not only for giving me his invaluable assistance, guidance and insightful suggestions during my graduate school experience in his lab, but teaching me how to be an independent scientist and a rational thinker. I greatly appreciated the time and helpful suggestions of my outstanding committee member s, Dr. Anna Lisa Paul, Dr. Curtis Hannah, Dr. William Gurley, and Dr. Jorg Bungert. Especially I want to thank Dr. A nna Lisa Paul one more time for giving me her support and encouragement not only scientifically but whenever I needed it most I a m also thankful to the current and past members of Ferl Lab including Beth J. Laughner, Dr. Agatha Zupanska, Dr. Fiona Denison, Claire Amalfitano Dr. Ann e Vissher, Dr. John Mayfield and Dr. Micheal Manak for being fantastic colleagues I n addition, I wan t to thank Beth Laughner for sharing her technical knowledge and suggestions with me and for our stimulating scientific morning discussi ons. I would also lik e to thank all PMCB faculty staff and all my fellow PMCB graduate students. Specifically I want to thank to Dr. Kevin Folta for letting me use his lab equipments and giving me his advice during the preparation of this manuscript. I thank my beloved friends Fikret and Tolunay Aydemir, Greg Maloney Mehmet Onur Baykan, Ferhat Aydemir, Sukru Gulec, Brent OBrien, Ali Sedighi Kerem Ok Mehmet Kara and Omer Gokcumen for their support whenever I needed. I also want to thank to Yue Su for everything she has done for me. No matter how far she is away I will never forget all the great moments we shared together. Finally, I want to thank all my family members my parents Ismail and Saadet Gokirmak and my brother Dr. Gokhan Gokirmak for always believing in me.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .............................................................................................................. 4 LIST OF FIGURES ....................................................................................................................... 8 LIST OF ABBREVIATIONS ...................................................................................................... 10 ABSTRACT ................................................................................................................................. 11 CHAPTER 1 THE 14 3 3 PROTEINS ..................................................................................................... 13 Introduction .......................................................................................................................... 13 Gene Organization an d Evolutionary History ................................................................. 17 Localization of 143 3 Proteins ......................................................................................... 19 Functions of 143 3 Proteins in Plants ............................................................................. 21 Role of 143 3 Proteins in Light Signaling ............................................................... 24 Role of 143 3 Proteins in Biotic and Abiotic Stress Response ........................... 25 Role of 143 3 Proteins in Chromatin mediated Gene Regulation ...................... 27 Conclusions .......................................................................................................................... 27 2 SER 62 OF ARABIDOPSIS 14 3 TARGET INTERACTIONS ................................................................................................ 38 Introduction .......................................................................................................................... 38 Materials and Methods ....................................................................................................... 41 3D Model and Protein Alignment ............................................................................... 41 Plasmids ........................................................................................................................ 42 Yeast Twohybrid System ........................................................................................... 43 Expression and Purification of Recombinant Proteins in Bacteria ....................... 44 Determining the Affinity and the Binding Kinetics of 14 3 3 ................................................................................................. 45 Subcellular Fractionation and Localization 143 .............................................. 45 Fluorescent Spectroscopy Analyses ......................................................................... 46 Cross linking Studies ................................................................................................... 47 SDS PAGE and Western Blot Analysis .................................................................... 47 Bimolecular Fluorescence Complementation (BIFC) ............................................. 48 Results .................................................................................................................................. 48 143 3 Dimerization Interface Shows Sequence Diversity .................................... 48 Arabidopsis 143 3 Exists as Dimers and Monomers in the Cytoplasm of Arabidopsis Tissue Culture Cells ........................................................................... 49 Effects of S62D Phosphorylation mimicking M utation on Arabidopsis 143 .............................................................................................................. 49 143 3 S62D Cannot Form Homodimers in Yeast .............................................. 51

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6 Heterodimerization of Arabidopsis 143 3s Can Be Regulated by Ser 62 Phosphorylation ........................................................................................................ 52 143 3 S62D Cannot Form Homodimers in Arabidopsis Protoplasts ................ 53 Phosphorylation of Ser 62 Regulates Target Interactions .................................... 53 Discussion ............................................................................................................................ 56 3 DIRECT INTERACTION BETWEEN ARABIDOPSIS 14 3 BOX BINDING FACTOR 3 (GBF3) ............................................................................................ 70 Introduction .......................................................................................................................... 70 Materials and Methods ....................................................................................................... 74 Yeast Strains and Media. ........................................................................................... 74 Plasmids ........................................................................................................................ 75 Monitoring Growth Curves and Cell Viability Assay ............................................... 76 Yeast His tag Pull down ............................................................................................. 77 Fluorescence Microscopy ........................................................................................... 78 Results .................................................................................................................................. 78 GBF3 Has A Toxic Phenotype in Yeast That Can Be Suppressed by 143 3s ................................................................................................................................ 78 GBF3 Directly Interacts with 143 ....................................................... 79 GBF3 Toxicity Is Not Related to Yeast Two hy brid Constructs and Is Likely Due to The Nuclear Function ................................................................................. 79 N terminal Proline rich Domain and C terminal Domain Are Required for GBF3 Toxicity ........................................................................................................... 80 Discussion ............................................................................................................................ 82 4 ARABIDOPSIS 14 3 3 EPSILON IS LOCALIZED IN THE PLASMA MEMBRANE A ND SECRETED IN RESPONSE TO FUNGAL ELICITOR ................ 92 Introduction .......................................................................................................................... 92 Materials and Methods ....................................................................................................... 94 Plant Mate rial and Elicitors ......................................................................................... 94 Specificity Test for Isoform Specific 143 3 Antibodies Using ELISA .................. 95 Cellular Fractionation .................................................................................................. 95 Western Blot Analysis ................................................................................................. 97 Plasmolysis and Detection of 143 3 EpsilonGFP by Fluorescence Microscopy ................................................................................................................ 97 Results and Discussion ...................................................................................................... 97 Polyclonal 14 3 3 Antibodies Recognize 14 3 3 Proteins with High Specificity ................................................................................................................... 97 Cellular Fractionation and Western Blot Analysis .................................................. 98 Arabidopsis 143 3 epsilon is secreted in response to fungal elicitation ............ 99 143 3epsilon GFP Is Localized in the Plasma Membrane .................................. 99 LIST OF REFERENCES ......................................................................................................... 104 BIOGRAPHICAL SKETCH ..................................................................................................... 117

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7 LIST OF TABLES Table page 1 1 143 3 protein binding motifs ........................................................................................ 30 1 2 Arabidopsis thaliana 143 3 protein gene family ....................................................... 32 1 3 Summary of recent studies investigating the involvement of plant 143 3 proteins in plant protein signaling pathways .............................................................. 37 2 1 Kinetic analysis of 143 3 and 143 3 S62D against non phosphorylated target s .............................................................................................................................. 69

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8 LIST OF FIGURES Figure page 1 1 The mode of 143 3/target interactions ..................................................................... 31 1 2 Whole protein sequence alignment of human (Hs), Arabidopsis (At), yeast (Sc), Drosophila (Dm) and C.elegans (Ce) 14 3 3s ................................................ 33 1 3 Unrooted phylogenetic tree of Arabidopsis 143 3 proteins ................................... 34 1 4 Chromosomal distribution of Arabidopsis 143 3 protein genes ............................ 35 1 5 3D struc ture model of 14 3 3 proteins ....................................................................... 36 2 1 143 3 proteins consist of 9 helices. ............................................................................ 58 2 2 Regulation of 143 3/target interactions by phosphorylation .................................. 59 2 3 Dimer/monomer status of 14 3 3 tion of Arabidopsis protoplasts ...................................................................................................................... 60 2 4 The effects of S62D mutation on the in trinsic fluorescence of 14 3 ............. 61 2 5 Phosphorylation mimic mutation S62D increases the hydrophobicity of 143 3 .................................................................................................................................. 62 2 6 Dimer status of 14 3 3 3 S62A expressed in yeast cells. ....................................................................................................................... 63 2 7 The effects of dimerization interface mutations of Ser 62 to Asp62 ( S62D) and to Ala 62 ( S62A) of Arabidopsis 143 3 3 iso forms in yeast two hybrid assay ....................................................................... 64 2 8 S62D phosphomimic mutation interferes with 143 3 homodimerization in Ara bidopsis protoplasts ................................................................................................ 65 2 9 Phosphorylation mimic mutation of Ser 62 to Asp62 of 14 3 ibits 143 3/target interaction ........................................................................................................ 66 2 10 Kinetic analyses of effect of Ser 62 to Asp ( S62D) phosphorylationmimic mutation in 143 to EYFP Difopein and EYF P R18 (Lys) ............ 67 2 11 Phosphorylation mimic mutation S62D in the dimerization interface of 143 3 3 /pho sphorylated target interaction. ........................................... 68 3 1 Three dimensional structure model of the GBF3 bZIP transcription factor ........... 84

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9 3 2 Arabidopsis GBF3 ( At2g46270) transcript profile in re sponse to different treatments. ....................................................................................................................... 85 3 3 Small colony formation in yeast transformed with GBF3 fused to GAL4 transcriptional activation domain (AD GBF3). ........................................................... 86 3 4 143 3 D interacts with GBF3 in yeast. ...................................... 87 3 5 Arabidopsis GBF3 and GBF3GFP fusion proteins cause growth inhibition in yeast l ikely through nuclear function ........................................................................... 88 3 6 Over expressin g GBF3 is toxic in yeast cells ............................................................ 89 3 7 Dissection of the GBF3 domains caus ing growth inhibition in yeast .................... 90 3 8 The model of G BF3 mediated toxicity in yeast .......................................................... 91 4 1 Specificity test for isoform specific 143 3 antibodies using ELISA ..................... 100 4 2 Localization analysis of 143 3 epsilon by western blot ......................................... 101 4 3 Elicitor induced secretion of 143 3 epsilon from A rabidopsis tissue culture cells ................................................................................................................................. 102 4 4 Localization of 143 3 epsilonGFP fusion pro tein in Arabidopsis root cells. .... 103

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10 LIST OF ABBREVIATION S PKA Protein Kinase A pThr phosphothreonine pSer phosphoserine GA G i bberellic A cid NR Nitrate R eductase GRF General Regulatory F actor GFP Green Florescent Protein ABA Abscisic A cid BR Brassinosteroids RSG REPRESSION OF SHOOT G ROWTH BRI1 BRASSINOSTEROID INSENSITIVE 1 BZR1 BRASSINAZOLERESISTANT 1 C O CONSTANS ROS Reactive Oxygen S pecies PCD Programmed Cell D eath HDAC Histone Deacetylases PKC Protein K in ase C D ifopein Dimeric Fourteen threethree Peptide I nhibitor GBF3 G box Binding Factor 3

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11 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 PHOSPHORYLATION MEDIATED REGULATION OF 14 3 3 PROTEIN DIMERIZATION IN Arabidopsis thaliana AND THE EFFECT OF DIM ERIZATION IN 143 3/TARGET INTERACTIONS By Tufan Gokirmak Decem ber 2010 Chair: Robert J. Ferl Major: Plant Molecular and Cellular Biology 143 3s are a family of regulatory proteins that are uniquely eukaryotic, evolutionarily conserved across all eukaryotes, and deeply involved in proteinprotein interactions that mediate diverse biological processes. The 143 3 proteins commonly bind to target proteins containing well defined phosphothreonine (pThr) or phosphoserine (pSer) motifs. Though evolutionaril y conserved, most eukaryotes have a range of 143 3 genes and proteins that provide functional divergence to the family. The families of 143 3 protei ns are present in all cell types as homo and hetero dimers. The143 3/ target interactions can be regulated at two different levels: First, the phosphorylation status of the target and second the phosphorylation status of 143 3 itself, which has been shown to regulate the dimer/monomer status of the 143 3s. The present study dem onstrated that in Arabidopsis there is phosphorylation dependent regulation of 143 3 dimerization that drastically affects the range of dimerization among 143 3s and severely affects target protein interactions. This phosphoylation dependent regul ation of 14 3 3 dimerization has been conserved throughout the other eukaryotic organism. The data in this dissertation

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12 demonstrated that phosphorylation of 143 3 at Ser 62 has the potential regulatory role in both dimeriation and target interactions. In addition, phosphorylation at this conserved serine residue has the capacity to influence homo and heterodimerization of 143 3 proteins

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13 C HAPTER 1 THE 14 3 3 PROTEINS Introduction 143 3s are a famil y of regulatory proteins that are uniquely eukaryotic and evolutionarily conserved across all eukaryotes Despite this conservation, most eukaryotes have a range of 14 3 3 genes and proteins that provides functional divergence to the family. The 143 3 proteins are deeply involved in proteinprotein interactions that mediate important biological process such as regulation of metabolic enzymes, signal transduction pathways, cell cycle regulation, cell differentiation and proliferation by interacting directly with numerous target proteins Some of these targets include histones, kinases, transcription factors and chromatin remodeling enzymes such as histone deaceylases, and naked DNA. The 143 3s were first discovered in mammalian brain tissue (Moore and Perez, 1968). The name 143 3 comes from the same study by Moore and Perez, in which they purified brain proteins with unknown function and gave each a numerical designation. The numbers corresponded to their ion exchange chromatography elution profiles and starc h gel electrophoresis mobility patterns. The first reported function of 143 3s was the activation of two enzymes, tyrosine hyroxylase and tryptophan hydroxylase, involved in the neurotransmitter pathway in the presence of calcium and calmodulin dependent kinase II or cAMP dependent kinase (Ichimura et al., 1987). Later, search es for regulatory proteins in diverse cellular processes such as cell cycle regulation, signaling, and metabolic pathways led to the re discovery of 143 3s as re gulatory proteins. Cu rrently, it is well know n that 143 3s play regulatory roles in

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14 vari ous biological processes by direct interacti o n with diverse target proteins both in animals and plants (Fu et al., 2000). 143 3s are character ized as acidic proteins with a pI in the range of 4 to 5.5. The 143 3 proteins are present in all cell types as homo and hetero dimers with a monomeric mass of 25 32 kDa. However, the dimer/monomer status of 143 3 proteins can be regulated by phosphorylation at the dimerization interface. In v itro and in vivo studies showed that several protein kinases phosphorylate human 143 58 located in helix H3 at the dimer interface leading to monomerization ( Hamaguchi et al., 2003; Megidish et al., 1998; Powell et al., 2003; Gu et al., 2006; Porter et al., 2006). Powell et al. (2003) and Gu et al. (2006) showed that the mutation at Ser 58 of 143 to mimic phosphorylation prevents homodimer formation. In addition, Woodcock et al., (2003) examined the effect of sphingosine dependent kinase, (SD K1) mediated phosphorylation and Gu et al. (2006) characterized protein kinase A (PKA) mediated phosphorylation at Ser 58 and showed that phosph o rylated Ser 58 also inhibits dimerization. The 143 3 proteins generally bind to target proteins containing well defined phosphothreonine (pThr) or phosphoserine (pSer) motifs (Ferl, 1996). Extensive study of the binding site for mammalian 143 3 proteins on serine/threonine kinase Raf 1 revealed t hat they recognize the consensus sequences Arg(Ser/Ar)X(pSer/pThr)XPro (mode 1: RSXpSXP ) and ArgX(Ar/Ser)X(pSer/pThr)XPro (mode 2: RXF/YXpSXP) in which Ar indicates an aromatic residue and X indicates any residue (Table 1 1) This interaction occurs wit hin the conserved core section of the 143 3 proteins, which encodes an amphipathic groove in each monomer (Muslin et al., 1996; Yaffe et al.,

