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GSK-3) in Dictyostelium Discoideum and the Role of GSK-3 Binding Partners in Dictyostelium Discoideum and Mammalian Cells

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GSK-3) in Dictyostelium Discoideum and the Role of GSK-3 Binding Partners in Dictyostelium Discoideum and Mammalian Cells Investigation into the role of Glycogen Synthase Kinase 3 (GSK-3) in Dictyostelium discoideum and the role of GSK-3 binding partners in Dictyostelium discoideum and mammalian cells Josephine E. Forde School of Biosciences Cardiff University PhD Thesis Submitted: November 2009 UMI Number: U585B60 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U585B60 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Ca r d if f UNIVERSITY PRIFYSGOL DECLARATION C a e RD yi §> This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed ........... d & c k . - . ...................... (candidate) Date ... ! H | Q s | a o \ p .......... STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of .................... (insert MCh, MD, MPhil, PhD etc, as appropriate) Signed .......d & 0 ( L : ...................... (candidate) Date ........... STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed d 6 < L - . ............................ (candidate) Date .. STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations. S ign ed ....................................(candidate) Date ...Jltl^.j.3.0.lQ .... STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loans after expiry of a bar on access previously approved by the Graduate Development Committee. Signed (candidate) Date Acknowledgements I could not have completed PhD project alone. I am indebted to my supervisors Professor Trevor Dale and Professor Adrian Harwood whose differing supervision styles complemented each other so well. Their guidance and enthusiasm, as well as their experimental knowledge, have been invaluable to me. I would like to thank all members, past and present, of the Dale and Harwood labs for their help and advice throughout the course of my project. In particular I would like to thank Ms Elizabeth Fraser and Dr Christine Humphreys for advice and for useful discussions, Miss Kirsty Richardson for her assistance with the FACS analysis, Dr Jonathan Ryves for his assistance with kinase assays and Dr Regina Teo for useful discussions on all things ‘Dicty’ related. I would also like to thank Dr Peter Watson for his advice, infectious enthusiasm and assistance in all things microscopy based. My parents, Pauline and Eamonn Forde have been a constant source of moral support throughout this project and Gareth, for being so understanding and putting up with the constant “Fll just be another hour.. Abstract. The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified as a key regulator of insulin-dependent glycogen synthesis. GSK-3 was subsequently shown to function in a wide range of cellular processes including differentiation, growth, motility and apoptosis. A number of mechanisms have been shown to allow differential regulation of GSK-3; these include modulation by upstream signals, control of substrate specificity and GSK-3 localisation. The aim of this project was to better characterise the role of GSK-3 in the social amoeba Dictyostelium discoideum and to investigate the possibility that GSK-3 activity is directed through interactions with specific GSK-3 binding proteins. In this study I generated both mammalian and Dictyostelium cell lines expressing GSK-3 homologues tagged with GFP. I demonstrated that the addition of this tag did not obviously alter cellular localization or the ability of GSK-3 to interact with known protein binding partners. I also confirmed that these fusion proteins retained kinase activity and that the levels of GSK-3 (3 over-expression used in this study were not detrimental to cell viability. I further characterized the role of GSK-3 in the social amoebae Dictyostelium, using both knockout and over-expression cell lines to demonstrate that this protein fulfils multiple roles during early development. Dark-field time-lapse microscopy techniques were employed to identify a role for GSK-3 in co-ordinating chemotaxis and cellular polarization. During the course of this project I also identified a requirement for GskA in cytokinesis when cells were grown in shaking culture. Finally, both the mammalian and Dictyostelium cell lines generated in this study were used for affinity chromatography experiments and a number of potential GSK-3 binding partners were identified. 