DISTINCT FUNCTIONS FOR THE HIGHLY RELATED PP2A B55 REGULATORY SUBUNITS (Bα AND Bδ) IN CELL CYCLE REGULATION AND TUMOURIGENESIS by Dominik Sommerfeld B.Sc., The University of British Columbia, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2018 © Dominik Sommerfeld, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Distinct functions for the highly related PP2A B55 regulatory subunits (Bα and Bδ) in cell cycle regulation and tumourigenesis submitted by Dominik Sommerfeld in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Examining Committee: Catherine Pallen, Ph.D. Supervisor Christopher Maxwell, Ph.D. Supervisory Committee Member Gregg Morin, Ph.D. Supervisory Committee Member Stephan Taubert, Ph.D. University Examiner Calvin Roskelley, Ph.D. University Examiner Additional Supervisory Committee Members: Samuel Aparicio, Ph.D. Supervisory Committee Member ii Abstract Progression through the phases of the cell cycle (G1, S, G2, and mitosis) is largely driven by the coordinated activities of protein kinases and phosphatases, which orchestrate the phase-specific phosphorylation of proteins. Protein Phosphatase 2A (PP2A) – a heterotrimeric holoenzyme complex composed of a scaffold (A), catalytic (C), and variable regulatory (B) subunit – has emerged as an essential cell cycle regulator. Specifically, PP2A complexes containing the B55 family of regulatory subunits (PP2A-B55) have been implicated in the control of various cell cycle phases. My studies investigated isoform-specific roles of the two highly related and abundantly expressed B55 subunits, Bα and Bδ, in the regulation of G1, S, G2 phase and mitotic progression. Synchronization of HeLa cell populations at various stages of the cell cycle revealed that the Bα and Bδ subunits differentially regulate the kinetics of all cell cycle phases. Specifically, these highly related isoforms exert opposing effects on G1 phase progression, and Bα but not Bδ regulates progression through S phase. Furthermore, my studies demonstrate that the Bα and Bδ subunits play distinct roles in the control of mitotic exit progression, where Bδ regulates early and Bα mediates late mitotic exit events. PP2A-B55 complexes are believed to negatively regulate entry into mitosis by antagonizing CDK1 activation and CDK1-mediated mitotic substrate phosphorylation. While I found that PP2A- Bδ indeed antagonizes mitotic entry, my studies surprisingly revealed that PP2A-Bα promotes proper mitotic entry and progression. Specifically, loss of Bα resulted in the degradation of mitotic regulators, defective activation of CDK1 and other mitotic kinases, and the collapse of a pre-mitotic state during G2 phase. Deregulation of CDK1 activation is associated with mitotic defects, reduplication of the genome, and genome instability in cancer cells. Therefore, I investigated whether loss of Bα may iii precipitate genome instability. Depletion of Bα (but not Bδ) increased the incidence of cytokinesis failure and genome reduplication, and lead to polyploidization. PP2A activity is frequently attenuated in human tumours, with recent studies demonstrating loss of Bα subunit expression in various cancers. My findings suggest that Bα loss may support tumourigenesis by predisposing cells to polyploidization and genome instability. iv Lay Summary Cancer is the second leading cause of death worldwide. In Canada, nearly 1 in 2 people will be diagnosed during their lifetime and 1 in 4 will die from cancer. Cancer is a disease caused by the uncontrolled growth and division of cells. In normal conditions, cells divide in a highly controlled manner by following a coordinated, multistep process. A protein called PP2A plays an important role in controlling the cell division process at various steps, and its function and activity is often compromised in cancer. In this study, I investigated how PP2A regulates various stages of the cell division process, and how the loss of specific functions of PP2A may contribute to uncontrolled cell division and cancer. v Preface I performed all of the experiments and data analyses presented herein. Except for a few sets of cell lysates that were prepared by A. Beigi, I also processed all samples for analysis. Dr. Catherine Pallen and I designed the study and experiments. Parts of the work presented in Chapters 3 and 4 have been composed into a manuscript that will be submitted for publication in June 2018. HeLa cells that stably express various fluorescent protein-tagged markers were provided to us by Dr. Christopher Maxwell’s groups and were generated by the indicated labs and institutes (see section 2.1.1). Cell cycle regulation in higher eukaryotes has been studied using various model systems, most notably including Drosophila melanogaster (fruit flies), Xenopus laevis (African clawed frog), and human immortalized and cancer cell lines (especially the HeLa cell line). Core cell cycle regulatory mechanisms are shared between these model systems, as are the names of key regulatory proteins. Please note that throughout this thesis, when referring to human proteins only, I have written abbreviated protein names in all CAPS. When referring to non-human (or both human and non- human) proteins, only the first letter of the abbreviated protein name is capitalized. No ethics approval was required for the research presented herein. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables .............................................................................................................................. xiii List of Figures ............................................................................................................................. xiv List of Symbols .......................................................................................................................... xvii List of Abbreviations ............................................................................................................... xviii Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ................................................................................................................1 1.1 The cell division cycle .................................................................................................... 1 1.1.1 A general overview of the cell division cycle ............................................................. 1 1.1.2 Cell cycle progression is regulated by reversible protein phosphorylation and targeted proteolysis ................................................................................................................. 3 1.1.2.1 Cyclin-dependent kinases in the driver’s seat ..................................................... 4 1.1.2.2 Protein phosphatases: modulators of cell cycle phosphorylation ....................... 6 1.1.2.3 Cell cycle ubiquitin ligases and targeted proteolysis: insurance against reversibility ......................................................................................................................... 7 1.1.3 G1 phase progression: control of the RB/E2F pathway by CDK-cyclin and PP1/PP2A ................................................................................................................................ 9 vii 1.1.4 Control of S phase progression by CDK-cyclin and PP1/PP2A ............................... 11 1.1.5 The expression and accumulation of mitotic regulators during G2 phase is regulated by phosphorylation-dependent mechanisms ......................................................................... 13 1.1.6 Mitosis: key phosphorylation- and proteolysis-dependent signaling networks ........ 16 1.2 Protein Phosphatase 2A: structure and function ........................................................... 27 1.2.1 The PP2A holoenzyme: a structural and functional centipede ................................. 28 1.2.2 The regulation of PP2A holoenzyme assembly ........................................................ 35 1.2.3 PP2A-B55: functions in interphase and mitosis ....................................................... 38 1.3 Deregulation of PP2A in cancer ................................................................................... 42 1.3.1 Tumour-promoting toxins and viral oncoproteins target PP2A ................................ 42 1.3.2 Overexpression of endogenous cellular PP2A inhibitory proteins ........................... 43 1.3.3 Genetic