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15 1997). Mutational and co crystallization studies with the mammalian 143 3 isoform and phosphorylated targets su ch as a phosphopeptide from Raf 1 kinase and a phospho peptide representing the 143 3 binding epitope of polyoma virus middle T antigen showed that the amino acids Lys49, Arg56, and Arg127 interact with the phosphorylated amino acids of the target protei n s (Petosa et al., 1998; Yaffe et al, 1997). Another biding motif, SWpTX (motif 3) was characterized by a genetic screen against the C terminus of the Kir2.1 potassium channel (Coblitz et al., 2005). However, 143 3s were also reported to bind to sequenc es that diverge from those socalled 14 3 3 binding modes N on phosphorylated target protein sequences have been identified such as Gly His Ser Leu (GHSL) of the glycoprotein IbIX V complex protein (Andrews et al., 1998) and Trp LeuAsp Leu Glu (WLDLE) of the R18 peptide, a synthetic 143 3 antagonist isolated by phage display assay against human 143 Co crystallization studies of 143 e core region of R18 peptide, WLDLE is located in the region of the amphipathic ligand binding groo ve of 143 3, where the phospho serine is located. In addition hydrophobic residues of R18 core sequence align with hydrophobic residues in the amphipathic ligand binding groove of 143 3. The structural features of the 143 3/R18 interaction suggest that R18 interacts with 143 3 in a very similar manner to natural phosphorylated target s (Ma s ters and Fu, 2001). Table 1 1 summarizes the major 143 3 binding motifs characterized in diverge nt organisms. For 143 3 target prot eins that undergo phosporylation, the interaction between the 143 3 and the target protein appears to be regulated by the phosphorylation status of the target protein. Binding of 143 3 to its target can have several mechanistic

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16 consequences (Figure 11) (Tzivion et al., 2002; Roberts, 2003): (1) The binding may induce conformational changes on the target : Crystal structure of 14 3 serotonin N acetyltransferase (AANAT) revealed that 143 3 binding to AANAT regulates the substrate affinity of AANAT by stabilizing the region that is involved in substrate binding (Obsil et al., 2001) (Figure 1 1A) (2) 14 3 3 binding change the nuclear vs. cytoplasmic location of the target protein: Nuclear vs. cytoplasmic localization of REPRESSION OF SHOOT GROWTH (RSG), a plant bZIP transcription factor involved in gibberellic acid (GA) hormone signaling is regulated by 143 3 interaction. Phosphorylation mediated binding of 143 3 sequesters RSG in the cytoplasm, which in turn lowers the GA level in the cell (Ishida et al., 2008) (Figure 1 1B) (3) 143 3s regulate the intrinsic catalytic activity of the partner protein : The plant plasma membrane H+A TPase enzyme activity is regulated by 143 3 binding. Phosphorylation mediated binding of 143 3s to the plasma membrane H+ATPase leads to the displacement of the conserved C terminal autoinhibitory domain which in turn increases the activity of the plasm a membrane H+ ATPase (Olsson et al. 1998; Fuglsang et al. 1999; Svennelid et al. 1999) (Figure 1 1C) (4) 143 3s control turnover of the target protein : Nitrate reductase (NR) is another plant enzyme regulated by 143 3 proteins in response to environm ental and internal signals. Regulation of NR b y phosphorylation 143 3s occurs at two levels: First, phosphorylation mediated binding of 143 3s at the hinge 1 region of nitrate reduc tase lowers the intrinsic enzymatic activity of the NR (Kanamaru et al., 1999). Second, 14 3 3 bi n ding regulates the proteolysis of nitrate reductase. Removal of 143 3s from plant extracts decreases the NR turnover. This suggests that 143 3 binding negatively regulates NR stability (Weiner and Kaiser, 1999) (Figure 11D) (5) 143 3 binding alters

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17 the affinity of the target to other proteins : Insulin receptor substrate 1 (IRS 1) is a key signaling protein that transmits insulin signaling through activating several signaling pathways such as PI 3 kinase and MAP kinase pathways (Kosaki et al., 1998 ). 14 3 3 binding to IRS 1 decreases the affinity of IRS 1 to PI 3 kinase, which in turn attenuates its ability to activate PI3 kinase (Tzivion et al., 2001) (Figure 1 1E) Gene Organization and Evolutionary History The members of the 143 3 protein family from the bovine brain were designated by Greek letters according to their order of elution during reversed phase chromatography. The Arabidopsis 143 3 protein family members were also assigned Greek letter designat ion, but the designation was based on the gene sequence similarity and the name designation starts from the end of the Greek alphabet (Ferl, 1996). The genes encoding the 143 3 proteins in Arabidopsis were given a three letter designation GRF (General Reg ulatory Factor) followed by a number. Table 12 summarizes the nomenclature used in Arabidopsis 143 3 protein gene family. Protein alignments of 143 3s from different species and isoforms from the same species showed that the middle core sections of the 143 3 proteins, which consist of the helices involved in dimerization and phosphorylated target binding are highly conserved (Figure 1 2). This high level of conservation makes 143 3 from divergent species fit into similar functional and structural model s. On the other hand, the amino terminus and the carboxyl terminus are highly divergent and this might contribute to isoform specificity of 143 3s (Sehnke et al., 2002). The plant 143 3 proteins cluster into two groups when analyzed phylogenetically: The epsilon group (Figure 1 3). The isoforms in the plant non epsilon group appear to be plant specific and are significantly different from the plant and animal epsilon groups (Ferl et al., 2002). In

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18 Arabidopsis the epsil on group has five members, and and the nonepsilon group has eight members, , Arabidopsis 143 3 proteins are located on all five chromosomes with at least one isoform on each (Figure 14). A similar chromosomal dis tribution was observed for other species that have more than one 143 3 isoform (Ferl et al., 2002). Early three dimensional structure models for 143 3 proteins were developed by X ray diffraction crystallography using mammalian and isoforms (Liu et al., 1995; Xiao et al., 1995). The more highly conserved core region of 143 3s makes these structural models valid for other 14 3 3s of diverse species (Ferl et al., 2002). Later, several X ray crystallography models for plant 14 3 3s were developed (Wurt ele et al., 2003; Ottmann et al., 2009). The 14 3 3 monomer consists of nine antiparallel helices that form an L sh aped structure (Figure 15) A concave amphipathic groove lies in the interior of the Lshape structure, where the ta rget protein interaction occurs. This highly conserved section of the 143 3 proteins (~70% conserved) is made up of four helices: Two of these are the H3 and H5 helices, which are primarily composed of charged and polar amino acids. The two others, H7 and H9, contain hydrophobic amino acids. The 143 3s can form both homodimers and heterodimers. The contact between the monomers occurs at the amino terminal of helix H1 of one monomer and helices H3 and H4 of the other monomer (Figure 1 5) The high amino sequence conservation of H1 and H3 also allows 143 3s to form hetorodimers (Jones et al., 1995; Rittinger et al., 1999) The 143 3 dimers can bind two or more target proteins simultaneously (Braselmann et al., 1995; Vincenz and Dixit, 1996) This capacity of 14 3 3s suggests that they may be able to act as adaptor proteins linking two or more different target

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19 proteins to one another. If so, they can influence the formation of protein complexes or change the structural conformation of the t arget proteins by binding to two different regions of the same protein. One example is the role identified for the 143 3 that functions as an adaptor protein in the interaction between the plant plasmamembrane proton ATPase and the plant toxin fusicoccin in the presence of magnesium. It was shown that a binding site for the fusicoccin was creat ed when the 14 3 3 protein binds to the carboxy terminal autoinhibitory (C TA) domain of the ATPase (Jahn et al. 1997; Wrtele et al. 2 003). Fusicoccin binding stab ilized the complex of 14 3 3 and the ATPase, as well as displacing the C TA domain, thus allowing the ATPase to become fully active (Chung et al., 1999). Localization of 14 3 3 P roteins The 143 3 proteins are localized throughout the entire cell, suggesting that they can be involved in diverse protein protein interactions. Subcellular localization of 143 3s can provide important clues to possible roles of 14 3 3s in eukaryotic organisms. The first reported mammalian 14 3 3s isolated from brain tissue w ere cytosolic (Moore and Perez, 1968). Later, 143 3s were found in the nucleus, mitochondria, plas ma membrane and chloroplast (Bihn et al., 1997; Bunney et al., 2001; Baunsgaard et al., 1998; Sehnke et al., 2000) Although 14 3 3s do not have nuclear targ eting sequences, Bihn at al (1997) showed that 143 3s are present in both Arabidopsis and maize nuclei. A localization study using four evolutionarily diverse Arabidopsis 143 3 isoforms; at 143 3s have distinct and differential subcelullar localization (Paul et al., 2005) Considering the lack of subcellular localization signals on 143 3s, differential sublocalization of the individual

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20 143 3 isoforms may be driven by target interactions rather than the intrinsic properties of 143 3s. Use of AICAR, a 5 AMP analog, and R18 peptide, a high affinity 143 3 target, showed that in the absence of the 143 3/ target protein interactions, 143 3s were found to localize throughout the cell withou t any clear subcellularization (Paul et al, 2005). Milton et al. (2006) reported that the mammalian 143 throughout the cell. The interaction between 143 family of transcription factors, localizes 14 3 that many 14 3 3 isoforms may have specific target s and distinct regulatory functions. Furthermore, as mentioned earlier, 143 3 can also form heterodimers, with a capacity to mediate concurrent interactions between two or more target proteins (Paul et al., 2005). In Arabidopsis a study with isoform specific antibodies showed that two 143 3s 3 3 isoforms from the non 3 3s promin ently located in the chloroplast (Sehnke et al., 2000). This finding suggests that phylogenetically different isoforms can share similar subcellular locations and functions. The expression profiles of 14 3 3 isoforms vary from tissue to tissue and organ to organ. For example, in Arabidopsis Therefore, the tissue s pecific expression of 143 3 isoforms increases the complexity of the 14 3 3 mediated regulation of the target proteins. Earlier GFP fusion studies in Arabidopsis also confirmed the isoform specific subcellular localization of 14 3 3 proteins. The 143 GFP fusion protein localizes to the plasma membrane, the 143 -

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21 GFP fusion tends to be in the cytosol, and the 143 GFP localizes at nuclear envelopes (Cutler et al., 2000; Sehnke et al., 2002). Localization of the 143 GFP fusion protein betwee n cytoplasm and nucleus was found to change during the course of the cell cycle. These proteins generally localized out of the nucleus, then entered the nucleus following nuclear division, and finally moved back out of the nucleus just before completion of cytokinesis (Cutler et al., 2000). The 143 3s are also implicated in regulation of subcellular localization for many mammalian target proteins. For example, 143 3 proteins regulate the activity of a type II tumor suppressor ING1 by targeting it to cytoplasm. The ING1 affects cell growth regulation, stress signaling, DNA repair and apoptosis by altering chromatin structure and transcriptional regulation. The interaction between 143 3 and ING1 is regulated by the phosphorylation status of ING1. Decreased expression and mislocalization of ING1 cause several different human cancers (Gong et al., 2006). The 143 3 proteins can also directly regulate subcellular localization of transcription factors such as FoxO forkhead type, which are involved in regulation of numerous genes that control cell proliferat ion and apoptosis. Interaction with 143 3 proteins affects the binding affinity of these transcription factors to their target DNAs (Obsilova et al., 2005). Functions of 14 3 3 P roteins in Plants In the early plant literature, 143 3s were recruited mor e heavily to central metabolic pathways, hormone signaling, carbohydrate metabolism and stress metabolism (reviewed in Ferl, 1996; Huber et al., 2002), whil e animal 143 3 interactions were more dedicated to signal transduction cascade players such as prot ein kinases, phosphatases and transcription factors (reviewed in (Mackintosh, 2004)). These include Raf (Freed et al., 1994; Irie et al., 1994; Li et al., 1995), Ras

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22 (Gelperin et al., 1995; Rommel et al., 1996), protein kinase C (Toker et al., 1992; Dellamb ra et al., 1995), and Bcr (Reuther et al., 1994; Braselmann and McCormick, 1995). In contrast to the early plant 143 3 literature where 143 3s were characterized as regulators of metabolic enzymes such as nitrate reductase (Bachmann et al., 1996), sucrose phosphate synthase (Moorhead et al., 1999), and starch synthase (Sehnke et al., 2001) recent in vitro and in vivo proteomics, genetics an d physiology studies have identified 143 3 proteins as essential players in plant signaling pathways. It is becomin g increasingly apparent that 143 3s are involved in the signal transduction associated with hormones, light responses, stress signaling as well as basic metabolism. Proteome and interactome studies indicate that 143 3s interact with a diversity of signal ing proteins and metabolic processes. Table 1 2 summarizes the recent studies of plant 143 3 binding proteins as signaling mediators. Role of 143 3 Protein s in Plant Hormone Signaling The active roles of 143 3s in plant hormone signaling have been descr ibed for gibberellic acid (GA), abscisic acid (ABA) and brassinosteroids (BR) (Ishida et al., 2008; Schoonheim et al., 2007; Gendron and Wang, 2007). In gibberellic acid mediated pathways, 143 3s are involved in negativefeedback regulation of GA homeostasis by altering the cytoplasmic/nuclear partitioning of a tobacco bZIP transcription factor, REPRESSION OF SHOOT GROWTH (RSG) (Ishida et al., 2004). RSG controls the GA level in cells through transcriptional regulation of genes encoding GA biosynthesis. An i ncreased level of GA causes the phosphorylation of RSG by a Ca+2 dependent kinase CDPK1. Upon phosphorylation, 143 3 binds to RSG and sequesters it in the cytoplasm, which in turn lowers the GA level in the cell (Ishida et al., 2008) by affecting

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23 transc ription. The removal of the 143 3 binding site leads to an accumulation of RSG protein in the nucleus thereby increasing the transcriptional activity of RSG in vivo Participation of the 143 3s in ABA signaling is also mediated through transcriptional regulation. 143 3s are shown to be present physically at the promoters of two Arabidopsis late embryogenesis genes, AtEm1 and AtEm6, which are induced by ABI3, an ABA regulated transcription factor (del Viso et al., 2007). 143 3s may act as an adaptor pr otein in the transcription complex found in Em gene promoters (del Viso et al., 2007). Another example of 143 3 being part of transcription complex is described in barley seeds where the ABI5 family of ABA regulated transcription factors in barley interac ts with 14 3 3s in yeast twohybrid assay s In the same study, it was shown that ABA induces the expression 143 3s in embryonic barley roots. Furthermore, RNAi mediated silencing of several 143 3 members in barley reduces the ABI5 mediated induction of A BA inducible reporter constructs (Schonnheim et al., 2007). Brassinosteroid (BR) hormone signaling is another well described signaling pathway in Arabidopsis that plays a crucial role in plant growth and development. The ligand, receptors, protein kinases and transcription factors of the BR signaling pathway have been characterized by several research groups (e.g. Kim and Wan g, 2010; Belkhadir et al., 2006). Recent reports showed that 143 3s play important regulatory function in BR signaling at several di fferent levels. Two 14 3 3 isoforms are present in a complex with membrane bound BR receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). The significance of this interaction is yet to be elucidated (Karlova et al., 2006). BR induced transcription factor B RASSINAZOLE RESISTANT 1 (BZR1) is also a target for 143 3 proteins in Arabidopsis and rice (Ryu et al., 2007; Bai

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24 et al., 2007). BZR1 is a highly phosphorylated transcription repressor whose phosphorylation status determines its nuclear/cytoplasmic locali zation (Ryu et al., 2007; He et al., 2005). BR induced dephosphorylation and nuclear accumulation of BZR1 causes brassinosteroidinduced growth and feedback regulation of brassinosteroid biosynthesis (Wang et al., 2002). Phosphorylation mediated BZR1/14 3 3 interaction causes cytoplasmic retention or nuclear export of the BZR1. Mutations on putative 14 3 3 interaction binding sites on BZR1s abolish the interactions an d increase the nuclear localization of BZR1s (Gampala et al., 2007) Role of 143 3 Protein s in Light Signaling Compared to hormone signaling, involvement of 143 3s in light signaling is less well understood. Phototropins are blue light specific plant protein kinase receptors that are autophosphorylated by blue light. They are involved in several different blue light responses such as phototropism, hypocotyl growth inhibition, stomatal opening and chloroplast movement (Christie, 2007). Autophosphorylation of a serine residue in the Hinge1 region of PHOT1 in response to blue light creates a 143 3 binding site (Inoue, et al., 2008). However, mutant phot1 plants without the 14 3 3 binding site did not show any phenotypic difference from wild type plants. A recent study also revealed that 143 3s interact with PHOT1 in an isoform specific ma nner (Sullivan et al., 2009). Only non epsilon isoforms were shown to interact with PHOT1. The same study also showed that 143 3s do not interact with PHOT2 due to the less conservation of the phosphomotifs in the Hinge 1 region of PHOT2 (Sullivan et al. 2008). A mechanistic link between 143 3s and redlight signaling was discovered using a reverse genetic approach. Arabidopsis T DNA insertion lines for 14 3 isoforms exhibited a delayed flowering time phenotype on long days. Plants also

