Mapping and Characterization of the Interaction Network of ALK Receptor Tyrosine Kinase using the Mammalian Membrane Two-Hybrid (MaMTH) Assay by Farzaneh Aboualizadeh A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Farzaneh Aboualizadeh 2018 Mapping and Characterization of the Interaction Network of ALK Receptor Tyrosine Kinase using the Mammalian Membrane Two-Hybrid (MaMTH) Assay Farzaneh Aboualizadeh Master of Science Graduate Department of Biochemistry University of Toronto 2018 Abstract Anaplastic Lymphoma Kinase (ALK) which belongs to the Receptor Tyrosine Kinases (RTKs) play a critical role in development and progression of many type of cancers. To design new treatments for ALK associated human diseases, it is essential to study the interactions of the protein to better understand the biological significance of its protein-protein interactions (PPIs) and how they contribute to ALK regulation and function. Novel high-throughput technology, the Mammalian Membrane Two-Hybrid (MaMTH), was used in this study to generate a targeted protein-protein interaction map (interactome) of full length ALK. Biased screening of 150 predicted and previously known ALK interactors were performed and 34 identified as novel ALK interactors. The active mutant cells stably expressing ALK F1245C, F1245I and L1196M have been generated which showed more phosphorylation of ALK (pALK1604) as compared to wild type. Moreover, these active mutants exhibited more phosphorylation of downstream signaling ERK1/2 as compared to wild type. ii Acknowledgments I would like to express my sincere gratitude to my supervisor, Dr. Igor Stagljar, for the useful advices, remarks and engagement through the learning process of this master thesis. His guidance helped me in all the time of research and writing of this thesis. I would like to thank him for his moral and emotional support during my study. Additionally, I would like to thank my committee members, Dr. Liliana Attisano, Dr. Daniela Rotin and Dr. Vuk Stambolic for their support on the way. A very special gratitude goes out to Dr. Jamie Snider who supported me in any way possible. I would like to thank him for helping me with my project and research and answering countless questions. I would like to thank all members of Stagljar lab for their never-ending support. They provided a friendly and cooperative atmosphere at work and useful feedback and insightful comments on my work. I wish to thank Dr. Tania Christova for her willingness to help whenever needed. I really appreciate it. Finally, I would like to express my profound gratitude to my spouse for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without him. My eternal gratitude goes to my parents and my brothers for their understanding, never-ending love, moral support, endless patience and encouragement as and when required. iii Table of Contents Acknowledgments…………………………………………………………………………….....iii Table of Contents………...………………………………………………………………….......iv Abbreviations…………………………………………………………………………………....ix List of Tables……..………………………………………………………………………………x List of Figures………...………………………………………………………………….............xi List of Appendices…………………………………………………………………………......xiii 1 Chapter 1: Introduction…………………………………………………………………1 1.1 Protein Kinases…………………………………………………………………...1 1.2 Protein tyrosine kinases……………………………………………………….....2 1.2.1 Receptor Tyrosine Kinases (RTKs)…………………………………...…..3 1.2.1.1 Classification and structure of RTKs……………………………...4 1.2.1.2 Mechanism of action of RTKs……………………….....................6 1.2.1.3 Regulation of RTKs…………….………………………………....8 1.2.1.4 RTKs and signaling pathways………………….………….……...8 1.2.1.5 RTKs in human diseases………………………….……….............9 1.3 Anaplastic Lymphoma Kinase (ALK) …………………………………….…...9 1.3.1 ALK function and structure…………………............................................10 1.3.1.1 Structure of ALK kinase domain…….……………………….….12 1.3.2 ALK activation and downstream signaling…………………………..........13 1.3.2.1 ALK phosphorylation and activation……….................................13 1.3.2.2 ALK downstream signaling….……………………………….….15 1.3.3 ALK in disease……………………………………………...……………...17 1.3.4 Inhibition of ALK activity…………..……………….………………….….21 1.3.5 Identification of human ALK interactors………………………….…….….23 Research objective……………………………………………………………………………...27 2 Chapter 2: Materials and methods…………………...…………………………….….28 2.1 General experimental methods……………….…………………………….….28 2.1.1 PCR amplification…………………………………………………….........28 2.1.2 Agarose gel electrophoresis……...………….……………………………...28 iv 2.1.3 Sequencing DNA from TCAG (The Center for Applied Genomics)………29 2.1.4 Competent E. coli preparation using Inoue method……………….