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Proteomic Analysis of Pp2a Mutants and Pp2a-Related Tumor Virus Proteins

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Proteomic Analysis of Pp2a Mutants and Pp2a-Related Tumor Virus Proteins PROTEOMIC ANALYSIS OF PP2A MUTANTS AND PP2A-RELATED TUMOR VIRUS PROTEINS by Yiwang Zhou A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Yiwang Zhou 2015 PROTEOMIC ANALYSIS OF PP2A MUTANTS AND PP2A-RELATED TUMOR VIRUS PROTEINS Yiwang Zhou Master of Science Graduate Department of Molecular Genetics University of Toronto 2015 ABSTRACT Protein phosphatase 2A (PP2A) is a tumor suppressor, whose function and activity is influenced by mutations of PP2A subunits and PP2A-related tumor virus proteins. In this work, protein-protein interaction changes in the normal PP2A interaction network caused by mutations of PP2A scaffolding A (PPP2R1A) and catalytic C (PPP2CA) subunits have been characterized by AP-SWATH. The results show that the normal function and activity of PP2A holoenzyme may be significantly altered and influenced by these mutations. This study also characterizes the interactomes of several PP2A-related tumor viral oncoproteins, including E4orf4. It is revealed for the first time that ASPP-PP1 complex subunits are among the major interactors of E4orf4, suggesting the involvement of E4orf4 in the regulation of Hippo signaling pathway. The results gained in my M.Sc. work provide a deeper understanding of the consequences of PP2A subunits mutations and the PP2A-dependent and PP2A-independent functions of the PP2A-related tumor virus proteins. ! ii! ACKNOWLEDGEMENT First and foremost, I would like to thank my supervisor, Dr. Anne-Claude Gingras for taking me as a master student, and for all the time and patience spent in providing me guidance throughout this study. Before joining Dr. Gingras' lab, I had no idea what real scientific research is and how to carry out an independent research project. These two years' experience develops my ability to work and think like a researcher, which is extremely worthwhile for my future career. I would also like to express my appreciation to the members of Dr. Gingras' lab: Jean-Philippe Lambert for answering my numerous questions and for helping me analyze the SWATH data; Zhen-yuan Lin for teaching me affinity purification and for always being there when I had trouble dealing with the mass spectrometers; James Knight for helping me with data processing and visualization; Frank Liu for solving all the problems I had in ProHits; Wade Dunham and Marilyn Goudreault for helping me find everything I needed in the lab; and the rest of the lab members for assistance through the years. For smoothly carrying out this study, I also want to thank my committee members, Dr. Philip Kim, Dr. Stéphane Angers and Dr. Vuk Stambolic for their guidance, as well as Dr. Philip Branton and Dr. Egon Ogris for their collaboration. In addition, I am indebted to my parents, who are always standing by me and offering me support and encouragement to pursue my dream. I am also grateful to have Huayun Hou as my roommate. Thanks for accompanying for these two years. Finally and most especially, I would thank to my boyfriend, Jiyuan Yang, for his understanding, support, patience and love during the past two years, no matter how far away we are separated. ! iii! TABLE OF CONTENTS ABSTRACT...................................................................................................................ii ACKNOWLEDGEMENT............................................................................................iii LIST OF TABLES........................................................................................................vi LIST OF FIGURES.....................................................................................................vii LIST OF ABBREVIATIONS.......................................................................................ix CHAPTER 1 INTRODUCTION 1.1 PP2A holoenzyme.............................................................................................1 1.2 The function of PP2A as a tumor suppressor....................................................6 1.3 Structure-function of the PP2A subunits under study 1.3.1 Scaffolding A subunit, PPP2R1A...........................................................8 1.3.2 Catalytic C subunit, PPP2CA..................................................................9 1.3.3 Regulatory B subunit, PPP2R2A..........................................................10 1.4 PP2A-related tumor virus proteins 1.4.1 SV40 small T antigen............................................................................11 1.4.2 Polyomavirus middle T antigen............................................................12 1.4.3 Adenovirus E4orf4 protein....................................................................13 1.5 Affinity purification and mass spectrometry...................................................14 1.6 Proximity-dependent biotin identification with BioID...................................19 1.7 Quantitative mass spectrometry with the Data Independent Acquisition method SWATH..............................................................................................22 1.8 Objectives of this project.................................................................................24 CHAPTER 2 MATERIALS AND METHODS 2.1 Plasmids..........................................................................................................26 2.2 Cell lines, culture, transfection and collection................................................29 2.3 Affinity purification 2.3.1 Anti-FLAG affinity purification............................................................30 2.3.2 Streptavidin affinity purification...........................................................32 ! iv! 2.4 Immunoprecipitation-Western Blot.................................................................33 2.5 Mass spectrometry 2.5.1 MS/MS..................................................................................................34 2.5.2 SWATH.................................................................................................35 2.6 Data analysis and visualization 2.6.1 DDA data search...................................................................................36 2.6.2 SAINT analysis.....................................................................................37 2.6.3 SWATH data analysis...........................................................................38 CHAPTER 3 RESULTS 3.1 Protein-protein interaction changes imparted by PPP2R1A mutations...........42 3.2 Validation of selected interaction changes for PPP2R1A mutations..............48 3.3 Protein-protein interaction changes imparted by PPP2CA mutations.............51 3.4 Validation of the interaction between ANKLE2 and PPP2CA mutants.........59 3.5 Interacting proteins of PPP2R2A....................................................................62 3.6 Interacting proteins of PP2A-related tumor virus proteins 3.6.1 SV40 small T antigen interacting proteins............................................67 3.6.2 Polyomavirus middle T antigen interacting proteins............................72 3.6.3 E4orf4 interacting proteins....................................................................72 CHAPTER 4 FUTURE DIRECTIONS AND DISCUSSION 4.1 Significance of the work.................................................................................82 4.2 Discussion.......................................................................................................83 4.3 Future directions..............................................................................................90 APPENDIX..................................................................................................................92 REFERENCES...........................................................................................................102 ! v! LIST OF TABLES Table 1. PP2A subunits..................................................................................................5 Table 2. Plasmids used in this study............................................................................27 Table 3. Mutations of PPP2R1A..................................................................................43 Table 4. Mutations of PPP2CA....................................................................................52 Table 5. Schematic representation of the various C subunit mutations.......................52 Table 6. Mutations of PPP2R2A..................................................................................63 Table 7. Mutations of E4orf4.......................................................................................75 ! vi! LIST OF FIGURES Figure 1. Crystal structure of PP2A and the formation of PP2A holoenzyme...............3 Figure 2. Workflow of affinity purification coupled with mass spectrometry (AP-MS).......................................................................................................................16 Figure 3. Model for application of BioID method.......................................................20 Figure 4. AP-SWATH data analysis pipeline..............................................................39 Figure 5. Dot-plot representation of protein-protein interaction changes for eight PPP2R1A mutations relative to wild-type PPP2R1A..................................................45 Figure 6. Validation of selected protein-protein interaction changes imparted by mutations of PPP2R1A.................................................................................................49
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