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25 show ed hyposensitive hypocotyl growth inhibition under red light but no difference under blue and far red light (Mayfield et al., 2007). In the same paper, a direct interaction between these two 143 3 isoforms and photoperiodic regulatory protein CONSTANS (CO ) was demonstrated using yeast twohybrid and coimmunoprecipitation assays. Although, 143 3s do not appear to interact with the photosensor phytochrome B (phyB) directly, the flowering delay phenotype and direct interaction with CO suggest that 143 3s a re part of the red light signaling pathway (Folta et al., 2008). Role of 143 3 Proteins in B iotic and Abiotic Stress R esponse There have been a number of studies suggesting that 14 3 3s play a role in plant stress responses (reviewed in Chevalier et al., 2009). Transcriptome analyses of plant biotic and abiotic treatments reveal that many 143 3 isoforms are consistently differentially expressed in response t o a variety of stressors (e.g. Chen et al., 2006; Lancien et al., 2006; Xu et al., 2006). Proteomic analyses also revealed an abundance of 143 3 partners that are associated with biotic and abiotic stress responses (Paul et al., 2009; Chang et al., 2009; Alexander and Morris, 2006). In addition to this indirect evidence several groups reported direct involvement of 143 3s in stress regulation. Reactive oxygen species (ROS) production by plasma membrane bound NADPH oxidases is an important event in response to both biotic and abiotic stress stimuli (Torres and Dangl 2005; Elmayan and Simon Pl as,2007). A yeast twohyb rid screen against the C terminal section of a plasma membrane NADPH oxidase in tobacco showed that NtrbohD interact s with Nt14 3 3h / omega1 isoform. In addition, stimulation of tobacco leaves with the fungal elicitor cryptogein ca used the accumulation of this 14 3 3 transcript. The significance of the 143 3/NtrbohD interaction was demonstrated by

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26 transforming the tobacco cells with antisense constructs for this Nt14 3 3h / omega1 isoform. Cells transformed with this antisense constr uct were not able to produce ROS in response to cryptogein elicitor (Elmayan et al., 2007) So, these results suggest that 143 3s may play a pivotal role in ROS mediated programmed cell death in plant defense signaling named hypersensitive response (HR) i n an isoform specific manner Plants usually trigger localized programmed cell death (PCD) as a defense response against pathogen attack (van Doorn and Woltering, 2005). Pseudomonas syringae pv tomato induced PCD in tomato and tobacco is positively regul ated by mitogen tomato 14 3 interacting protein in a yeast two of the expre enhancement in PCD response in tobacco. by a site specific P h ysiological evidence for 143 3 mediated abiotic stress respon se was shown by over expression of Arabidopsis 143 t ype cotton plants, these transgenic cotton plants had a slow wilting phenotype and higher photosynthesis rate under water stress conditions. Furthermore, under normal growth conditions, these transgenic cotton plants displayed a late leaf senescence phenot ype (Yan et al., 2004).

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27 Role of 14 3 3 Proteins in C hromatin mediate d Gene R egulation In addition, there is evidence to indicate that 143 3 proteins are involved in chromatin mediated gene regulation through interaction with histones (Chen and Wagner, 1994) and histone deacetylases (HDACs ) (Grozinger and Schreiber, 2000) Cross linking experiments and affinity chromatography revealed that 143 3 proteins bind to histones in rat cell line PC12. The 143 3 proteins enhance the histone phosphorylarion me diated by protein kinase C (PKC), which leads to increasing secretion of catecholamine in chromaffin cells. The 143 3 proteins were also found to inhibit the rate of histone dephosphorylation (Chen and Wagner, 1994). Transcription is partly controlled by the acetylation status of the histones. Histone deacetylation is mediated by histone deacetylases (HDACs). The interaction between 143 3 proteins and HDACs was demonstrated by Grozinger and Schreiber (2000). This interaction triggers nuclear export of HDA Cs, which permits gene expression (Chang et al., 2005). Conclusions In contrast to the early plant 143 3 literature where 14 3 3s were characterized as metabolic regulators, recent in vitro and in vivo proteomics genetics and physiology studies have pla ced 143 3 proteins as essential components of plant signaling pathways. It is becoming increasingly apparent that 14 3 3s are involved in signal transduction associated with hormones, light responses, stress signaling as well as basic metabolism Proteome and i nteractome studies indicate that 143 3s interact with a diversity of over hundred proteins involved in signaling and metabolic processess. Considering that 143 3s are extensively involved in protein protein interaction, understanding the regulation of 14 3 3/ target interactions is essential both at cellular and organismal levels.

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28 143 3/ target interactions can be regulated at two different levels: first the level of phosphorylation on the 14 3 3 recognition motif on the target protein; second, the phosphorylation status of 14 3 3 itself The regulation of 143 3/ target interactions by phosphorylation of 143 3s has been reported previously. Phosphorylation of human 143 3 at Ser 58 by PKA and B/Akt kinases has been shown to affect target interacti ons by regulation of the dimer status of the 143 3 (Gu et al., 2006, Powell et al., 2002). Two other residues of 14 3 3 Ser 184 and Thr 232, have been also demonstrated to be phosphorylated and involved in 14 3 3/ target interactions (Aitken et al., 1995; Dubois et al., 1997). The chapters of this dissertation mainly focus on the regulation of 143 3 dimerization and the effect of dimerization in Arabidopsis 143 3/ target interactions. Phosphorylation of a conserved serin e residue, Ser 62, in the dimerization interface of 143 3 interferes with the dimer ization of the 143 3 protein. In vitro and in vivo studies showed that Ser 58 of human 14 3 3 62 in Arabidopsis 143 3 located in helix H3 at the dimer interface, can be phosphorylated by several different kinases (Hamaguchi et al., 2003; Megidish et al., 1998; Powell et al., 2003; Gu et al., 2006; Porter et al., 2006). These studies showed that the phosph orylation of Ser 58 inhi bits dimerization of 143 3 In addition, phosphorylation mimic mutations, Glu 58 and Asp 58, in the dimerization interface of 143 were shown to prevent homodimer formation ( Powell et al. 2003; Sluchanko et al., 2008). The present study showed that a similar mechanism also exists in plant 143 3s, specifically in the model plant organism Arabidopsis 143 3s. In addition, this study demonstrated that phosphorylation of 143 3 in the dimerization interface not only disrupts the dimerization, but it can also change the affinity of a specific isoform to

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29 another 143 3 isoform as a dimer ic partner. Arabidopsis has 13 different 143 3 isoforms and they can form 91 potential 143 3 dimers (13 homodimers, 78 heterodimers). Considering the differential affini ties of 14 3 3 isoforms for the 143 3 target s, this large number of 143 3 dimer combinations may explain the functional divergence of the 143 3 protein families. Therefore, an understanding of t he regulation of hetero or homodimer formation is crucial for describing how 143 3s are involved in dynamic protein protein interactions with different target affinities.

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30 Table 1 1. 143 3 protein binding motifs 14 3 3 binding motifs Protein Sequence Reference Mode 1 RSXpSXP Yaffe et al., 1997 Mode 2 RXF/YXpSXP Yaffe et al., 1997 Mode 3 SWpTX Coblitz et al., 2005 H(+) ATPase YpTV Fuglsang et al., 1999 Exoenzyme S DALDL Henriksson et al., 2002 Glycoprotein Ib IX V complex GSHL Andrews et al., 1998 R18 WLDLE Petosa et al., 1998

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31 A B C D E Figure 11. The mode of 143 3/ target interactions. Binding of 143 3 to its target may A) induce conformational changes, B) alter the nuclear vs. cytoplasmic distribution, C) regulate the intrinsic catalytic activity, D) control turnover of the target protein, or E) associate multiple targets together. (F igure adapted from Gkirmak et al., 2010).

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32 Table 1 2. Arabidopsis thaliana 143 3 protein gene family Gene Name Protein Name Genomic Locus GRF1 At4g09000 GRF2 At1g78300 GRF3 At5g38480 GRF4 At1g35160 GRF5 At5g16050 GRF6 At5g10450 GRF7 At3g02520 GRF8 At5g65430 GRF9 At2g42690 GRF10 At1g22300 GRF11 At1g34760 GRF12 Iota ( ) At1g26480 GRF13 At1g78220

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33 Figure 12. Whole protein sequence alignment of human (Hs), Arabidopsis (At), yeast (Sc), Drosophila (Dm) and C.elegans (Ce) 14 3 3s. Protein sequences were aligned using online MultAlin software ( http://multalin.toulouse.inra.fr/multalin/multalin.html ) (Corpet, 1988). High consensus (= 90 %) residues are in red, low consensus (= 50 %) residues are in blue and non conserved residues are in black. C onsensus symbols: is either I or V; $ is either L or M; % is either F or Y; # is either N, D, Q, E, B or Z.

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34 Figure 13. Unrooted phylogenetic tree of Arabidopsis 143 3 proteins. Whole protein sequences of 13 Arabidopsis 143 3s were downloaded from TAIR website ( http://www.arabidopsis.org/ ) by their genomic locus numbers (Table 1 2). Protein sequences were aligned by ClustalW ( www.ebi.ac.uk/clustalw ) and the phylogeneti c tree was drawn with TreeView (Page, 1996) using the UPGMA method. Epsilon group Non epsilon group

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35 Figure 14. Chromosomal distribution of Arabidopsis 143 3 protein genes. Arabidopsis 143 3 genes encoding 143 3 proteins were placed on Arabidopsis chromosomes by plugging in Arabidopsis 143 3 genomic locus numbers (Table 1 2 ) into the Arabidopsis Information Resource ( TAIR) Chromosome Map Tool ( http://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp). The tool allows the display of alternative names by entering the alternative name after the genomic locus name (i.e, entering: At5g65430 Kappa will display only Kappa on the chromosome).

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36 A B Figure 15. 3D structure model of 143 3 proteins. A) Side view of the 143 3 dimer. B) Top view of 143 3 dimer. The model is derived from Nicotiana tabacum 143 3 in complex with the differentiation ind ucing fungal agent Cotylenin A (PDB3E6Y) (Figure adapted from Ottmann et al., 2009) using PDB SimpleViewer 3.8 software. Figures were captured by screen shots. Each helix is labeled with a different color. Helix H1 represents the first helix starting from amino terminus. Helix H9 is the last helix near to the carboxy terminus.

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37 Table 1 3. Summary of recent studies investigating the involvement of plant 143 3 proteins in plant protein signaling pathways Signaling Pathway Target Mode of Action Reference Gibberellic acid (GA) RSG Localization Ishida et al., 2008; Ishida et al., 2004 Abscisic acid (ABA) ABI3, ABI5 Association Schoonheim et al., 2007; del Viso et al., 2007 Brassinosteroids (BR) BRI1, BZR1 Localization Karlova et al., 2006; Ryu et al., 2007; Bai et al., 2007 Blue light signaling PHOT1 Unknown Inoue et al., 2008; Sullivan et al., 2009 Red light signaling CO Unknown Mayfield et al., 2007 Hypersensetive response (HR) NtrbohD Activity Elmayan et al., 2007 Hypersensetive response (HR) Stability Oh et al., 2010

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38 CHAPTER 2 SER 62 OF ARABIDOPSIS 14 3 ATION AND TARGET INTERACTIONS Introduction The 143 3 proteins are phospopeptide binding regulatory proteins that constitute conserved and ubiquitous eukaryotic protein families 143 3 proteins were first discovered in mammalian brain tissue in the late 1960s Later, 143 3 proteins were shown to be involved in important biological processes such as signal transduction cell cycle regulation, cell differentiation and proliferation and regulation of metabolic enzymes through interacting with target proteins containing well defined phosphothreonine or phosphoserine motifs (Ferl et al., 1996). Binding of 143 3s can affect their target s in a va riety of different ways: 143 3 binding may increase or decrease the enzymatic activity of the target keep the target in a specific subcellular location, act as an adapter protein by binding to two target s at same time or regulate the stability of the tar get protein (reviewed i n G kirmak et al., 2010; Tzivion and Avruch, 2002). Phylogenetic analysis of 143 3 proteins from diverse organisms showed that plant 143 epsilon group. Non epsilon 143 3 isoforms from different plant species form a separate distinct phylogenetic cluster which is very different from animal 143 3 isoforms and plant epsilon isoforms (Ferl et al., 2002). In Arabidopsis, the epsilon group has five members, (Ferl et al., 2002) The 143 3 proteins have a monomeric mass of 2532 kDa and they can form homo and hetero dimers. Each 143 3 monomer consists of nine antiparallel helices

PAGE 39

39 that form an L shaped structure (Figure 21A) 143 3 dimerization occurs between t he amino terminal of the H1 helix of one monomer and the H3 and H4 helices of the other monomer (Figure 2 1). The sequence conservation of H1 and H3 helices between isoforms allows 14 3 3s to form hetero dimers as well as homodimers (Jones et al., 1995; Rittinger et al., 1999). However, there is still substantial sequence diversity in Helix 1 and Helix 3, which might be responsible for the differential affinity of one 143 3 isoform to another as a dimer ic partner (Figure 21B). For instance, w ith 13 different 143 3 isoforms, Arabidopsis can form 91 potential 143 3 dimers (13 homodimers, 78 heterodimers). This large number of 143 3 dimer combinations may explain the functional divergence of the 143 3 protein families. Considering the differential affinities of 143 3 isoform to the 14 3 3 target s, each 143 3 dimer combination may exhibit distinct affinity to target proteins. Therefore, understanding t he regulation of heteroor homo dimer formation is crucial for describing how 143 3s are involved in dynamic protein protein interactions in response to specific time and input. The 143 3/ target protein interacti ons occur in a concave amphipathic groove that lies in the interior of the Lshape structure in each 143 3 monomer (Muslin et al., 1996; Yaffe et al., 1997) (Figure 21A) Mutational and co crystallization studies with the mammalian 14 3 3 isoform and ph osphorylated target s such as a phosphopeptide from Raf 1 kinase and a phospho peptide representing the 143 3 binding epitope of polyoma virus middle T antigen showed that the amino acids Lys 49, Arg 56, and Arg 127 of 143 3 interact with the phosphoryl ated amino acids of the target proteins (Petosa et al., 1998; Yaffe et al, 1997). However, 14 3 3s were also reported to bind to nonphosphorylated target protein sequences such as Gly His Ser Leu (GHSL) of the

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40 glycoprotein IbIX V complex protein (Andrews et al., 1998) and TrpLeu Asp Leu Glu (WLDLE) of the R18 peptide, a synthetic 143 3 antagonist isolated by a phage display assay against human 143 crystallization studies of 14 3 with R18 revealed that the core r egion of the R18 peptide, WLDLE, is located in the amphipathic ligand binding groo ve of 143 3, where the phospho serine of a phosphorylated target is normally located. Furthermore, hydrophobic residues of the R18 the core sequence align s with hydrophobic residues in the amphipathic ligand binding groove of 143 3. The structural features of the 143 3/R18 interaction suggest that the R18 peptide interacts with 14 3 3 in a manner similar to natural phosphorylated target s ( Wang et al., 1999; Master s and Fu, 2001). A synthetic higher affinity 14 3 3 antagonist difopein ( di meric fo urteen three three pe ptide in hibitor) was designed based on the dimeric R18 peptide (Masters and Fu, 2001). Expression of difopein in human cell lines led to apoptosis, indicating that 14 3 3s are involved in the regulation of anti apoptotic pathways. 143 3/ target interactions can be regulated by phosphorylation at two fundamentally different levels: f irst, level of phosphorylation at the 143 3 binding motif of the target protein (Figure 2 2A ); second, level of the phosphorylation status of 143 3 itself, which can determine the dimer ic status of the 14 3 3 protein (Figure 2 2B). In vitro and in vivo studies showed that human 143 58 located in helix H3 at the dimer interface can be phosphorylated by several different kinases (Hamaguchi et al., 2003; Megidish et al., 1998; Powell et al., 2003; Gu et al., 2006; Porter et al., 2006). Woodcock et al. (2003) and Gu et al. (2006) examined the effect of sphingosine dependent kinase (SDK1) mediated phosphorylation and protein kinase A (PKA)

PAGE 41

41 mediated phosphorylation at Ser 58, respectively. They showed that phosph orylation of Ser 58 inhibits dimerization of 143 3 In addition, phosphorylationmimic mutations, Glu 58 and Asp 58, in the dimer interface of 143 were shown to prevent homodimer formation ( Powell et al. 2003; Sluchanko et al., 2008). Alignment of human 143 Arabidopsis 143 3s showed that the Ser 58 residue is conserved in all Arabidopsis isoforms exce pt 143 3 4A ). In this the study the effect of phosphorylation of Ser 62 in Arabidopsis 143 dime rization was investigated This residue correspon ds to the Ser 58 of human 143 The present study showed that the 143 62 ( S62D) phospho mimic mutation disrupts 143 3 homodimerization, which in turn inhibits the 143 3 / target interaction s. The data also showed that mutant S 62D can form heterodimers with 143 3 3 3 3 3 3 b ut not w ith 14 3 3 143 14 3 3 3 3 It was also demonstrated that when Ser 62 is phosphorylated, the affinity of 143 3 hetero dimerization to certain 143 3 isoforms is significantly increased. The present study characterized a phosphorylation mediated de novo regulation of 143 3 hetero dimerization that clearly affects target interactions in plants Material s and Methods 3D Model and P rotein Alig nment The 143 3 dimer model was derived from Nicotiana tabacum (PDB3E6Y) (Ottmann et al., 2009) using PDB SimpleViewer 3.8 software. Whole protein sequences of all 13 Arabidopsis 143 3 proteins were aligned using online MultAlin software ( http://mu ltalin.toulouse.inra.fr/multalin/multalin.html ) (Corpet, 1988).

