NEGATIVE REGULATION OF CYTOKINE SIGNALLING IN THE MYELOID LINEAGE Investigating the Role of CBL and SH2B1 by Mojib Javadi Javed A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Mojib Javadi Javed, 2012 NEGATIVE REGULATION OF CYTOKINE SIGNALLING IN THE MYELOID LINEAGE Investigating the Role of CBL and SH2B1 Mojib Javadi Javed Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto 2012 ABSTRACT Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. ii Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL- C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells. iii ACKNOWLEDGMENTS The past number of years have had their fill of challenges and moments of excitement. I was very fortunate to have had the support of colleagues, family and friends, throughout this process. I would like to thank Dr. Dwayne Barber for all his support and mentorship. You have always challenged me to ask the right scientific questions, and have been patient and receptive as I worked towards the answers. I greatly appreciate the exceptional training I have received under your supervision. I would also like to thank my committee members, Dr. Jane McGlade and Dr. Vuk Stambolic for their guidance. Your critique, input and advice have been an integral part of my advancement as a scientist. To my lab mates in the Barber lab, past and present, thank you for your sense of humor and encouragement. Thank you for listening to all my rants. Sharing this time with all of you has been an amazing experience. I look forward to our continued friendship and camaraderie. To my family and friends, thank you for your relentless support. Especially to my parents, thank you for instilling me with the passion to learn, and the drive to achieve anything I set my mind to. Finally to Sara Zokaei, thank you for helping me stay positive, motivated and inspired. Your support has made the tough “all my experiments failed” moments bearable and the moments of success even sweeter. iv TABLE OF CONTENTS ACKNOWLEDGMENTS................................................................................................. iv TABLE OF CONTENTS .................................................................................................. v LIST OF TABLES......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix ABBREVIATIONS ......................................................................................................... xii 1 INTRODUCTION........................................................................................................ 1 1.1 HEMATOPOIESIS ............................................................................................... 2 1.1.1 Erythropoiesis ................................................................................................ 4 1.2 ERYTHROPOIETIN SIGNALLING ...................................................................... 6 1.2.1 Activation of the Erythropoietin Receptor....................................................... 6 1.2.2 Downstream Signalling Pathways.................................................................. 7 1.2.3 Attenuation of EPO Signalling...................................................................... 13 1.3 GM-CSF SIGNALLING...................................................................................... 15 1.3.1 GM-CSF Receptor Complex ........................................................................ 15 1.3.2 Downstream Signalling ................................................................................ 16 1.3.3 Attenuation of GM-CSF Signalling ............................................................... 23 1.4 SH2B Family of Adaptor Proteins .................................................................. 25 1.4.1 SH2B Family................................................................................................ 25 1.4.2 SH2B1 Regulation of Signalling................................................................... 25 1.4.3 Role of SH2B family in EPO signalling ........................................................ 29 1.5 Casitas B-lineage Lymphoma (Cbl)................................................................ 30 1.5.1 Structural Organization of Cbl...................................................................... 30 1.5.2 Cbl as an E3 Ligase..................................................................................... 31 1.5.3 Adaptor Protein Functions of Cbl................................................................. 36 1.5.4 CBL Associated Oncogenesis ..................................................................... 38 1.6 RATIONAL AND HYPOTHESIS........................................................................ 42 1.7 THESIS OBJECTIVES ...................................................................................... 44 v 2 THE SH2B1 ADAPTOR PROTEIN ASSOCIATES WITH A PROXIMAL REGION OF THE ERYTHROPOIETIN RECEPTOR .................................................................... 46 2.1 ABSTRACT ....................................................................................................... 47 2.2 INTRODUCTION................................................................................................ 48 2.3 EXPERIMENTAL PROCEDURES:.................................................................... 50 2.4 RESULTS .......................................................................................................... 55 2.4.1 COLT Screening Identifies SH2B1β Interacting with EPO-R Y343.............. 55 2.4.2 SH2B1 Associates with the EPO-R in Hematopoietic Cell Lines................. 55 2.4.3 SH2B1 Binds Specifically to pY343 and pY401 of the EPO-R upon EPO Stimulation .............................................................................................................. 57 2.4.4 SH2B1 Associates with Unphosphorylated EPO-R ..................................... 61 2.4.5 SH2B1 is Phosphorylated in Response to EPO Stimulation........................ 65 2.4.6 SH2B1 Associates with the EPO-R in Primary Splenic Erythroblasts ......... 68 2.4.7 SH2B1 is a Negative Regulator of Downstream EPO Signaling.................. 71 2.5 DISCUSSION..................................................................................................... 73 3 CBL linker region and RING finger mutations lead to enhanced GM-CSF signalling via elevated levels of JAK2 and LYN. ...................................................... 78 3.1 ABSTRACT ....................................................................................................... 79 3.2 INTRODUCTION................................................................................................ 80 3.3 MATERIALS AND METHODS .......................................................................... 83 3.4 RESULTS .......................................................................................................... 87 3.4.1 Enhanced and Prolonged Phosphorylation of the GM-CSFR βc in CBL Mutant Expressing Cells. ........................................................................................ 87 3.4.2 Elevated levels of JAK2 kinase in CBL-Y371H and CBL-C384R expressing TF-1 cells ................................................................................................................ 89 3.4.3 Increased expression of LYN kinase in CBL mutant cells. .......................... 91 3.4.4 Enhanced phosphorylation of SHP2 in