Plk4 Regulates Cell Motility through Arhgef1 and Rho GTPase Activation by Olga Brashavitskaya A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Olga Brashavitskaya 2019 Plk4 Regulates Cell Motility through Arhgef1 and Rho GTPase Activation Olga Brashavitskaya Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto 2019 Abstract Cell migration is a fundamental process essential for embryogenesis, immune cell function and maintenance of homeostasis. It is driven primarily by polymerization of the actin cytoskeleton, which is regulated by a family of small Rho GTPases. Rho GTPases Rac1 and Cdc42 are known as the major regulators of actin polymerization and RhoA is mostly thought of as the regulator of actin stress fiber formation and actomyosin contraction. Rho GTPases are activated by GEFs and deactivated by GAPs. Polo-like kinase 4 (Plk4) is well-acknowledged as the master regulator of centriole duplication. Nevertheless, our laboratory has shown that Plk4 activates RhoA during cytokinesis to ensure proper cleavage furrow positioning. We also identified a novel role for Plk4 in regulation of spreading, migration and invasion of normal and cancer cells. However, the mechanisms through which Plk4 regulates these processes are not completely understood. I hypothesized that Plk4 regulates the activation of RhoA and possibly other Rho GTPases to indice cell migration and spreading. In support of this hypothesis, I show here that Plk4 acts as an activator of Rho GTPases RhoA and Rac1 in cycling cells. I also identify 12 GEFs that possess Plk4 consensus phosphorylation motif and show that Plk4 physically interacts with the GEFs Arhgef1 and P-Rex2. Plk4 functionally interacted with Arhgef1 to affect cell migration, showing at least partial dependence of Plk4 migration phenotype on Arhgef1. Further delineation of their interaction showed that Plk4 binds the DH/PH domains and phosphorylates the L- ii DH/PH fragment of Arhgef1, and that Plk4 enhances Arhgef1 GEF activity. Finally, I show that Plk4 is not likely to activate Arhgef1-RhoA pathway to enhance cell spreading but may act through P-Rex2-Rac1. These results identify a novel mechanism through which Plk4 regulates cell migration. Plk4 has emerged as a potential oncogene, promoter of tumour progression and metastasis formation, prompting the creation of Plk4 inhibitors for therapeutic purposes. The data presented here deepen our understanding of the pathways that may be affected by the Plk4- targeted therapy. iii Acknowledgments I would like to thank my supervisor Dr. Carol Swallow, for her continuous support, guidance, patience and for taking a chance on me when I first applied to the lab as a fourth-year undergraduate student. Carol, your high expectations and encouragement have made me strive for and achieve my best in the lab and in life. Your kindness, sense of humour and compassion has created a truly wonderful and supportive environment in the lab. Throughout my future career I will aim to achieve the highest level of professionalism, integrity and scientific curiosity that you have always demonstrated. I will measure my work by your standards and always ask myself: “Would Carol approve?” before finalizing a draft. I am honoured to have you as my role model and you are the best mentor that anyone could ask for. A sincere thank you for guiding me, believing in me and supporting me all these years. A special thank you to Dr. Jim Dennis for thought-provoking scientific discussions and providing direction and support throughout my studies. Your love for science is truly inspiring and admirable. I would also like to extend my sincere gratitude to the members of my advisory committee, Dr. Katalin Szaszi and Dr. Jim Rutka. Thank you for always finding the time in your busy schedules to give me guidance, timely and valuable advice, continued encouragement and support. Thank you to Dr. Szaszi and her lab for sharing your technical expertise that has been invaluable in my studies. My experience in the lab would have never been the same, without the wonderful past and present members of the Swallow and Dennis labs, who have become my teachers and friends. In particular, I would like to thank Dr. Rosario for teaching me and supporting my scientific endeavours from the very beginning. Thank you to Dr. Karineh Kazazian, Dr. Wendy Johnston, Dr. Yosr Haffani, Dr. Anita Johswich, Dr. Ryan Williams, Dr. Francis Zih, Dr. David Berger- Richardson, Karina Pacholczyk, Judy Pawling, Aldis Krizus, Dr. Shelly Luu, Roland Xu, Michelle Lee and Alexandra Chirila, your support and encouragement mean the world to me. I would like to express my profound gratitude to my family, who have always been there for me, without you none of this would ever be possible. Mama and papa thank you for sacrificing so much to give me the best opportunities in life and for supporting me in all my decisions. Lena, you are more than my big sister, you are my best friend for life. Through thin and thick, I know you’ll always have my back and it gives me the strength and courage to shoot for the stars. A huge thank you to my boyfriend, Alex (a.k.a. Dr. Falkenhagen) for your care, understanding, humour and unconditional love and support. I have this degree to thank for meeting you and you to thank for getting to the end of this degree. I could not have dreamed of a better person to share this journey with, I love you. Finally, I would like to thank my friends, especially Polina, Maria and Sasha who kept me sane throughout the years and brought the life into my life/study/work balance. iv Contributions The author performed all experiments described in this thesis with the following contributions: Dr. Karineh Kazazian: made the shPlk4, shLuciferase and U2OS T-REx YFP-Plk4 stable cell lines used in the experiments in this thesis; made the Plk4 truncation fragments used in the experiments described in figure 4.7 and 4.8a. Created figure 6.1 from the author’s data. Qinghong Dan (under supervision of Dr. Katalin Szaszi): performed the active Arhgef1 pulldowns described in figure 4.9. v Table of Contents Table of Contents Acknowledgments.......................................................................................................................... iv Contributions....................................................................................................................................v Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations ................................................................................................................... xiv Chapter 1 ..........................................................................................................................................1 Introduction .................................................................................................................................1 1.1 Family of Polo-like kinases .................................................................................................1 1.1.1 Plk1 ..........................................................................................................................3 1.1.2 Plk2, 3 and 5 ............................................................................................................4 1.1.3 Plk4 regulation and structure ...................................................................................5 1.1.4 Plk4 in cell cycle ......................................................................................................8 1.1.5 Plk4 in centrosome duplication ................................................................................8 1.1.6 Plk4 in cell motility, invasion and spreading .........................................................12 1.1.7 Plk4 in carcinogenesis and cancer progression ......................................................13 1.2 Cell Migration ....................................................................................................................21 1.2.1 Individual cell migration ........................................................................................22 1.2.2 Collective cell migration ........................................................................................32 1.3 Rho GTPases ......................................................................................................................35 1.3.1 Rho GTPase structure and guanine nucleotide switch regulation .........................38 1.3.2 Rho GTPase signaling in cell motility ...................................................................40 1.3.3 Rho GTPases in cell spreading and cell shape.......................................................50 vi 1.3.4 Rho GTPases in cancer ..........................................................................................54 1.4 GEFs for Rho GTPases ......................................................................................................57 1.4.1 GEFs structure .......................................................................................................57