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42 Plasmids The full open reading frames ( ORF ) of all thirteen Arabidopsis 143 3 isoforms and the N termina l half of nitrate reductase (NR 1 562aa) (residues 1 562) were PCR amplified with Gate way tagged primers from a n Arabidopsis leaf and flower cDNA library, a gift from Dr. Kevin Folta (University of Florida). EYFP difopein and EYFP R18 (Lys) in pEYFP were kindly provided by Dr. Haian Fu (Emory University). EYFP Difopein and EYFP R18 (Lys) we re PCR amplified from these vectors with Gateway tagged primers PCR fragments were subcloned i nto pDONR221 vector using Gateway BP Clonase II (Invitrogen, CA). The phosphorylationmimic mutation S62D and nonphosphorylatable S62A in 14 3 and S534L mutation that distrupts 143 3 binding to the nitrate reductase NR1 562aa (residues 1562) ( NR1 562aaS534L) (Kanamura et al., 1999) were created by site directed mutagenesis and overlapping PCR (Ho et al., 1989). Mutant PCR fragments were subcloned into the pDONR221 vector as described above. All thirteen wild type Arabidopsis 143 3s and the point mutation ( S62D and S62A) cDNAs in pDONR221 were cloned into the pDEST22 yeast twohybrid prey vector via Gateway LR Clonase II (Invitrogen, CA) reacti on s. The143 3 143 and NR1562 and NR1562S534L cDNAs in pDONR221 were cloned into the pDEST32 yeast twohybrid bait v ector via Gateway LR Clonase II reactions. EYFP Difopein and EYFP R18(Lys) fragments in pDONR221 were transferred int o E.coli expression vector pDEST15 by LR Clonase II recombination reaction The 143 tagged recombinant protein in E.coli was described previously (Lu et al., 1992; Wu et al., 1997). 14 3 and 143 codin g sequences were PCR amplified from pDONR221143 and

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43 pDONR221143 vectors with primers containing NdeI Bam HI restriction sites and cloned into the pET15b E.coli expression vector. The constitutive yeast expression vector p415GPD ( Ampr LEU2 ARS/CEN ) (Mumberg et al., 1995) was a gift from Dr. Paul van Heusden (Leiden University). Arabidopsis 143 143 and 143 coding sequences were cloned into Bam HI and Hind III restriction sites of p415GPD using primers with Bam HI and Hind III restriction sites Split YFP vectors, pHNYFP (YFPN: N terminal fragment) and pFCYFP (YFPC: C terminal YFP fragment), for bimolecular fluorescence complementation (BiFC) assay s were developed by Akhtar et al. (2008). 143 3 and 143 3 S62D fragments were PCR am plified with primers that introduce NdeI/NcoI restriction sites. Digested PCR fragments were ligated into NdeI/NcoI sites of pHNYFP and pFCYFP plasmids to create C terminal 14 3 3/split YFP peptides. Yeast T wo hybrid S ystem S. cerevisiae str ain AH109 ( MATa, trp1901, leu23, 112, ura352, his3200, gal4gal80LYS2::GAL1UASGAL1TATAHIS3,GAL2UASGAL2TATA ADE2,URA3::MEL1UASMEL1TATAlacZ, MEL1) was co transformed with bait (pDEST 22) and prey (pDEST32) vectors by using the standard lithium a cetate/polyethylene glycol method (Gietz and Woods, 2006). Cotransformed yeast cells were selected on synthetic complete dropput, SC LeuTrp plates. T he spot assays were performed using yeast cells cultured overnight in SC Leu Trp medium at 30C. Concentrations were adjusted to A600 of 0.2 and 10 l of the culture and its ten fold serial dilutions were spotted on SC LeuTrp and SC Leu Trp His plates. Due to the self activation of 143 -

PAGE 44

44 3 DNA binding domain, background yeast growth on SC Leu Trp His plates was inhibited by inclusion of 0.5 mM 3AT in the media. SC Leu Trp p lates were incubated at 30 C for 36 hr, and SC Leu Trp His plates were incubated at 30 C for 48 hr Expression an d Purification of Recombinant Proteins in Bacteria E. coli strain BL21AI (Invitrogen) was transformed with pET15b14 3 143 pET15b143 pDEST1 5 EYFP difopein and pDEST15EYFP R18 (Lys) plasmids. Transformed cells were cultured i n Luria Bertani (LB) medium containing 50 g/ml of carbeniciline until A600 between 0.5 and 1 was reached. Recombinant protein expression was induced with 0.2% arabinose (final concentration) and induced cultures were incubated for 3 h at 37C and harvested by centrifugation at 5,000 x g for 30 minutes. Pellets of cells expressing GST tagged EYFP difo pein and EYFP R18 (Lys) were re suspended in GST binding buffer (25mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA 1 mM dithiothreitol (DTT) and proteaseinhibitor mixture (Calbiochem)). Pellets of cells expressing H is tagged 143 3 3 suspended in equilibration buffer (50 mM sodium phosphate, pH 8.0, with 0.3 M sodium chl oride and 10 mM imidazole and proteaseinhibitor mixture (Calbiochem)). Cells were lysed by French press at 1260 psi. Cellular debris was removed by centrifugat ion at 12,000 X g for 30 min at 4 C. The recombinant GST fusion proteins were affinity purified using a column loaded with glutathione Sepharose resin (Calbiochem) and eluted from the beads by adding the elution buffer (100mM Tris HCl, pH8.0, containing 20mM glutathione). The His tagged recombinant proteins were affinity purified using column loaded with HIS Select HF Nickel Affinity Gel resin

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45 (Sigma ) and eluted from the column by adding the elution buffer (50 mM sodium phosphate, pH 8.0, with 0.3 M sodium chloride and 250 mM imidazole). Samples were d ialyze d overnight at 4C in p hosphate buffered s aline (PBS). Determining the Affinity and the Binding K inetics of 143 3 S62D for T arget s The interaction kinetics of 143 d 143 S62D with two analy tes; EYFP difopein and EYFP R18(Lys) were investigated using the Octet QK platform with amine reactive biosensors (Fortebio). R ecombinant 14 3 3 ( 35 g/ml ) in MES buffer (pH 6) were immobilized onto the amine reactive biosensors surface as ligands. The kinetics parameters of 143 3 GST tag ged analytes, EYFP Difopein and EYFP R18(Lys) were measured agai nst four two fold dilutions of 2 M of analytes u sing the Octet QK platform at 30 C Subcellular F ractio nation and Localization 143 Arabidopsis suspension cells were harvested o n day 3 by filtration through MiraCloth (Calbiochem ). S uspension cells (10g ) were treated with g ently overnight (12 14 h) with 50 ml of protoplasting solution (0.1% celluysin, 0.1% macerase and 0.1% pectolyase in 10% mannitol, 0.5X MS salts, pH 5.7) on a horizontally rotating platform (Belly D ancer ) Protoplasts were filtered through the Miracloth and pelleted by centrifugation at 900 rpm for 5 min in a swinging bucket centrifuge. Pelleted cells were washed and resuspended gently in 10 ml MMS (10% mannitol with 0.5 X MS salts, ph 5.7) in the presence of phospha tase and pro tease inhibitors (Calbiochem). Protoplasts were re pelleted an d re suspended in 5 ml of 1X nuclear isolation buffer ( NIB ) (Sigma) in th e presence of 25 mM NaF, phosphatase and protease inhibitors. An aliquot of protoplasts, representing the whole cell fraction was removed. The cell membrane of

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4 6 the remaining protoplats was disrupted with 0.15% Triton X 100 on ice for 5 min and the nu clei pelleted at 2000xg, ( 4 C ) for 10 min. The supernat ant representing the cytoplasmic fraction was removed and kept on ice. The nuclear pellet was resuspended in 5 ml of NIB buffer and an aliquot representing the whole nuclear fraction was removed. The nucle i were re pelleted and resuspended in 2 ml of NIB A portion ( 500 ) of nuclear fraction was pelleted at 2000xg, ( 4 C ) for 5 min and resuspended in 450 of NHB ( 5 mM Hepes, pH7.4, 2 mM EDTA, 2 mM K ) dii odosalicylate (LIS) was added to the nuclear fraction to a concentration of 5 mM to extract the soluable proteins and gently roc ked on ice for 15 min. The insoluble nuclear fraction was pelleted at 12,000xg, ( 4 C ) for 5 min and the supernatant was kept as the soluble nuclear fraction. The pellet was washed and resuspended in 500 the insoluble fraction. Fluorescent Spectroscopy Analyses The e ffect of the phosphorylation mimi c mutation S62D on the structure of 14 3 3 was studied by measuring the changes in the intrinsic tryptophan fluorescence of 143 using a fluoro meter (Jobin Yvon HORIBA FluoroMax 3). E.coli expressed 143 S62D and 295 nm (slit width of 5 nm) in PBS buffer and the intrinsic tryptophan fluorescence was recorded in the range of 300 400 nm (slit width of 2.5 nm). Hydrophobicity of wild type 143 3 3 S62A was measured with bis ANS (Invitrogen) a fluoresce nt hydrophobic probe that binds to hydrophobic residues in proteins (Takashi et al., 1977) R ecombinant proteins (25

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47 in PBS buffer (pH 7.5) were incubated with indicated concentrations of bis ANS (Figure 2 5) and were excited at 295 nm (slit width o f 5 nm) F luorescence values were recorded in the range of 305575 nm (slit width of 5 nm). Cross linking S tudies S. cerevisiae strain INVSc1 ( MATa his3D1 leu2 trp1289 ura352 MATAlpha his3D1 leu2 trp1289 ura352) was transformed with p415GPD 143 143 143 the standard lithium acetate/polyethylene glycol method (Gietz and Woods, 2006). Transformed cells were selected on SC Leu plates. A single colony from eac h transformation was picked and cultured overnight in SC Leu media to express recombinant 143 3 and 143 suspended in yeast breaking buffer (50 mM sodium phosphate, pH 7.4, 5% glycer ol, 1 mM PMSF 1mM DTT, phosphataseinhibitor mixture and proteaseinhibitor mixture (Calbiochem) ) to OD600 of 75. Cell free yeast lysates were prepared by homogenization with acidwashed glass beads and subsequently centrifuged at maximum speed for 10 minu tes. Recombinant 143 3 3 free yeast lysates and e ndogenous Arabidopsis 143 were cross linked with varying concentrations of bis[sulfosuccinimidyl] suberate (BS3) (Pierce) at room temperature for 30 minutes. The Cross linking reaction was quenched by adding Tris HCl, pH 7.5 to a final concentration of 20 50 mM. SDS PAGE and Western Blot A nalysis R ecombinant proteins and plant and yeast lysates were resolved us ing a discontinuous SDS PAGE gel (4% stacking ge l, 12% resolving gel). Gel resolved proteins were transferred to nitrocellulose membranes for w estern blot analysis using a

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48 Mini Trans Blot cell (BioRad). M embranes were blocked overnight with PBS containing 0.05% (v/v) Tween 20 and 5% (w/v) non fat milk powder. Blocked membranes were washed with PBS containing 0.05% Tween 20 and incubated for 1 h with polyclonal anti 143 3 antibody anti ADH antibody and anti Histone H3 antibody (1:2000) in PBS containing 0 .05% Tween 20. Membranes were washed with PBS containing 0.05% Tween 20 and incubated for 45 min with horseradish peroxidaseconjugated donkey anti rabbit IgG (1:4000) in PBS containing 0.05% Tween 20. Protein bands were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Pierce). Bimolecular Fluorescence C omplementation (BIFC) Protoplasts isolated from Arabidopsis suspension cells were transformed with b imolecul ar fluorescence complementation ( BIFC ) vectors as described in Akhtar et al. (2008). Briefly, protoplasts were prepared as described in the subcellular fractionation method. Protoplasts were incubated in W5 solution (154 m M NaCl, 5 m M KCl, 125 m M CaCl2, 5 m M Glc, 2 m M MES KOH, pH 5.7) for 30 mi n on ice before the transformation according to the protocol described in Yoo et al., (2007). Results 143 3 Dimerization Interface S hows Sequence D iversity The 3D structure models developed for both animal and plant 143 3s (Liu et al., 1995; Xiao et al ., 1995; Wurtele et al., 2003; Ottmann et al., 2009) indicate that th e dimerizati o n of 143 3s occurs between helix H1 of one monomer and helices H3 and H4 of the other monomer (Figure 21A). Protein sequences around the dimerization interface in Helix H1, Helix H 3 and Helix H4 of Arabidopsis 143 3s were aligned with online MultAlin software ( http://multalin.toulouse.inra.fr/multalin/multalin.html ) (Corpet, 1988) The alignment showed generally high conservation around the dimerization

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49 domains but a substantial level of amino acid sequence diversity i s also present, which might be responsib le for differential affinity in heterodimer formation (Figure 2 1B ). Arabidopsis 143 3 Exist s as Dimers and Monomers in the C ytoplasm of Arabidopsis Tissue Culture C ells The dimeric status of 143 3 in protoplasts isolated from Arabidopsis tissue culture cells was investigated by cross linking with BS3. Western blot analysis with subcellular fr actionations of Arabidopsis protoplasts showed that 143 3 predominantly cytoplasmic. However, the evolutionarily distinct 14 3 3 isoform was localized in the non soluble nuclear fraction of Arabidopsis protoplasts (Figure 23A). These data suggest th at heterodmerization of some 143 3 isoform combinations may not occur in vivo due to the different subcellular compartmentalization. To study the dimer/monomer status of the 143 3 the cytoplasmic fraction was cross linked with indicated concentrations of bis(sulfosuccinimidyl) suberate ( BS3) and western blotting was performed with polyclonal isoform specific 14 3 3 antibody ( Figure 2 3B ). This experiment showed that 14 3 3 wa s present in both monomeric and dimeric conformations. Considering that previously the native forms of 143 3 proteins were reported to be homoand hetero dimers (Ferl. 1996), this result suggests that there might be a cellular mechanism that regulates th e monomer/dimer status of 143 3s in Arabidopsis. Effects of S62D P hosphor ylation mim icking M utation on Arabidopsis 143 S tructure Several groups showed that the phosphorylation of S er 58 in Helix 3 inhibits dimerization of human 143 2006). 143 3 p rote in sequence alignment of human 143 3 (zeta) with Arabidopsis 143 3s showed that this ser ine residue is conserved in eleven Arabidopsis 143 3 isoforms