….……29 2.1.5 E. coli transformation using heat shock method…………………...………29 2.1.6 Glycerol stock preparation…………………………………………………30 2.1.7 DNA isolation from bacteria……………………………………………….30 2.1.8 Co-Immunoprecipitation (Co-IP) ………………………………………….30 2.2 Gateway cloning…………………………...………………………………...….31 2.2.1 Purification of entry clones…………………………….……...…………...31 2.2.2 Gateway LR cloning……….………….…………………….…………......31 2.3 Site-directed mutagenesis……….………………………………..………….…31 2.3.1 Primer design guidelines……….…………….…………………………….31 2.3.2 PCR amplification using site-directed mutagenesis primers………………32 2.3.3 DpnI digestion and transformation of mutagenized PCR product…………32 2.4 Tissue culture protocols…………………………………………..…………….33 2.4.1 Growing up cells from liquid nitrogen storage.…………………….…...…33 2.4.2 Freezing cells…….…………….…………………………...……….……...33 2.4.3 Cell splitting…….….…………….……………………………...................33 2.4.4 Counting cells …….….…………….………………………………….…...34 2.4.5 Seeding cells in plate……….…………….……………….………………..34 2.4.6 Transient transfection of cells using standard CaCl2 method……………....35 2.4.7 Transfection of stable cells using standard CaCl2 method…………………35 2.4.8 Transfection of cells using FLP-In TREx system………………………….36 2.4.9 Transfection of cells using lipofectamine 3000……………………………36 2.4.10 Picking foci………….……………………………………………………37 2.4.11 Luciferase assay…….…………………………………………………….37 2.4.12 Cell viability assay….…………………………………………………….37 2.5 Western immunoblotting protocols…………………………………..………..38 2.5.1 Protein extraction…………………………………………………………..38 2.5.2 Measuring protein concentration using Bradford assay……………………38 v 2.5.3 Preparation of stacking and resolving SDS polyacrylamide gel for western immunoblotting…………………………….……………….……………………38 2.5.4 Gel electrophoresis of protein samples, transfer and western immunoblotting…………………………………………………...……………...39 2.6 ALK Bait generation…………………..………………..…………………..…..40 2.6.1 Gateway cloning of ALK gene into MaMTH bait vector………………….40 2.6.2 Transfection of C-tagged ALK bait into reporter cell line………………....40 2.6.3 Testing the expression of ALK-Cub-TF in the presence and absence of tetracycline……………………………………………………………………….40 2.7 Prey generation…………………………………...……………………………..41 2.7.1 Gateway cloning of preys into MaMTH prey vectors……………………...41 2.7.2 Testing the expression of MaMTH prey constructs………………………..41 2.8 Testing the interaction of bait and prey using MaMTH………………..…….41 2.9 Testing the interaction of bait and prey in serum starvation media………....42 2.10 Testing the interaction of bait and prey in the presence of drug using MaMTH………………………………………………………………………….42 2.11 Preparation of FAM150A and FAM150B ALK ligands……………...……...42 3 Chapter 3: Results……………………………………………………………………...44 3.1 Generation of stably integrated ALK-Cub-TF………………………..….…...44 3.2 Generation of N- and C-terminally tagged preys…………………..…….…...45 3.3 Targeted screening for ALK interactors using MaMTH in media +/- tetracycline…………………....…………………………………………………47 3.4 MaMTH experiment in serum starvation media…………………….……….49 3.5 Targeted screening for ALK interactors using MaMTH………………..…...50 3.6 Targeted screening for SH2/PTB domain containing prey library using MaMTH………………………………………………………………………….53 3.7 Summary of preliminary targeted screening of ALK wild type using MaMTH………………………………………………………………………….56 3.8 Validation of novel ALK interactors using Co-IP……………………..……...58 3.9 Active and kinase-dead forms of ALK……………………..………..………...59 3.9.1 Generation of ALK active and kinase-dead mutants………………......…..59 vi 3.9.2 Expression and phosphorylation status of mutant ALK baits……………...60 3.9.3 Interaction of active and kinase-dead ALK mutants with ALK known interactors…………………………………………………………………...……61 3.9.4 Characterization of downstream signaling of ALK wild type and mutants using Western immunoblotting…...………………………………………..…….62 3.9.5 Characterization of tyrosine phosphorylation of ALK wild type and mutants in the presence of Brigatinib……………………………………..………………64 3.10 Investigating phosphorylation-dependent interactions of ALK wild type using MaMTH…………………………………………………………………..66 3.11 Assessment of cell viability in the presence of brigatinib……………........….67 3.12 Testing phosphorylation-dependent interactions of ALK wild type at different concentrations of brigatinib, using MaMTH……………………….68 3.13 Characterization of ALK bait expression and downstream signaling in the presence of Brigatinib……………………………………..……………………69 3.14 Testing phosphorylation-dependence of ALK wild type interactions at 25nM concentration of brigatinib……………………...……………………………...70 3.15 Functional characterization