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Protein Tyrosine Phosphorylation in Haematopoietic Cancers and the Functional Significance of Phospho- Lyn SH2 Domain

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Protein Tyrosine Phosphorylation in Haematopoietic Cancers and the Functional Significance of Phospho- Lyn SH2 Domain Protein Tyrosine Phosphorylation in Haematopoietic Cancers and the Functional Significance of Phospho- Lyn SH2 Domain By Lily Li Jin A thesis submitted in conformity with the requirements for the degree of Ph.D. in Molecular Genetics, Graduate Department of Molecular Genetics, in the University of Toronto © Copyright by Lily Li Jin (2015) Protein Tyrosine Phosphorylation in Haematopoietic Cancers and the Functional Significance of Phospho-Lyn SH2 Domain Lily Li Jin 2015 Ph.D. in Molecular Genetics Graduate Department of Molecular Genetics University of Toronto Abstract Protein-tyrosine phosphorylation (pY) is a minor but important protein post-translational modification that modulates a wide range of cellular functions and is involved in cancer. Dysregulation of tyrosine kinases (TKs) and protein-tyrosine phosphatases (PTPs) have been observed in multiple myeloma (MM) and acute myeloid leukemia (AML) and is a subject of study. Using recently developed mass spectrometry-based proteomics techniques, quantitative PTP expression and cellular pY profiles were generated for MM cell lines and mouse xenograft tumors, as well as primary AML samples. Integrated comprehensive analyses on these data implicated a subset of TKs and PTPs in MM and AML, with valuable insights gained on the dynamic regulation of pY in biological systems. In particular, I propose a model that describes the cellular pY state as a functional output of the total activated TKs and PTPs in the cell. My results show that the global pY profile in the cancer models is quantitatively related to the cellular levels of activated TKs and PTPs. Furthermore, the identity of the implicated TK/PTPs is system- ii dependent, demonstrating context-dependent regulation of pY. To further understand pY regulation, I studied the phosphorylation of a conserved tyrosine in the Src homology 2 (SH2) domains of Src family kinases, which was frequently observed in human cancer specimens and regulated in cancer-derived cell lines. Using the Lyn SH2 domain as a model, I discovered that when this tyrosine (Y194) is phosphorylated, the domain has reduced ability to interact in vitro with phosphopeptides and phosphoproteins. Sequence analysis of the binding motifs revealed that phosphorylation at Y194 decreased the selectivity of Lyn SH2 for the third residue C-terminal to the pY of the ligand. Together, these data show that tyrosyl phosphorylation alters the substrate binding profile of Lyn SH2 and may potentially affect Lyn kinase signaling. This may be a general mechanism for all Src family kinases and represents another layer of the complexity of pY regulation. iii List of Abbreviations AML acute myeloid leukemia AP affinity purification ATP adenosine triphosphate CLL chronic lymphocytic leukemia EGF epidermal growth factor EGFR epidermal growth factor receptor ESI electrospray ionization FGFR3 fibroblast growth factor receptor 3 HER2 human epidermal growth factor receptor 2 HRG HER2/heregulin IGF-1 insulin-like growth factor 1 IGF1R IGF-1 receptor IL-6 interleukin 6 IMAC immobilized metal affinity chromatography INSR insulin receptor IP immunoprecipitation IPI international protein index ITD internal tandem duplication Kd dissociation constants KMS Kawasaki Medical School LC MS/MS liquid chromatography tandem mass spectrometry MAPK mitogen-activated protein kinase iv MGUS monoclonal gammopathy of undetermined significance MM multiple myeloma MS mass spectrometry nRTK non-receptor tyrosine kinase NSCLC non-small cell lung cancer PDGF platelet derived growth factor PLSR partial least squares regression PTB protein tyrosine binding PTP protein tyrosine phosphatase pY tyrosine phosphorylation ROS reactive oxygen species rt room temperature RTK receptor tyrosine kinase SA Streptavidin SFK Src family kinases SH2 Src homology 2 SILAC stable isotope labelling by amino acids in cell culture SRM selected reaction monitoring STAT signal transduction and activator of transcription TK tyrosine kinase XIC extracted ion current chromatography v Table of Contents List of Abbreviations ................................................................................................. iv Table of Contents ....................................................................................................... vi List of Tables .............................................................................................................. x List of Figures ............................................................................................................ xi List of Appendices .................................................................................................... xii Chapter 1 Introduction .................................................................................................... 