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50 (Figure 2 4 A ). Here, the structural effects of phosphorylationmimic mutation S62D in Arabidopsis 143 were examined. The S62D residue corresponds to Ser 58 in human 14 3 mimic mutations, including Ser 58 to Glu 58 (S58E), on the structure of human 143 3 They showed that S58E increased the monomeric status and the susceptibility of 143 3 to proteolysis. Arabidopsis 143 63 and W 234), one of which, W 63, is in the dimerization interface. T he intrinsic tryptophan fluorescence properties of 143 3 were compared by exciting at 295 nm The florescent emission was recorded in the range of 300400 nm. Both proteins showed the same emission maxima at 341 nm, but 143 almost 2 fold the relative fluorescence intensity of wild type 143 the intrinsic tryptophan fluorescence of wild type 14 3 3 and 143 3 S62A wer e essentially the same (Figure 2 4B ). The higher intrinsic fluorescence of the S62D mutation sugges ts that tryptophans in 143 are more accessible to the solvent than those in wild type 143 If the S62D point mutation causes structural changes in 143 3 it may affect th e hydrophobic nature of the protein as well. T he effect of S62D point mutation o n the hydrophobicity of 143 was investigated by bisANS fluorescence. BisANS is a molecule that binds to hydrophobic residues on the surfaces of p roteins and becomes flu orescent (Andley et al., 2008). The mutant 143 exhibi ted higher bis ANS fluorescence compared to wildtype 143 5A and 2 5B) and a significant blue shift of the emission maximum from 520 nm to 480 nm (Figure 25B). These data suggest that the S62D phosphorylation mimic mutation results in higher

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51 exposure of hydrophobic residues to bis ANS binding (Figure 25B ). The intensity of the fluorescence and the emission maxima of the nonphosphorylationmimic control mutant 143 protein were not significantly different than wild type 14 3 3 5C) 143 3 S62D Cannot Form H omo dimer s in Y east T he impact of the S62D mutation in dimerization of 14 3 was investigated by two different approaches. First, we employed the yeast twohybrid assay by cloning 143 3 S 62D and 143 S 62A each into bait (pDEST32) and prey (pDEST22) vectors. Yeast twohybrid assay s for homodimer ization of 14 3 3 3 S 62A were performed by cotransforming S. cerevisiae AH109 yeast cells with bait and prey vectors. A single colony from each co transform ation was picked and cultured overnight in liquid SC Leu Trp me dia. H omodimerization was indicated by yeast growth on SC Leu Trp His dropout media. This assay showed that 143 and 14 3 S 62A can each fo rm a homodimer, whereas 143 can not (Figure 2 6A ). Second, the effect of the S62D mutation on the ability of 143 to homodimerize was studied by cross linking. The 14 3 3 and 143 coding sequences were cloned into p415GPD constitutive yeast expression vectors and transferred into the yeast strain INV Sc 1 a fastgrowing diploid strain used for protein expression. Transformed cells were selected o n Sc Leu plates. A single colony from each transformation was picked and cultured overnight in SC Leu liquid media. Cell lysates were cross linked with indicated concentrations of BS3 (Figure 2 6B ) This c ross linking study showed that the S62D mutation interfered with dimer for mation dramatically compared to levels exhibited by wild type 143 3 -

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52 reduced band at 29 kDa (Figure 2 6B ). These results show that 14 3 3 dimerization has the potential for regulation in vivo through a mechanism involving p hosphorylation at the conserved Ser 62 of 143 Heterodimer ization of Arabidopsis 14 3 3s Can Be Regulated by Ser 62 P hosphorylation With thirteen 14 3 3 isoforms, Arabidopsis can have potentially 91 different (13 homo and 78 hetero) 143 3 dimer combinations However, the regulation of how isoforms chose their dimer partners is still a mystery in 143 3 biology. Here we discovered the potential for phosphorylation mediated regulation of 143 3 dimer partner selection. The coding sequences of 14 3 3 3 were cloned into the DNA binding domain fusion (bait) vector, pDEST32, and all thirteen Arabidopsis 143 3 isoforms were cloned in to the activation domain fusion (prey) vector pDEST22. Y east twohybrid assays were performed to characterize the dimerization profiles of 143 3 3 Colony growth on triple dropout plates (Sc LeuTrp His ) demonstrated that the wild type 143 thirteen isoforms with various interaction affinities (Figure 2 7A ). However, phosphorylation mimic mutation S62D was not able to form dimers with 14 3 3 143 3 14 3 3 3 and 143 143 3 S62D monomer has an increased dimerization affi nity for 14 3 143 3 4 3 3 3 2 7B ). These results showed that phosphorylation of Ser 62 may influence both the selection of a dimerization partner and dimer affinity. On the other hand, the dimerization profile of 143 was not significantly different than wild type 143 2 7C )

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53 143 3 S62D Cannot Form Homodimers in Arabidopsis P rotoplasts T he interference of the S62D mutation with 143 3 homodimerization was further investigated by a biomolecular fluorescence complementation (BiFC) assay in Arabidopsis protoplasts using a transient expression system (Akhtar et al., 2008). The principle of BiFC assay is summarized in Figure 28A. 143 3 coding sequences fused to N terminal (YFPN) and C terminal (YFPC) fragments of th e yellow fluorescent protein (YFP) were co transformed into Arab idopsis protoplasts (Figure 2 8B ). YFP signal was detected when 143 YFPN and 143 YFPC were co transferred into protoplasts indicating 143 protoplast s. In contrast, protoplasts cotransferred with 14 3 YFPN and 143 YFPC did not have strong YFP fluorescence, indicating that the mutation interferes with 143 n protoplasts (Figure 28B). T he dimerization of 143 3 3 hybrid assay was also tested (Figure 27B) with the BiFC assay in protoplasts. We detected a very strong YFP fluorescence when Arabidopsis protoplasts were co transferred with 14 3 YFPN and 143 YFPC (F igure 2 8B). This result suggested that 143 with non phosphorylated 14 3 3 Phosphorylation of Ser 62 R egulates T arget I nteractions Dimerization is essential for most 143 3 interactions. There are several reports showing that 143 3/ target interactions are regulated by the dimer status of the 143 3s (Tzivion et al., 1998; Shen et al., 2003). Recently, Zhou et al. (2009) showed that phosphorylation of mammalian 14 3 58 interferes with the 143 3/ASK1 interaction, which leads to ASK1mediated oxidant stress induced cell death. In this part

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54 of the study, the effect of the S62D mut ation o n 143 3 / target interactions was investigated using two known 143 3 target s, difopein ( di meric fo urteenthree three pe ptide in hibitor) and nitrate reductase, representing the non phosphorylated and phoshorylated target s respectively. A non phosphorylated synthetic 143 3 target R18 peptide, was isolated from a phage display screen against mammalian 143 (Wang et al., 1999). Later, another high affinity 143 3 target difopein, was created by Masters and Fu (2001) by combining two R18 peptides separated by a short linker sequence. In the same study, they also developed a negative control, R18 (Lys), by changing two acidic residues (D12 and E14) to lysine, which cause d a disruption of 143 3 binding. We received EYFP fusions of difopein and R18(Lys) in the pEYFP vector as gifts from Dr. Haian Fu (Emory University) and inserted the coding sequences into the yeast two hybrid bait vector, pDEST32 and GST tagged pDEST15 u sing Gateway cloning. First, the impact of the S62D mutation o n the 143 was studied using a yeast twohybrid assay. T he yeast twohybrid assay indicated that wild type 143 and 143 S62A mutant proteins did interact with EYFP difopein in yeast. However, the S62D mutation disrupted the 14 3 3/difopein interaction (Figure 2 9A ). In contrast, 143 3 3 did not interact wi th EYFP R18 (Lys) the negative control (Figure 2 9B). Second, the effect of S62D mutation o n the kinetics of 14 3 difopein and 143 R1 8 (Lys) (the negative control) interactions were characterized Wild type 143 3 we re covalently immobilized onto Amine Reactive Biosensors and the real time interaction with EYFP difopein a nd EYFP R18 (Lys) were recorded using the Octet QK platform (ForteBio) (Figure 2 10) The kinetic

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55 values were calculated using Octet software (Table 2 1 ) Both 143 3 show ed no signif icant interaction with the negative control peptide EYFP R18 (Lys), indicating that interaction between 14 3 3 and EYFP difopein was specific (Figure 2 10C and 10D, Table 21 ). The sensogram obta ined with wild type 143 3 and EYFP difopein (0.25 M 2 M) interaction demonstrated a strong binding affinity (Figure 210A) with an association rate ( kon) of 1.07 0.01 x103 M 1 S 1 and a dissociation rate ( koff) of 5.96 0.05 x 105 S 1. The equilibrium dissociation constant ( KD = koff/ kon) for the 143 3 /EYFP difopein interaction was 55.9 nM (Table 2 1 ). On the other hand, monomeric 14 3 showed a lower affinity to EYFP difopein (Figure 210B). Although the association rate of 143 FP difopein, 1.47 0.05 x103 M 1 S 1, was very similar to wild type 143 3 from EYFP difopein 12fold faster than 143 a dissociation rate of 5.05 0.06 x 106 S 1,resulting in an equilibrium dissociation constant KD o f 345 nM (Table 2 1 ). In summary, the kinetics data showed that the monomeric 14 3 3 S62D has 6 times lower affinity than wildtype 143 3 to EYFP difopein. Finally, the effect of S62D mutation on binding of 143 3 to the N terminal half of nitrate redu ctase (NR1 562aa) was characterized. Nitrate reductase is a well studied phosphorylated 14 3 3 target (Kanamaru et al., 1999) by yeast twohybrid assays. Nitrate reductase is phosphorylated at Ser 534, and this phosphorylation is required for 143 3 interaction (Kan amaru et al., 1999). The point mutation S534L on NR1562aa was created to use as a negative control for the yeast twohybrid interaction assay. The greatly reduced growth of colonies in the y east twohybrid a ssay showed that the

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56 S62D mutation significantly decreased the affinity of 14 3 3 NR1 562aa (Figure 2 11A). As expected, the negative control, the S534L mutation of NR1562aa did not interact with 143 3 or 143 3 S62D (Figure 211B ). Discussion The 14 3 3 proteins are essential regulatory proteins that are involved in multiple essential cellular processes through interacting with over one hundred different phosphorylated target proteins both in plants and animals. Some of the important 143 3 target proteins include transcription factors, metabolic enzymes, cell cycle regulators, proteins involved in programmed cell death and signaling proteins (Mackintosh, 2004; Jin et al., 2004; Oecking and Jaspert, 2009; Paul et al., 2009; Chang et al., 2009). Regulation of 143 3/ target interaction can happen at two different levels: 1) phosphorylation at the level of the target (Figure 2 2A), and 2) phosphorylation at the level of 14 3 3 pr otein itself (Figure 22B). The regulation of 14 3 3/ target interactions by phosphorylation of 143 3s has been reported previously. Phosphorylation of human 143 3 at Ser 58 by PKA and B/Akt kinases has been shown to affect target interactions by influencing the capacity of 143 3 to dimerize (Gu et al., 2006, Powell et al., 2002). Two other residues of 14 3 3 Ser 184 and Thr232, have been shown to be phosphorylated and to affect 14 3 3/ target interactions (Aitken et al., 1995; Dubois et al., 1997). T his study demonstrated that Arabidopsis 143 3 is mainly localized in cytoplasm and is present both in dimer and monomer conformati ons (Figure 2 3B). P ossible role of Ser 62 phosphorylation in the dimerization interface of Arabidopsis 143 3 in the regul ation of dimerization and target interactions was investigated. A site directed mutagenesis approach was used to imitate the phosphorylation at Ser 62 by replacing

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57 this residue with Asp. The phospho rylation mimic mutant S62D caused significant biophysical changes to the structure of 143 3 as evidenced by the higher intrinsic tryptophan fluorescence increased hydrophobicity of 143 3 S64D might be due to the destabilization of the dimer conformati on. This hypothesis was confirmed by several different in vivo studies such as yeast twohybrid, cross linking and BiFC in Arabidopsis protoplasts. Although the S62D point mutation abolished the homodimerization ability of 143 yeast two hybrid assay showed that 143 dimer s with seven Arabidopsis 143 3 isoforms (Figure 27B). In addition, the dimerization affinity of the mutant 143 type 143 The capacity of Ser 62 phosphorylation to influence dimerization may explain how 1 4 3 3s choose their dimer partner in response to certain signals and in different cellular locations. Finally, inhibition of dimerization of 14 3 3s by phosphorylation significantly reduced the interaction of 143 3 with phosphorylated and non phosphoryla ted target s such as nitrate reductase and EYFP difopein respectively. In summary, phosphorylation of Ser 62 has a potential regulatory role in both dimerization and target interactions.

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58 A B Figure 21. 143 3 proteins consist of 9 helices. A) Top view of 143 3 dimer model derived from Nicotiana tabacum 143 3 in complex with the differentiation inducing fungal a gent Cotylenin A (PDB3E6Y) ( Figure adapted from Ottmann et al., 2009) using PDB SimpleViewer 3.8 software. B) Whole protein sequence alignment of Arabidopsis 143 3 protein family. Protein sequences were aligned using online MultAlin software ( http://multalin.toulouse.inra.fr/multalin/multalin.html ) (Corpet, 1988). The helices in dimerization interface were indicated with boxes. High consensus (= 90 %) residues are in red, low consensus (= 50 %) residues are in blue and non conser ved residues are in black. Consensus symbols: is anyone of I and V; $ is anyone of L and M; % is anyone of F and Y; # is anyone of N, D, Q, E, B and Z.

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59 A B Figure 22. Regulation of 143 3/ target inter actio ns by phosphorylation. A) The phosphorylation state of the target protein carrying the 143 3 binding motif re gulates the 143 3 interactions. B) Phosphorylation of 143 3 at the dimerization interface disrupts dimer structure, which also inhibits target interaction.

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60 A B Figure 23. Dimer/monomer status of 14 3 3 protoplasts. A) Subc ellular fractionations of Arabidopsis protoplasts and loca li zation of 14 3 3 and 143 3 1. Total protoplast, 2. Cytopl asmic fraction, 3. Total nuclear fraction, 4. Soluble nuclear fraction, 5. Insoluble nuclear fraction. Arabidopsis alcohol dehydrogenase (AtADH) and histone H3 antibodies are used as cytoplasmic and nuclear controls, respectively B) The cytoplasmic fracti on of Arabidopsis protoplasts was crosslinked with indicated concentrations of BS3. 14 3 3 was detected with western blotting using 14 3 3 specific antibody.

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61 A B Figure 24. The effects of S62D mutation on the intrinsic fluorescence of 143 3 A) Protein sequence alignment of Helix H3 of human 143 3 and Arabidopsis 143 3 protein family (At143 3). The phosphorylatable Ser 58 that is conserved in human 143 3 11 Arabidopsis 14 3 3 isoforms is indicated. B) Intrinsic tryptophan fluor escence o f At14 3 3 ) and 143 (green) R ecombinant protein 295 nm and recorded from 300 nm to 400 nm.

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62 A B C Figure 25. Phospho rylation mimic mutation S62D increases the hydrophobicity of 143 3 Interaction of 143 3 and its mutations with the hydrophobic probe bis ANS. The Y axis represents f luorescence resonance energy transfer (FRET) from excited Trp residues at 295 nm to bis ANS interacting with the hydrophobic surfaces of A ) 143 143 and C) 14 3 All recombinant proteins were at 25 and bis ANS concentrations were indicated.

