Molecular mechanisms controlling SH2 domain- containing inositol 5’phosphatase (SHIP) function in B cells by Samantha Dawn Pauls A thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry and Medical Genetics University of Manitoba Winnipeg Copyright © 2016 by Samantha Dawn Pauls TABLE OF CONTENTS Table of Contents ............................................................................................................................ ii Thesis Abstract................................................................................................................................ v Acknowledgement ........................................................................................................................ vii List of Figures ................................................................................................................................ ix List of Tables ................................................................................................................................ xii List of Copywrited materials for which permission was obtained .............................................. xiii Abbreviations ............................................................................................................................... xiv CHAPTER 1: GENERAL INTRODUCTION ............................................................................... 1 1.1 Discovery of B cells ......................................................................................................... 1 1.2 The benefits of B cells: immune response to pathogens .................................................. 3 1.3 The hazard of B cells: role in inflammatory and autoimmune conditions ....................... 5 1.4 B cell development ........................................................................................................... 7 1.5 Immune receptor signaling basics: ITAM versus ITIM ................................................. 11 1.6 Signalling through the B cell receptor............................................................................ 12 1.6.1 Signal initiation ....................................................................................................... 12 1.6.2 PLCγ pathway ......................................................................................................... 19 1.6.3 MAPK pathways ..................................................................................................... 20 1.6.4 PI3K pathway.......................................................................................................... 21 1.6.5 BCR signal modulation by co-receptors ................................................................. 28 1.6.6 Abnormal BCR signaling and disease .................................................................... 30 1.7 SH2 domain-containing inositol 5’phosphatase (SHIP) ................................................ 32 1.7.1 Discovery of SHIPc ................................................................................................ 32 1.7.2 SHIP knock-out models .......................................................................................... 32 1.7.3 Structure and function ............................................................................................. 33 1.7.4 Regulation of SHIP protein levels .......................................................................... 41 1.7.5 SHIP in autoimmune and inflammatory disorders.................................................. 42 1.8 The actin cytoskeleton and B cell signaling ................................................................... 44 1.8.1 Signal initiation ....................................................................................................... 44 1.8.2 Branched actin filament formation ......................................................................... 46 1.8.3 PI3K signaling and actin ......................................................................................... 48 1.9 Thesis overview.............................................................................................................. 49 1.9.1 Study Rationale ....................................................................................................... 49 ii 1.9.2 General hypothesis .................................................................................................. 50 1.9.3 Objectives ............................................................................................................... 50 CHAPTER 2: DEFINING THE INTERACTION NETWORK OF SHIP ................................... 52 2.1 Specific Introduction ...................................................................................................... 52 2.2 Materials and Methods ................................................................................................... 54 2.2.1 Cell lines and reagents ............................................................................................ 54 2.2.2 Co-immunoprecipitation, western blotting and multiple reaction monitoring ....... 55 2.2.3 Bioinformatics prediction tools............................................................................... 56 2.2.4 Recombinant proteins ............................................................................................. 56 2.2.5 Biacore screening .................................................................................................... 56 2.2.6 Biacore steady-state affinity analysis...................................................................... 59 2.3 Results ............................................................................................................................ 59 2.3.1 Evidence for an interaction between Bam32 and SHIP in B cells .......................... 59 2.3.2 Identification of potential SHIP interaction partners using prediction tools .......... 62 2.3.3 Identification of SHIP interaction partners by a Biacore screening assay .............. 66 2.3.4 Biacore affinity analysis of SHIP-FcγRIIB and SHIP-Nck interactions ................ 70 2.4 Discussion ...................................................................................................................... 71 CHAPTER 3: A NOVEL PHOSPHORYLATION SITE IN SHIP INDUCES BINDING TO NCK AND REGULATES ACTIN DYNAMICS IN B CELLS .................................................. 75 3.1 Specific Introduction ...................................................................................................... 75 3.2 Materials and Methods ................................................................................................... 78 3.2.1 Cell culture and reagents ......................................................................................... 78 3.2.2 Plasmids and transfections ...................................................................................... 78 3.2.3 Generation of stable cell lines ................................................................................. 79 3.2.4 GST pull-downs ...................................................................................................... 79 3.2.5 FRAP assay for actin turnover ................................................................................ 80 3.2.6 Membrane recruitment assay .................................................................................. 80 3.2.7 Intracellular staining ............................................................................................... 81 3.2.8 Spreading assay ....................................................................................................... 81 3.2.9 Statistical Analysis .................................................................................................. 82 3.3 Results ............................................................................................................................ 82 3.3.1 SHIP interacts with the cytoskeletal adaptor protein Nck ...................................... 82 3.3.2 SHIP inhibits actin turnover by a mechanism seemingly dependent on Tyr944 .... 84 3.3.3 Effect of SHIP-EGFP on actin turnover is overcome by co-overexpression of Nck 87 iii 3.3.4 SHIP membrane recruitment and enzymatic activity are not diminished by Y944F mutation ................................................................................................................................ 87 3.3.5 Overexpression of SHIP-EGFP has no measurable effect on cell spreading in a transient transfection system ................................................................................................. 89 3.3.6 Cell viability after transfection is variable and vector-dependent .......................... 90 3.4 Discussion ...................................................................................................................... 92 CHAPTER 4: CONTROLLING THE INTRACELLULAR LOCALIZATION DYNAMICS OF SHIP .............................................................................................................................................. 97 4.1 Specific Introduction ...................................................................................................... 97 4.2 Materials and Methods ................................................................................................... 99 4.2.1 Cell culture and reagents ........................................................................................