1 1.1 Protein Tyrosine Phosphorylation: Function and Dysregulation in Haematopoietic Cancers ............................................................................................. 1 1.1.1 Enzymes and Domains Involved in pY-Mediated Functions ........................ 1 1.1.1.1 Tyrosine Kinases ..................................................................................... 2 1.1.1.2 Src Family Kinases ................................................................................. 3 1.1.1.3 Protein Tyrosine Phosphatases ............................................................... 4 1.1.1.4 Src Homology 2 Domain ........................................................................ 5 1.1.1.4.1 Structure and Functions of SH2 Domains ........................................... 5 1.1.1.4.2 Genetic Mutations of SH2 Domains in Human Diseases .................... 7 1.1.2 Biological Function of Phosphorylated Tyrosines ......................................... 9 1.1.3 Dysregulation of Protein Tyrosine Kinases and Phosphatases in Haematopoietic Cancers ....................................................................................... 11 1.1.3.1 Multiple Myeloma ................................................................................ 11 1.1.3.2 Acute Myeloid Leukemia ..................................................................... 13 1.2 Mass Spectrometry Based Proteomics ................................................................ 15 1.2.1 Mass Spectrometry for Proteomics Studies ................................................. 16 vi 1.2.2 Label-Free Quantification by Mass Spectrometry ....................................... 16 1.2.2.1 Measurement by Intensity of MS1 Ion Current .................................... 17 1.2.2.2 Selected Reaction Monitoring ............................................................... 18 1.2.3 Peptide Enrichment ...................................................................................... 18 1.2.3.1 Enrichment of Tyrosyl Phosphorylated Peptides .................................. 18 1.2.3.2 Enrichment of Oxidized Cysteine-Containing Peptides (qPTPome) .... 19 1.3 Key Statistical Analysis Used in this Thesis ....................................................... 20 1.3.1 Partial Least Squares Regression ................................................................ 20 1.4 Specific Aim for this Thesis ............................................................................... 21 Chapter 2 Comprehensive Analysis of Protein Phosphotyrosine in Multiple Myeloma ....................................................................................................................................... 22 2.1 Abstract ............................................................................................................... 23 2.2 Introduction ......................................................................................................... 23 2.3 Western Analysis Showed Distinct Expression, Glycosylation, Phosphorylation of pY Signaling Molecules in MM Samples ............................................................. 27 2.4 pY Profiling Revealed Distinctive Differences between MM Cultured Cell lines and Xenograft Tumors .............................................................................................. 30 2.5 Co-Variance Analysis of PTP and pY Profiles Implicated a Subset of PTP in pY Regulation ................................................................................................................. 39 2.6 Correlation Heatmap Revealed Regulation “Hotspots” ...................................... 42 2.7 Discussion and Conclusion ................................................................................. 46 2.8 Materials and Methods ........................................................................................ 54 vii Chapter 3 Comprehensive Analysis of Protein Phosphotyrosine in Acute Myeloid Leukemia ....................................................................................................................... 62 3.1 Abstract ............................................................................................................... 63 3.2 Introduction ......................................................................................................... 63 3.3 pY Profiling Revealed Distinctive Features in Patient Subgroups and Implicated a Subset of TKs in pY Regulation ............................................................................ 66 3.4 Integrated Analysis of PTP Expression and Cellular pY Implicated PTPs in AML pY Regulation ................................................................................................. 74 3.6 Discussion and Conclusion ................................................................................
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