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63 A B Figure 2 6. Dimer status of 14 3 143 14 3 yeast cells. A) Yeas t two hybrid assays show ed that 14 3 and 143 S62A monomers interact in yeast (top and bottom panel s respectively ). H owever, 143 not interact (middle panel). B) 143 143 and 143 over expressed in INV Sc1 yeast strain d were cross linked with indicated concentrations of BS3. The Dim er status of the proteins was detected by western blot using 143

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64 A B C Figure 27. The effects of dimerization interface mutations of Ser 62 to Asp62 ( S62D) and to Ala 62 ( S62A) of Arabidopsis 143 with thirteen Arabidopsis 143 3 isoforms in yeast two hybrid assay A) Dim erization profile of wild type 14 3 D imerization profile of 143 Dimerization profile of 143 S62A. pDEST22 pDEST22 pDEST22 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 14 3 3 SC Leu Trp 36h SC Leu Trp His+0.5mM 3AT 48h SC Leu Trp 36h SC Leu Trp His 48h SC Leu Trp 36h SC Leu Trp His+0.5mM 3AT 48h

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65 A B Figure 28. S62D phosphomimic mutation interferes with 143 3 homodimerization in Arabidopsis protoplasts. A) Schematic representation of the of BIFC assay for 143 3 dimerization. N terminal YFP fragment (YFPN) and C terminal YFP fragment (YFPC) in blue and orange, respectively, were fused to the C terminal ends of 14 3 3 143 3 proteins. B) BIFC fluorescence and bright field photos of Arabidopsis protoplast transiently expressing 143 3 YFPN/14 3 3 YFPC, 143 3 YFPN/14 3 3 YFPC and 143 3 YFPN/14 3 3 YFPC fusion proteins.

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66 A B Figure 29. Phosphorylation m imic mutation of Ser 62 to Asp 62 of 143 3 3/ target interaction. A) Protein protein interaction using yeast tw o hybrid assay with 143 143 and 143 in activation domain fusion vector and EYFP difopein in DNA binding domai n fusion vector interaction and B) EYFP R18(Lys). pDEST22 represents the empty control vector.

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67 A B C D Figure 210. Kinetic analyses of effect of Ser 62 to Asp ( S62D) phosphorylation mimic mutation in 143 to EYFP Difopein (2000250 nM) and EYFP R18 (Lys) (2000250 nM) A) 14 3 /EYFP Difopein binding kinetics, B) 143 S62D/EYFP Difopein binding kinetics, D ) 143 /EYFP R18(Lys) binding kinetics C) 143 S62D/EYFP R18(Lys) binding kinetics. 14 3 3 Difopein 14 3 3 S62D Difopein 14 3 3 R18 (Lys) 14 3 3 S62D R18 (Lys)

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68 A B Figure 211. Phosphorylation mimic mutation S62D in the dimerization i nterface of 14 3 3 3 /phosphorylated target int eraction. A) Yeast two hybrid assay to show p rotein protein interaction s between 143 3 ( in the activation domain fusion vector ) and A) NR1 562aa ( in the DNA binding domain fusion vector) or B) NR1 562aaS534L ( in the DNA binding domain fusion vector) pDEST22 represents empty control vector.

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69 Table 2 1. Kinetic analysis of 143 3 and 14 3 3 S62D against non phosphorylated target s Ligand Analyte K on (M1s1) K off (s1) K D a (nM) 14 3 3 EYFP Difopein (1.07 0.01) x 10 3 (5.96 0.05) x 10 5 55.9 14 3 3 S62D b EYFP Difopein (1.47 0.05) x 10 3 (5.05 0.06) x 10 6 345 14 3 3 c EYFP R18(Lys) 14 3 3 S62D d EYFP R18(Lys) aKD values of each interaction are calculated as koff/ kon. bKinetic parameters of this interactions are calculated with 2000nm and 1000nm concentration of analytes. Lower concentrations (500nm and 250nm) of anaylte did not give any significant kinetics parameters.c andd no significant interaction determined.

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70 CHAPTER 3 DIRECT INTERACTION B ETWEEN ARABIDOPSIS 14 3 BOX BINDING FACTOR 3 (GB F3) Introduction Gene regulation is a complex multistep cellular task that involves integration of several cellular processes such as signal transduction pathways, pr oteinprotein interactions, subcellular compartmentalization of proteins, chromatin structure remodeling, RNA synthesis and RNA processing. Transcription factors have funda mental roles in gene regulation, which controls all major events of the organisms su ch as cell cycle, development, metabolism and environmental responses. The availability of the full sequence for the Arabidopsis genome has led to the realization that there are more than 1500 transcription factors which represents approximately 5% of the Arabidopsis genome. These have been classified into several different families one of which is the basic leucine zipper (bZIP) family which is the eighth largest in Arabidopsis (Riechmann et al., 2000). With more than eighty members, bZIPs participate i n the regulation of diverse biological processes such as light and stress signalling, pathogen response, hormone response, seed maturation and flower development (Marc Jakoby et al. 2002). G box binding factor 3 (GBF3) is a member of the bZIP transcr iption factor family that binds to a cis acting element called G box (5' CCACGTGG 3'). The G box is present in many plant gene promoters (Yamaguchi Shinozaki and Shinozaki, 2006; Lu et al., 1996) and is involved in the regulation of the response to diverse environmental stimuli, such as cold, salt, light, dehydration, hypoxia and abscisic acid (ABA) ( Mallappa et al., 2008; Menkens and Cashmore, 1999,

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71 Shinozaki et al., 1997). In addition to GBF3, the G box sequence is recognized by variety of other bZIP and also basic helix loophelix ( bHLH ) transcription factors (de Pater et al., 1997; Martinez Garcia et al., 2000; Malaba et al., 2006; Kim et al., 2007, Alanso et al., 2009). The binding affinity of bHLH transcription factors is not exclusive to the whole G b ox, but rather to the central CANNTG sequence ( Siberil et al., 2001). T he flanking sequences of the G box have a significant role in determining the specificity and affinity for the range of the GBF interactions with diverse to the entire Gbox sequence s (Williams, et al., 1992; Schindler et al., 1992a; Izawa et al., 1993). The term GBF is therefore closely linked to these bZIP factors that bind to the G box. GBF proteins have three major domains: The N terminal proline rich domain, the central bZIP domain and an undefined C terminal domain (Figure 3 1). The N terminal proline rich domain is involved in either in transactivation, as in GBF1 (Sc hindler et al., 1992b), or repression as with soybean SGBF 2 (Liu et al., 1997). The central bZIP domain consis ts of a basic region for DNA binding and a nuclear localization signal (Hurst, 1996; Terzaghi et al., 1997) together with a leucine zipper domain for dimerization (Figure 3 1 ). The C terminal region does not as yet have an assigned function. Transcripts encoding Arabidopsis GBF3 and several G box binding bZIP s, from other plant species such as bean ROM1 and ROM2, rice OsBZ8, and maize ZmBZ 1 and EmBP 2 have been shown to be up regulated in response to ABA treatment (Lu et al., 1996; Chern et al., 1996a; Chern et al., 1996b; Nakagawa et al., 1996; Nieva et al., 2005; Suzuki et al., 2003). Ano ther study also showed that

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72 GBF3 transcript is up regulated in the dark (Schindler et al., 1992c) Arabidopsis GBF3 transcript profile using the Genevestigator Respons e Viewer ( http://www.genevestigator.com ) Arabidopsis microarray database showed that GBF3 transcription is highly induced by osmo tic stress, drought, ABA, salt stress, cold and light (Figure 32) These data suggest that as a transcription factor GBF3 may play a very important role in regulation of expression of genes involved in environmental response The G box of alcohol dehydrogenase ( Adh) is among the well studied cis elements in plants and is known to be occupied by a multiprotein complex that includes 143 3 proteins (Ferl and Laughner, 1989; DeLisle and Ferl, 1990; McKendree et al., 1990; Lu et al., 1992; Lu et al., 1994). In addition, GBF3 can interact in vitro with the G b ox in the Adh promoter specifically. The in vivo footprint profile of the Adh G box and in vitro footprint profile of the GBF3/Gbox were essentially the same (Lu et al., 1996). However, t he 14 3 3 proteins do not directly interact with the G box element ( Lu et al., 1992). I t was proposed that 143 3s can be part of the G box protein complex through interaction with GBF3 or another protein (Lu et al., 1996; Paul and Ferl, 1997). The abscisic acid VIVIPAROUS1 (VP1) response complex that interacts with a Gbo x in the rice Em promoter is another example where 143 3 proteins are part of the G box binding protein complex ( Schultz et al., 1998). In addition, an i n vitro crosslinking study showed that 14 3 3 proteins directly interact with EmBP1, a GBF like bZIP transcription factor, and VP1, a tissue specific regulatory factor. It has been

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73 proposed that 143 3 protein has a scaffolding type of function in the assembly of transcription complexes involvi ng VP1 and EmBP1 (Schultz et al., 1998). The 143 3 proteins are phosphopeptidebinding proteins that have been associated with many important biological processes in eukaryotes such as signal transduction, cell cycle regulation, apoptosis, metabolic pathways and cell differentiation (Fu et al., 2000; Ferl et al., 2002). 1 4 3 3 s exi st in multiple isoforms and can form homo and hetero dime rs 14 3 3s were first discovered in plant s to be part of G box binding complexes and thought to regulate the Adh promoter (Lu et al., 1992) L ater they were sho wn to be involved in regulation of important plant metabolic enzymes such as nitrate reductase, ATP synthase, sucrose phosphate synthase, glutam ate synthase starch synthase and ascorbate peroxides (Ferl, 1996). Although much of the 14 3 3 related plant res earch is focused on the role of 14 3 3s in the regulation of metabolic enzymes, s everal studies have revealed a role of 14 3 3s in plant signaling and gene regulation (reviewed in Oecking and Jaspert, 2009; Gokirmak et al., 2010). Several studies showed that 143 3 proteins may be actively involved in stress management in plants. Four out of eight rice 14 3 3 genes, GF14b, GF14c, GF14e and Gf14f, are differentially regulated by abiotic stresses such as salinity, drought, wounding and abscisic acid (Chen e t al., 2006). In tobacco, T14 3 3 is induced by salt stress (Chen et al., 1994). T he 14 3 3 proteins function as regulators of target proteins that are involved in several abiotic stress responses In Arabidopsis the activity of mitochondrial and chloroplast ATP synthases are regulated by 143 3 proteins during the light/dark transition

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74 (Bunney et al., 2001). A transgenic study with cotton showed that constitutive expression of a cotton 143 erred drought tolerance, probably through controlling the stomatal opening by H+ ATPase regulation (Yan et al., 2004). The 14 3 3 proteins also play an essential role in ABA signaling pathway. Schoonheim et al. (2007) showed that 143 3s are intermediate p layers in ABA signaling during barley seed germination. The interaction between 143 3 proteins and ABI5 family members increases the trans activation capacity of ABI5. Considering the well established role of both GBF3 and 143 3s in stress regulation in plants the present study was designed to demonstrate a genetic and di rect interaction between 143 and GBF3 using a heterologous model system S cerevisiae Expression of GBF3 in Saccharomyces cerevisiae causes cellular toxicity. Serial N terminal and C terminal deletions showed that t his toxicity can be released by deletion of an N terminal proline rich domain or the C terminal undescribed domain. This suggests these two domains are essential for GBF3 function. Coexpres sion of 143 3s in yeast rescue GBF3 mediated cellular toxicity through direct 143 3/GBF3 interaction. This suggests that 143 3 proteins have an antagonistic role in GBF3 mediated regulation of transcription in plants Material s and M ethods Yeast Strains and M edia. S. cerevisiae str ain INVSc1 ( MATa his3D1 leu2 trp1289 ura352 MATAlpha his3D1 leu2 trp1289 ura352) was used for galactose inducible protein expression. S. cerevisiae strain AH109 ( MATa, trp1 901, leu23, 112,

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75 ura352, his3200, gal4gal80LYS2::GAL1UASGAL1TATAHIS3,GAL2UASGAL2TATA ADE2,URA3::MEL1UASMEL1TATAlacZ, MEL1) was used for yeast twohybrid assay s. Yeast cells cultured in rich medium (YPD: 1% yeast extract, 2% bactopeptone, and 2% glucose) were transformed by the standard lithium acetate/polyethylene glycol method (Gietz and Woods, 2006). Transformed cells were selected on synthetic dropout media (SD) (Clontec h, CA) plates deficient in specific amino acids for plasmid selection. Spot assays as a measure of growth were performed with yeast cells cultured overnight in appropriate SD glucose medium. Cells were pelleted and washed in sterile water. Concentrations of overnight cultures were adjusted to OD600 of 0.2. Ten fold serial dilutions of each normalized culture were prepared. Aliquots of 10 l from e ach culture and dilutions were spotted on SD glucose or SD galactose plates and incubated at 30 C for the indicated times. Plasmids The full length ORF of Arabidopsis GBF3 (At2g46270) and 143 ( At1g78300) ORFs were PCR amplified with Gateway tagged primers (supplemented table 1) from a flower and leaf cDNA library (a gift fr om Dr. Kevin Folta). The point mutation 14 3 3 3 was generated by sitedirected mutagenesis and overlapping PCR (Ho et al., 1989). PCR fragments were first cloned into the pDONR221 vector to create entry vectors using Gateway BP Clonase II (Invitrogen, CA ). N terminal and C terminal deletions of GBF3 were PCR amplified with Gatewa y tagged primers using GBF3 in pDONR221 as template and subcloned into pDONR221 as

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76 described earlier. The 143 3s, full length GBF3 cDNA and its N terminal deletions were cloned i nto pDEST22 ( GAL4 AD), pDEST32 ( GAL4 DBD) and Gal inducible pYES DEST52 vectors from pDONR221 clones via Gateway LR Clonase II (Invitrogen, CA) reactions. The GBF3 GFP C terminal fusion construct was created by sequential cloning. GFP was PCR amplified wi th a forward primer containing a multi cloning tag and GFP attB2 primer. The multi cloning tagged primer had the following sequence and the restriction sites: 5 AAGCTT GAGCTC GAATTC ATGGTGAGCAAGGGCGAG 3 (HindIII site is underlined, SacI site is bold and E coRI site is italic). The second round of PCR was performed with a forward primer ( mcsGFP attB1 a primer specific to the multi cloning site and the first codon of GFP ) and the GFP attB2 primer The PCR product was gel purified and cloned into the pDONR221 vector using Gateway BP Clonase II. GFP with the mu lti cloning site was cloned into the pYES DEST52 vector via the Gateway LR Clonase Reaction II. GBF3 was cloned into HindIII and EcoRI sites upstream of the GFP in pYES DEST52. Monitoring Growth Curves and Cell Viability Assay Three colonies from yeast cells transformed with pYES DEST52 empty vector and GBF3 in pYES DEST52 (pYES GBF3) were picked and cultured overnight in SD Ura liquid medium containing 2% glucose at 30 C. Concentrations were normalized f or OD600 = 0.2 in SD Ura containing 2% galactose and growth at 30 C was monitored at every 12 hr Cell viabi lity was measured every 24 hr after induction of GBF3 by galactose The s ame numbers of cells transformed with pYES GBF3 and pYES DEST52 (vector control) were

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77 plated o n three SD Ur a containing 2% glucose plates to repress GBF3 expression. After three days of inc ubation at 30C, the colonies were c ounted and the percent ratios determined. Yeast Histag P ull down The his tag pull down protocol for GBF3 and 14 3 3 interaction was modified from Gong et al., 2006. Briefly, INVSc1 cells cotransferred with pYES GBF3/p415GPD 143 GBF3/p415GPD 143 in SC LeuUra with 2% gluco se overnight at 30C. Cells were washed in sterile water and resuspended in 30 ml of SC LeuUra with 2% galactose to induce protein expression from th e GAL1 promoters. After 48 hr of culturing at 30C with shaking, cells were crosslinked with 1% formaldeh yde (final concentration) at room temperature for 20 mins. Cross linking was quenched with 0.5 M glycine (final concentration) at room temperature for 10 mins. After cross linking, the cells were washed first in cold Tris buffered saline (TBS) and then in cold lysis buffer (50 mM HEPES KOH (pH 7.5), 200 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 10% (v/v) glycerol and 1% (v/v) Triton X 100). Finally, cells were resuspended in 1 ml of cold lysis buffer containin g 1mM PMSF, 1mM DTT, phosphataseinhibitor mixture and proteaseinhibitor mixture (Calbi ochem). The cells were broken open by vortexing with an equal volume of acidwashed glass free extract was mixed with 0.1 ml Ni NTA agarose beads (Sigma) and incubated overnight at 4C with agitation The Ni NTA agarose beads were gently washed three times with lysis buffer and three times with lysis buffer with 20 mM imidazol and twice with 1 X PBS buffer. Beads were resuspened in 1X SDS PAGE buffer and the samples were boiled for 10 min.