2 Table of contents CHAPTER 1 7 INTRODUCTION 7 1.1 Introduction 8 1.2 GSK-3 genes 9 1.3 GSK-3 substrates 13 1.4 Inhibitory phosphorylation of GSK-3 16 1.5 Activating phosphorylation of GSK-3 17 1.6 Regulation of glycogen synthesis 19 1.7 Regulation of the canonical Wnt pathway 20 1.8 Regulation of Hedgehog signal transduction 24 1.9 Regulation of the cytoskeleton by GSK-3 27 1.10 Regulation of apoptosis and cell viability 29 1.11 Regulation of transcription factors 29 1.12 The role of GSK-3 in Dictyostelium 30 1.13 Dictyostelium chemotaxis 33 1.14 Dictyostelium Cytokinesis 34 1.15 Aims of this study 38 CHAPTER 2 39 MATERIALS AND METHODS 39 2.1 Cloning 40 2.1.1 Dictyostelium Vectors 40 2.1.2 Mammalian Vectors 41 2.2 PCR 43 2.3 Cell culture 44 2.3.1 Dictyostelium cell culture 44 2.3.2 Mammalian cell culture 44 3 2.4 Transformations 45 2.4.1 Dictyostelium transformations 45 2.4.2 Mammalian transformation 46 2.5 Screening of clones 48 2.5.1 Screening o f Dictyostelium clones 48 2.5.2 Screening of T-REx 293 GSK3P-GFP and GFP cell lines 48 2.6 Immunofluorescence 49 2.6.1 Dictyostelium immunofluorescence 49 2.6.2 Mammalian immunofluorescence 49 2.7 Western blot 50 2.7.1 Dictyostelium western blots 50 2.7.2 Mammalian western blots 50 2.8 FACS sorting of Dictyostelium cells 51 2.9 Mammalian LacZ Assay 52 2.10 Mammalian Wst-1 Assay 52 2.11 Kinase Assay 53 2.11.1 Dictyostelium kinase assay 53 2.11.2 Mammalian kinase assay 54 2.12 Dictyostelium Cell Sizing 54 2.13 Dictyostelium Multinucleate Assay 54 2.14 Dictyostelium Cell Proliferation Assay 55 2.15 Dictyostelium Development assay 55 2.16 Dictyostelium cAMP pulse assay 56 2.17 Affinity purifications 56 2.17.1 Dicytostelium affinity' purifications 56 2.17.2 Mammalian affinity purifications 57 2.18 SILAC 59 2.19 Mass Spectrometry Analysis 60 2.19.1 In-gel d igestion 60 2.19.2 LC-MS/MS 60 2.20 SILAC coupled Mass Spectrometry Analysis 61 2.20.1 Gel electrophoresis and in-gel digestion 61 2.20.2 LC-MS/MS 61 2.20.3 Quantification and Bioinformatic Analysis 62 4 CHAPTER 3 64 CHARACTERIZATION OF GSK3B-GFP AND GSKA-GFP CELL LINES 64 3.1 Generation of Dictyostelium GskA-GFP cell lines 65 3.2 Generation of T-REx GSK30-GFP cell lines 70 3.3 Localization of GskA-GFP and GSK3P-GFP 75 3.4 Assessment of kinase activity 78 3.5 Conclusions 82 CHAPTER 4 83 THE ROLE OF GSK-3 IN DICTYOSTELIUM DISCOIDEUM 83 4.1 GskA performs a role during early development 84 4.2 gskA null cells do not stream or polarize 89 4.3 GskA performs a role during cytokinesis 92 4.4 Ax2/gsk- cells are smaller than their Ax2 counterparts 96 4.5 Conclusions 98 CHAPTER 5 101 IDENTIFICATION OF GSK-3 BINDING PARTNERS 101 5.2 Isolation of potential GSK-3 interaction proteins 105 5.2.1 Initial affinity purifications 105 5.2.2 Utilization of chemical cross-linking 107 5.3 Analysis of proteins that co-purified with GskA 108 5.4 SILAC-linked Mass Spectrometry 112 5.3 Analysis of SILAC results 115 5.3.1 STRING analysis 117 5.4 Validation of interactions 120 5.5 Conclusions 122 5 CHAPTER 6 125 DISCUSSION 125 6.1 The role of GskA in Dictyostelium 127 6.1.1 Potential mechanisms linked to direct action of GskA 128 6.1.2. Potential mechanisms linked to changes in gene expression 129 6.2 The role of key residues of GskA 131 6.2.1 Separation of the functions performed by GskA during development 131 6.2.2. Regulatory roles of Y214 and R96 132 6.3 Identification of potential GSK-3 interaction partners 133 6.3.1 Identification of ribosomal proteins as GSK-3 binding partners 134 6.3.2 Potential GskA interaction partners 134 6.3.3 Potential GSK3f5 interaction partners 136 6.4 Conclusions 140 APPENDIX 141 GO term analysis 146 BIBLIOGRAPHY 155 6 Chapter 1 Introduction 1.1 Introduction The aim of this project was to better characterise the role of the serine/threonine kinase Glycogen Synthase Kinase 3 (GSK-3) in the social amoeba Dictyostelium discoideum, and to investigate the possibility that GSK-3 activity is directed through interactions with specific protein binding proteins. GSK-3 is a highly conserved kinase that functions in multiple signalling pathways including Wnt and Hedgehog signal transduction, protein synthesis, glycogen metabolism, mitosis and apoptosis.
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