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78 Western blots were performed using a 143 orm specific polyclonal antibodies and the protein bands visualized by ECL chemifluorescence substrate (Thermo Scientific). Fluorescence M icroscopy Yeast cells co transferred with pYES GFP and pYES GBF3 GFP were cultured overnight in SC Leu Ura with 2% glucose at 30C. Cells were washed in sterile water and protein expression was induced in equal volume of SC LeuUra with 2% galactose for 24 hours. For fluorescence microscopy, cells were fixed in 70% ethanol for 1 hour at room temperature. Nuclei w ml1 DAPI (Sigma). GFP and DAPI localizations were examined with Olympus BX51 fluorescent microscope coupled to an Evolution MP cooled chargecoupled device camera with Q capture 2.60 s oftware (Quantitative Imaging, Burnaby, British Columbia, Canada). R esults GBF3 H a s A T oxic P henotype in Y east That Can Be S uppressed by 143 3s Expression of GBF3 severely inhibits yeast growth. Colonies of yeast cells transformed with GAL4 AD GBF3 were significantly smaller than colonies of cells transformed with vector control s (Fig ure 3 3 A ). However, co expression of certain Arabidopsis 143 3s suppressed GBF3 toxicity. Y east cells co transformed with AD GBF3 and Arabidopsis 143 3 14 3 3 or 143 to the GAL4 DNA binding domain in the pDEST32 vector (DBD Omega, DBD Mu and DBD Upsilon) had normal size d colonies compared to cell s co transformed with empty pDEST22 and pDEST32 vectors (Figure 3 3 A ). On t he other hand, yeast cells co trans formed with AD GBF3 and DBD 143 had intermediate

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79 size d colonies (Figure 33A). These result s suggested that suppression of GBF3 mediated toxicity in yeast by 143 3 s is isoform specific. When the cells were cotransformed with AD GBF3 and DBD 143 3 S6 2D (DBDS62D), the colonies were only slightly larger than the cells expressing only AD GBF3 (Figure 33B) sug g esting that the dimeric status of the 143 3 is essential for supression of the GBF3 toxicity phenotype GBF3 Directly I nteracts with 14 3 east The direct interaction of 143 tag pull down assay in yeast. Yeast cells co expressing GBF3 or GBF3 C terminal his tag (GBF3 his tag) and 143 3 linked with formaldehyde and lys ed. The cell lysates were incubated with nickel his re sin overnight at 4C. T he nickel his resin was then excessively washed and the proteins were eluted C rosslinks were reversed with boiling in SDS sample buffer. 143 3 bound to GBF3 were analyzed with w estern blotting (Figure 34). As a negative control, GBF3 with no his tag was used to show that the 143 3 pulled from the resin was attached to GBF3 and not due to nonspecific binding to the nickel his resin (Figure 34 ). These data showed that 143 3 GBF3 Toxicity Is Not Related to Yeast Two hybrid Constructs and Is Likely Due to The Nuclear F unc tion To confirm that GBF3 is toxic in yeast cells without the GAL4 AD, full len gth GBF3 and GBF3GFP were cloned into pYES DEST52, a galactose inducible high copy yeast expression vector. INV Sc1 cells were transformed with pYES DEST52, pYES GBF3 and pYES GBF3 GFP vectors and plated on Sc Ura

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80 plates containin g 2% glucose to select the transformed cells. A single colony for each transformation was picked and cultured overnight in glucose containing SD Ura medium. Cells were spotted on SD Ura containing glucose and galactose plates. Spot assays on galactosecontaining agar plates showed that cell growth was severely inhibited upon galactose induction compared with cells transf ormed with an empty plasmid and a plasmid expressing GFP alone (Figure 3 5A ). Fluorescence microscopy showed that GBF3 was localized into nucleus (Figure 3 5B) suggesting that the toxic phenotype of GBF3 may be linked to its nuclear functions. We further analyzed GBF3 med iated toxicity by comparing cell growth and cell viability of c ells expressing GBF3 versus cells transformed with the empty pYES DEST52 vector in gal actose containing liquid media culture. The presence of GBF3 not only severely inhibited cell growth but affected cell survival as well (Fig ure 3 6A ). After 48 hr, 56 % and after 72 hr only 15 % of the cells expressing GBF3 could form colonies on glucosecontaining agar plates (Figure 3 6B ). N terminal Proline rich D omain and C terminal Domain Are Required for GBF3 T oxicity GBF3 consists of several domains: 1) an N terminal proline rich domain, which is thought to be involved ei ther in transactivation or repression; 2) a central bZIP domain consisting of a basic region for DNA binding, a nuclear localization signal (Hurst, 1 996; Terzaghi et al., 1997), and a leucine zipper domain for dimerization; and 3) a C terminal region that does not have an assigned f unction (Figure 3 7A ). To determine which regions are necessary for GBF3 toxicity a

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81 panel of N terminal and C terminal GBF3 deletions were generated and these deletion series were expressed in yeast cells using the galactose in ducible pYES DEST52 vector (F igure 3 7A ). S pot assay were performed to determine which domains are required for the GBF3 mediated toxicity (Fig ure 3 7B ). Dele tion of the first 70 aa from the N terminal end, which partially removes the proline rich domain, caused a dra matic drop in ce llular toxicity compared to th e fulllength GBF3 (Figure 37B, construct II). However, deletion of the whole prolinerich domain completely ( 1 108 aa) completely remove d the GBF3 toxicity in yeast cells (Figure 3 7B, construct III). This result suggest s t hat the N terminal proline rich domain is essential for GBF3 mediated cellular toxicity. This observation was f urther confirmed with several other N terminal deletions which removed additional residues up to th e bZIP domain. These deletions also showed no toxic ity (Figure 3 7B, constructs IV and V). To identify other domains that may be c ritical for GBF3 toxicity, several C terminal deletions were performed. Removal of last 27 residues ( 355382 a a) had no effect on GBF3 toxicity However furthe r removal of the C terminal region up to the bZIP domain ( 329382 aa) completely abolished (Figure 37B, constructs VII) demonstrading that this region (aa 329355) is important for GBF3 toxicity. Finally, the role of the basic region localized in the bZI P domain of GBF3 in cellular toxicity was studied with an internal deletion construct (Figure 3 7B, construct IX) Removal of the DNA binding region ( basic region, 261280 aa) only slight ly repressed GBF3 toxicity (Figure 3 7B, constructs IX ) This result

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82 suggest s that GBF3 toxicty is not mediated by specific interactions w ith yeast DNA, but through an unknown mechanism. Discussion The 143 3 proteins have been shown to be involved in cell cycle progression, cell proliferation and differentiation by directly interacting with histones, chromatin modifying enzymes, kinases and cruciform DNA (Chen and Wagner, 1994; Grozinger and Schreiber, 2000) In addition, they are also found to be part of transcriptional G box DNA binding complexes in several plant species, including Arabidopsis rice and maize (Lu et al., 1992) Our current knowledge of 143 3 proteins suggests that they play a central role in eukaryotic gene regulation. The presence of 14 3 3s in transcription protein comp lexes ha s been reported previously. 143 3 proteins interact with several group s of plant transcription factors such as the PHDf HD family in Arabidopsis maize, and parsley (Halbach et al., 2000), and bZIP transcription factors such as RSG in tobacco, which cont rols shoot development by regulating the genes involved in gibberellin biosynthesis (Igarashi et al., 2001). In addition, 143 3s bind to general transcription factors including human and Arabidopsis TATA box binding protein (TBP), transcription factor IIB (TFIIB) and human TBP associated factor hTAFII32 (Pan et al., 1999). There is also evidence indicated that 143 3 proteins are involved in chromatin mediated gene regulation through interaction with histones (Chen and Wagner, 1994) and histone deacetylases (HDACs) (Grozinger and Schreiber, 2000; Chang et al., 2005) GBF3 transcription has been shown to be upregulated by several environmental stimu li and stress responses such as osmo tic stress, drought,

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83 ABA, salt stress, cold and light (Figure 3 2) In agreement with the previous literature discussing the role of 143 3 proteins and GBF3 in gene regulation and in stress management, the present study showed that there is a direct interaction between 143 3 proteins and GBF3. Heterologous ex pressi on of GBF3 in yeast caused severe cellular toxicity that was partially suppressed by co expression of 143 3s (Figure 32). In addition, the his tag pull down assay showed that 143 3 and 143 3 interact directly with GBF3 (Figure 34 ). Although 143 3 S62D was pulled down with GBF3, the level of repression by 143 3 S62D was not significant (Figure 3 3B). These data suggest that the dimeric status of 143 3s is required for the antagonistic role of 143 3s in GBF3 toxicity. These findings confirm the previous model that 14 3 3s may indirectly interact with the G box through binding to GBF3. GBF3 has a poteintial 143 3 binding motif (265RKQS 268) localized in the DNA binding domai n (Aitken, 1996). Although not directly confirmed, the binding of 143 3 proteins to this site has the potential to alter GBF3 function by keeping GBF3 out of the nucleus, decreasing the half life, or changing the activity of GBF3. F uture work needs to focus on the mechanism o f the rescue of GBF3 toxicty by 14 3 3s A model describing how direct interaction between 143 3s and GBF3 may antagonistically affect function is given in Figure 3 8. We also utilized this toxic phenotype in yeast to characterize the domains of GBF3 protein. Deletions of N terminal prolinerich and C terminal uncharacterized domains release the toxic phenotype, suggesting that these domains are essential for GBF3 function.

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84 Figure 31 Three dimensional structure model of the GBF3 bZIP transcription factor The model is derived from Schizosaccharomyces pombe bZIP transcription factor PAP1. PAP1 is in complex with the DNA oligomer 5 TTACGTAA 3 (PDB 1GD2) (Figure adapted from Fujii et al., 2000) using RCSB Protein Workshop Viewer for PDB software. Figure was captured by screen shot Pro rich domain is located in the aminoterminus. Leucine zipper is involved in dimerization and basic region binds to the target DNA sequence.

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85 Figure 32 Arabidopsis GBF3 ( At2g46270) transcript profile in response to different treatments. G enevestigator Response Viewer ( http://www.genevestigator.com ) Arabidopsis microarray database showed that GBF3 transcript ion is significantly up regulated by environmental stimuli such as osmatic stress, drought, ABA, salt stress, cold and light. Screen shot shows only the treatments that show two fold changes. Fold changes on the figure is in log scale.

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86 A B Figure 33. Small colony formation in yeast transformed with GBF3 fused to GAL4 transcriptional activation domain (AD GBF3). A) AD GBF3 mediated toxicity is suppressed by cotransformation with Arabidopsis 143 3s fused to GAL4 DNA binding domain (DBD) in pDEST32 vector. B) Monomeric 14 3 3 does not rescue AD GBF3 mediated toxicity as effective as 14 3 3 AD/DBD: empty pDEST22 and pDEST32 vectors respectively DBD Lambda: 143 Mu: 143 3, DBD Pi: 14 3 Upsilon: 143 SC Leu Trp SC Leu Trp

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87 Figure 34. 143 3 tag pull down assay was performed in yeast cells co transformed with pYES GBF3 his tag /empty p415GPD vector, pYES GBF3 his tag /14 3 and pYES GBF3 his tag /14 3 GBF3 had a stop codo n, which prevents the fusion the C terminal 6x His tag fusion. Pull down experiment was analyzed with W estern blottin g using 14 3 y ( 143 3 )

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88 A B Figure 35. Arabidopsis GBF3 and GBF3GFP fusion proteins cause growth inhibition in yeast like ly through nuclear function. A) Spot assays of yeast expressing GBF3. Yeast cells were transferred with galactoseinducible empty pYES DEST52, pYES DEST52/GBF3 (pYES GBF3), pYES DEST52/GFP (pYESGFP) or pYES DEST52/GBF3 GFP (pYES GBF3 GFP) vectors. Equal volume of tenfold serial dilutions of transformed cells were spotted on glucose or gal actose containing SD Ura plates and were incubated at 30 C for 2 days or 3 days respectively. B) GBF3 is localized to the nucleus. Yeast strain INV Sc1 expressing GFP or GBF3GFP from galactose inducible GAL1 promoter was grown in synthetic complete media w ithout uracil, stained with DAPI and exami ned by a florescent microcopy. BF, bright field.

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89 A B Figure 36. Over expressing GBF3 is toxic in yeast cells. A) Yeast growth curves in SD Ura liquid media containing 2% galactose. Cells were cultured overnight in SD Ura containing 2% glucose, washed and resuspended in SC Ura containing 2% galactose to an initial OD600 of 0.1. OD600 was monitored every 12 hours. Eac h point represents the mean OD600 of three different cultures SE. B) Cell viability assay. After galactose induction, every 24 hours OD600 of 1/30.000 cells per ml were plated on SC Ura containing 2% glucose to determine w he ther GBF3 slows down the cell growth or it affects the cell viability. The percent survival refers the ratio of number of colonies harboring pYES GBF3 relative to those harboring empty pYES DEST52.

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90 A B Figure 37. Dissection of the GBF3 domains causi ng growth inhibition in yeast. A) Schematic representation of the domain structures in full length GBF3 and various deletion constructs expressed. B) Spot assays of yeast expressing GBF3. GBF3 deletion constructs showed that N terminal prolinerich domain is required for growth inhibition in yeast. Partial deletion of the proline rich domain (construct II) yields reduced growth inhibition. A

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91 A B Figure 38. The model of GBF3 mediated toxicity in yeast. A) Over expression of GBF3 in yeast causes severe cellular toxicity. The potential 143 3 binding site is located in DNA binding domain (265RKQS 268). B) Co expression of 143 3s with GBF3 in yeast partially rescues the toxic phenotype through direct interaction. Pro rich dom ain (in orange) and an uncharacterized C terminal domain (in red) are essential for GBF3mediated toxicity in yeast

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92 CHAPTER 4 ARABIDOPSIS 14 3 3 EPSILON IS LOCALIZED IN THE PLASMA MEMBRANE AND SECRETED IN RESP ONSE TO FUNGAL ELICI TOR Introduction T he 143 3 proteins are highly conserved, ubiquitous eukaryotic proteins 143 3s are characterized as acidic, dimeric proteins. They form homo and heterodimers with a monomeric mass of 2532 kDa. The 143 3 proteins usually bind to target proteins containi ng well defined phosphothreonine or phosphoserine motifs (Muslin et al., 1996; Rittinger et al., 1999). 143 3s are involved in a diversity of proteinprotein interactions, which allows them to mediate a range of biological functions. In plants, 14 3 3 pro teins have been shown to play important biological roles through regulation of certain key enzymes such as nitrate reductase, sucrose phosphate synthase, starch synthase, glutamate synthase, ATP synthase and ascorbate peroxides. In mammals, 14 3 3s have been shown to be involved in cell cycle progression, cell proliferation and differentiat ion by directly interacting with histones, chromatin modifying enzymes, kinases and naked DNA (reviewed in Ferl, 1996; Huber et al., 2002, Oecking and Jaspert, 2009; Gokirmak et al., 2010) In addition they are also part of transcriptional G box DNA bindi ng complexes in several plant species, including Arabidopsis, rice and maize (Lu et al., 1992) The plant 143 3 proteins cluster into two groups when analyzed epsilon group. The isoforms in the plant non epsilon group appear to be significantly different from the plant and animal epsilon groups (Ferl et al., 2002). In Arabidopsis, the epsilon epsilon group has eight

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93 3 3s can provide important clues to possible roles of 143 3 family members. The first reported mammalian 143 3s isolated from brain tissue were cytosolic (Moore and Perez, 1967). Later, 143 3s were foun d in the nucleus, mitochondria, plasma membrane and other organelles. Although 143 3s do not have nuclear targeting sequences, Bihn et al. (1997) showed that 143 3s are present in both Arabidopsis and maize nuclei. A localization study using four evoluti onarily diverse Arabidopsis 143 3 isoforms; fused to green florescent protein (GFP) revealed that 14 3 3s have distinct and differential subcelullar localization. Considering that 14 3 3 proteins lack subcellular localization signals differential sublocalization of the individual 14 3 3 isoforms may be driven by target interactions rather than the intrinsic properties of 14 3 3s. Use of AICAR, a 5AMP analog, and R18 peptide, a high affinity 143 3 target, showed that in the absence of the 14 3 3/ target protein interactions, 143 3s were found to localize throughout the cell without any clear subcellularization (Paul et al, 2005). These results suggest that many 143 3 isoforms may have a defined subset of target s and distinct functions. In Arabidopsis, a study with isoform specific antibodies showed that two 143 3 3 isoforms from the non 3 3s prominently located in the chloroplast (Sehnke et al., 2000). This finding suggests that phylogenetically different isoforms can share similar subcellular locations and functions. The expression profiles of 143 3 isoforms vary from tissue to tissue and organ to

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94 organ (Chung et al., 1999 and Daugherty et al., 1996). Therefore, the tissue specific expression of 14 3 3 isoforms increases the complexity of the 143 3 mediated regulation of the target proteins. Several studies reported the presence of 143 3s in the extracellular space. 143 3s are shown to be secreted into the extracellular space of Chlamydomonas and involved in cross linking of hydroxyproline ( Hyp ) rich glycoproteins in the Chlamydomonas cell wall ( Voigt and Frank, 2003). Another study showed that 1 4 3 3s are secreted from human fibroplasts (Ghahary et al., 2005). 143 3s were also identified in a pea root cap secretome analysis in response to inoculation with a pea pathogen, Nectria haematococca (Wen et al., 2007). This study suggested that extracel lular 143 3s may be involved in plant pathogen response. The present stu dy also showed that a 143 3 ( epsilon ) is secreted from Arabidopsis suspension cells by fungal elicitation probably from the plasma membrane. Material s and Methods Plant Material a nd Elicitors Arabidopsis Col 0 hypocotyl suspension cell cultures were generated and maintained according to Ferl and Laughner, 1989. Cells were transferred to fresh media every three days to ensure that they were in log growth phase. Arabidopsis Col 0 ecotype seeds were surface sterilized with 40% house hold bleach with 2% Tween 20 and washed five times with sterile water. Sterile seeds were cold treated at 4 C for 3 days to enhance germination and plated on vertical 0.5X Murashige and Skoog (MS) medium agar plates containing 0.05% MES hydrate, 0.6 % sucrose, Gamborg vitamins (Sigma) and 0.45% p hytagel (Sigma).

PAGE 95

95 Roots of 2 weeks old plants were used for fluorescence microscopy. Yeast elicitors were prepared by dissolving 0.5% yeast extract (YE) (Difco) in MMS media (1 X MS salts, 10% mannitol, pH 5.7) and autoclaving. Specificity Test for Isoform Specific 143 3 Antibodies U sing ELISA the pET15b expression vector and the recombinants hi stagged proteins were expressed in E.coli and purified through h is tag/nickel column s (Sigma) as previously described (Wu et al., 1997). The wells of an ELISA plate were coated with 10ng/ l of recombinant 14 3 3 proteins ov ernight at 4 C. The next day, the plate was washed four times with ELISA wash buffer (1X PBS with 0.02% sodium azi de and 0.5% Tween 20) and blocked with ELISA blocking solution (1% milk powder in 1X PBS wi th 0.02% sodium azide) for 1 hr at room temperature followed by 4 washes ith ELISA wash buffer Plate was washed with ELISA wash buffer four times. Seven isoform specific polyclonal rabbit anti At14 3 3 antibodies (143 ; diluted 1:2000) were applied for 1 hr followed by 4 washes. The wells were incubated with h orseradish peroxidase conjugated secondary antibodies ( diluted 1:4000) for 45 min and washed as before. Labeled wells were identified using SuperSignal West Pico Chemiluminescent Substrate according to the suppliers instruct ions (Thermo Scientific). Cellular F ractionation Arabidopsis suspension cells were harvested on day 3 by filtration through MiraCloth ( Calbiochem ) S uspension cells (10g) were treated gently overnight (12 14 h r ) with 40 ml of protoplasting solution (0.1% c elluysin, 0.1% macerase

PAGE 96

96 and 0.1% pectolyase in 10% mannitol, 0.5X MS salts, pH 5.7) on a rotating platform (Belly D ancer ) Protoplasts were fi ltered through Miracloth and pelleted by centrifugation at 900 rpm ( 120 x g) for 5 min in a swing ing bucket centrifuge. The supernatant was kept as the cell wall fraction. Pelleted cells were washed and resuspended gently in 10 ml MMS (10% mannitol with 0.5 X MS salts, ph 5.7) in the presence of phospha tase and protease inhibitors (Calbiochem, CA). Proto plasts were repelleted and resuspended in 5 ml of NIB buffer (Sigma) in th e presence of 25 mM NaF, phosphatase and protease inhibitors. An aliquot representing the whole cell fraction was removed. The cell membrane was disrupted with 0.15% Triton X 100 on ic e for 5 min and the nuclei pelleted at 2000 x g, ( 4 C ) for 10 min. The supernatant representing the cytoplasmic fraction was removed and kept on ice. The nuclear pellet was resuspended in 5 ml of NIB buffer and an aliquot representing the whole nuclear fraction was removed. The nuclear fraction was stained with Trypan blue and visualized with a light microscope. The nuclei were repelleted and resuspended in 2 ml of NIB. The nuclear fraction (500 l) was pelleted at 2000 x g, ( 4 C ) for 5 min and resuspen and 0.1% digitonin) with phospha 50 mM lithium 3,5 diiodosalicylate (LIS) was added to the nuclear fraction to a concentration of 5 mM and gently r ocked on ice for 15 min. The insoluable nuclear fraction was pelleted at 12,000 x g, ( 4 C ) for 5 min and the supernatant kept as the soluable nuclear fraction. The pellet was washed and resuspended in the insoluable nuclear fracti on.

PAGE 97

97 Western Blot A nalysis Loadings of all cellular fraction protein samples except the digested cell wall fraction (8 fold more dilute) were adjusted against each other according to their starting volume and mixed with 2X sample buffer The proteins were run in discontinuous SDS PAGE and transferred to nitrocellulose using a Minifold I Dot Blot system (Schleicher & Schuell BioScience). 143 3 epsilon specific polyclonal antibody was used to detect the localization of each 143 3 isoform in cellular extrac ts and l abeled bands were identified using SuperSignal West Pico Chemiluminescent Substrate according to the suppliers instructions (Thermo Scientific). AtADH1 and histone H1 antibodies were used as cytoplasmic and nuclear control antibodies respectivel y. Plasmolysis and D etection of 14 3 3 Epsilon GFP by Fluorescence M icroscopy Transgenic Arabidopsis plants constitutiv ely expressing 143 3 epsilonGFP fusion protein were planted on vertical plates. GFP fluorescence signal in root cells of two week old plants was visualized before and after plasmolysis with an Olympus BX51 fluorescence microscope. Plasmolysis was induced in 0 .5 M NaCl solution for 5 min Results and Discussion Polyclonal 143 3 Antibodies Recognize 143 3 Proteins with High S pecificity Since antibody cross reactivity can lead to false interpretation of the western blot and confocal microscopy data, we decided to characterize the specificity of each antibody against recombinant 14 3 3 isoforms using ELISA. The assay showed that isoform s pecific antibodies, chi, epsilon, iota, omega and

PAGE 98

98 upsilon have very specific affinity for the corresponding recombinant 143 3. Nu and mu antibodies bind their corresponding isoform but they also have some level of cross reactivity to other isoforms. The nu ant ibody cross reacts with 143 3 kappa isoform. The 143 3 mu ant ibody cross reacts with 143 3 omega and psi isoforms ( Figure 41). These results were further investigated with western blot analysis and showed that the cross reactivity was not as promi nent as that seen with ELISA (data not shown). Cellular Fractionation and Western Blot A nalysis Intact protoplasts from Arabidopsis hypocoty l tissue culture c ells were isolated by protoplastin g enzyme solution. T reatment of protoplasts with 0.15% Triton X 100 disrupts the cell membrane but it keeps the nuclear envelope intact at this concentration. The insoluble nuclear fraction was separated from the solu ble fraction using 50 mM of lithium 3,5 diiodosalicylate (LIS), a hypotonic deter gent The identit iy of the c ellular fractionation was confirmed with SDS PAGE/w estern blot assays using histone H3 and alcohol dehydrogenase p olyclonal antibodies ( Figure 42 A). These western blots showed that the cellular fractions were separated from each other without a ny major contamination. Western blot with 143 3 epsilon specific antibody showed that this isoform is present in al l cellular fractions ( Figure 4 2 B). Considering the 8fold more dilute loading of the cell wall fraction, the 143 3 epsilon is mainly local ized in the digested cell wall fraction ( Figure 4 2 B, lane1). The large amount of the 143 3 epsilon in protoplast is present in the cytoplasmic fraction ( Figure 4 2 B, lane 2 and 3). A small amount of isoform is also found in the nuclear fractions ( Figure 4 -

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99 2 B, lane 4) and the majority of these proteins are shown to be present in the solu ble nuclear fraction ( Figure 4 2 B, lane 5). Arabidopsis 143 3 epsilon is secreted in response to fungal elicitation 143 3 epsilon was detected in the digested cell wall fraction of Arabidopsis suspension cells during the isolation of Arabidopsis protoplasts for subce llular fractionation ( Figure 4 2 B). Considering the fact that the protoplasting enzymes cellulose, macerase and pectolyase were isolated from fungi and they a re not completely pure, we inquired whether the 143 3 epsilon is localized in the cell wall or secreted in response to fungal elicitors present in protoplasting enzyme cocktail. We detected the 143 3 epsilon secretion after incubation of cells with boiled enzyme cocktail overnight (data not shown). To support the secretion hypothesis, suspension cells were also treated with 0.5% yeast extract (YE) overnight and a substantial amount of 14 3 3 epsilon secretion was detected w ith Western blotting ( Figure 4 3 ). Finally, 143 3epsilon GFP Is L ocalized in t he Plasma M embrane Two week old transgenic Arabidopsis plants expressing 143 3 epsilonGFP fusion protein were used to characterize the plasma membrane and/or cell wall localizatio n of 14 3 3 epsilon GFP. GFP fluorescence was detected at the plasma membrane /cell wall interface ( Figure 4 4 A). To determine the localization of 143 3 epsilonGFP in the plasma membrane or cell wall, Arabidopsis roots were plasmolysed with 0.5 M NaCl for 5 minutes. After plasmolysis, GFP fluorescence signal and plasma membrane was pulled awa y from the cell wall ( Figure 44C and 4D). This result suggested that 143 3 epsilon is localized in plasma membrane but not in cell wall.

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100 Figure 4 1. Specificity test for isoform specific 143 3 antibodies using ELISA. Each column was coated with 12 recombinant 143 3 proteins and each row was incubated with 7 isoform specific polyclonal antibodies.

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101 Figure 42 Localization analysis of 14 3 3 epsilon by western b lot Arabidopsis cell suspensions were used to create sub cellular and sub nuclear fractions: lane1 cell wall, lane2 whole cell (protoplast), lane3 cytoplasm, lane 4 whole nuclei, lane5 soluble nuclei, lane6 halos. A) W estern blot with control antibodies, alcohol dehydrogenase 1 (AtADH1) and histone H3. B ) Western blot with 143 3 epsilon specific polyclonal antibody. Loadings of proteins samples in lane 2, 3, 4, 5 and 6 were approximately equal, but the l oading of lane 1 was 8 fold more dilute.

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102 Figure 43 Elicitor induced secretion of 14 3 3 epsilon from Arabidopsis tissue culture cells. 8 grams of Arabidopsis tissue culture cells were treated with 40 ml of 0.5% YE in MMS media overnight. Western blot was performed with 143 3 epsilon isoform specific antibody. YE, yeast extract (Difco).

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103 A B C D Figure 44 Localization of 14 3 3 epsilon GFP fusion protein in Arabidopsis root cells. A) Detection of the localization of 14 3 3 epsilon by GFP fluorescent, B) Bright field image of A), C) Detection of the localization of 143 3 epsilon after plasmolysis by GFP fluorescent, D) Bright field image of B) PM stands for plasma membrane. CW stands for cell wall. PM/CW PM

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112 Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357 358 Pan S, Sehnke PC, Ferl RJ, Gurley WB (1999) Specific interactions with TBP and TFIIB in vitro suggest that 143 3 proteins may participate in the regulation of transcription when part of a DNA binding complex. Plant Cell 11: 15911602 Paul AL, Liu L, McClung S, Laughner B, Chen S, Ferl RJ (2009) Comparative interactomics: analysis of arabidopsis 143 3 complexes reveals highly conserved 143 3 interactions between humans and plants. J Proteome Res 8: 19131924 Paul AL, Sehnke PC, Ferl RJ (2005) Isofor m specific subcellular localization among 143 3 proteins in Arabidopsis seems to be driven by client interactions. Mol Biol Cell 16: 17351743 Pei DS, Wang XT, Liu Y, Sun YF, Guan QH, Wang W, Yan JZ, Zong YY, Xu TL, Zhang GY (2006) Neuroprotection against ischaemic brain injury by a GluR69c peptide containing the TAT protein transduction sequence. Brain 129: 465479 Petosa C, Masters SC, Bankston LA, Pohl J, Wang B, Fu H, Liddington RC (1998) 143 3zeta binds a phosphorylated Raf peptide and an unphosphor ylated peptide via its conserved amphipathic groove. J Biol Chem 273: 1630516310 Porter GW, Khuri FR, Fu H (2006) Dynamic 14 3 3/client protein interactions integrate survival and apoptotic pathways. Semin Cancer Biol 16: 193202 Powell DW, Rane MJ, Chen Q, Singh S, McLeish KR (2002) Identification of 143 3zeta as a protein kinase B/Akt substrate. J Biol Chem 277: 2163921642 Powell DW, Rane MJ, Joughin BA, Kalmukova R, Hong JH, Tidor B, Dean WL, Pierce WM, Klein JB, Yaffe MB, McLeish KR (2003) Proteomic identification of 143 3zeta as a mitogen activated protein kinaseactivated protein kinase 2 substrate: role in dimer formation and ligand binding. Mol Cell Biol 23: 53765387 Reuther GW, Fu H, Cripe LD, Collier RJ, Pendergast AM (1994) Association of the protein kinases c Bcr and Bcr Abl with proteins of the 143 3 family. Science 266: 129133 Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genomewide comparative analysis among eukaryotes. Science 290: 21052110 Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, Gamblin SJ, Yaffe MB (1999) Structural analy sis of 14 3 3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 143 3 in ligand binding. Mol Cell 4: 153166

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117 BIOGRAPHICAL SKETCH Tufan Gokirmak was born in I stanbul, Turkey, in 1979. He received his undergraduate degree from Bogazici University, Department of Molecular Biology and Geneti cs, in the Summer of 2002. After receiving his B achelor of S cience degree, he moved to Oregon, U nited States to pursue a Master of Science degree at Oregon State University (OSU) Department of Horticulture under the supervision of Dr Shawn Mehlenbacher. After he received his M.S. degree from OSU in June 2005, he moved to Florida to start his Ph.D. at the University of Florida, Plant Molecular and Cellular Biology Program under the supervision of Dr. R obert J. Ferl. During his doctoral studies he investigated the role of phosphorylation mediated regulation of 143 3 protein dimerization in Arabidopsis thaliana and the effect of this regulation in 143 3/target interactions.