Identification of novel regulatory mechanisms for Cdc42 GTPase-activating protein CdGAP/ARHGAP31, a protein involved in development and cancer Ali Ben Djoudi Ouadda Department of Anatomy & Cell Biology McGill University, Montréal, Québec, Canada Submitted October, 2016 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Ali Ben Djoudi Ouadda, 2016 Acknowledgments I would like to express my deepest thanks and appreciation to my supervisor, Dr Nathalie Lamarche-Vane, who has opened the door of her laboratory and gave me the opportunity to pursue an excellent research training. Without her kindness, support, guidance and persistent help this thesis would not have been possible. I would like to thank my mentor Dr. Carlos Morales, who has supported, encouraged and guided me with valuable advice from the beginning. I would like also to thank my advisory committee members, Dr. Isabelle Rouiller and Dr. Peter Siegel for their encouragement and precious scientific inputs and feedbacks. I would like to acknowledge the Fonds de Recherche du Québec-Santé (FRSQ) which awarded me a Doctoral Training Scholarship and the McGill Faculty of Medicine/Department of Anatomy & Cell Biology which granted me a Doctoral Internal Scholarship, GREAT Travel and Merit Awards. In addition, a thank you to my colleagues in the Department of Anatomy & Cell Biology, RI-MUHC, IRCM and IRIC for their help and collaboration, either with reagents or scientific discussion and troubleshooting. Special thanks to Martin, Yi and Vilayphone for their precious help and support during my early days in the lab, and to Philippe, Sadig, Fereshteh, Hidetaka, Tristan, Jonathan and Judith for their help, kindness and availability. A special word of thanks goes to my mother Kaissa, and siblings Moussa, Djamila, Ouiza, Zohra, Saida, Djilali, Naima, Hassina and Massinissa for their indefectible support and encouragement. Without their love, patience and sacrifice, I could not have completed this work. I would specifically like to thank my late father Tayeb who assisted and encouraged me throughout my life, and I am sorry that he had not lived to see me complete my Ph.D. Finally, but most importantly, I wish to thank my wife Biba and children Ales and Aya for their love, support, assistance and faith in me. ii | P a g e Abstract The small Rho GTPase proteins act as molecular switches that regulate diverse cellular processes linked mostly to the actin-cytoskeleton remodeling making them essential regulators of cell adhesion, migration and invasion. Dysregulation of their activities can result in different abnormal phenotypes particularly, tumor progression and metastasis. Hence, regulators of Rho GTPases such as Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase-activating proteins (RhoGAPs), are critical for normal cellular responses and are targets for subversion during oncogenic transformation. CdGAP (Cdc42 GTPase-activating protein) is a member of a well-conserved subfamily of RhoGAP proteins and a negative regulator of the small Rho GTPases, Rac1 and Cdc42. Associated with a rare developmental disorder (AOS, Adams-Oliver Syndrome) and required for a normal angiogenesis, CdGAP plays important roles in the regulation of cell migration and proliferation during early development. In addition, recent findings characterize CdGAP as an essential synergistic component between TGFβ and HER2/Neu/ErbB-2 signaling pathways which play a positive role in cancer, particularly breast cancer. CdGAP is regulated by lipid binding, protein-protein interactions and phosphorylation, still these mechanisms are not well understood. In this work we first investigate the interaction between CdGAP and its negative regulator, the endocytic protein Intersectin. Using an in vitro approach, we identify a novel, atypical xKx(K/R) (SKSKK) motif in the basic rich (BR) region of CdGAP that directly interacts with the Intersectin-SH3D domain. Moreover, the well-conserved motif is required for the regulation of CdGAP activity following Intersectin binding. Next, we investigate CdGAP phosphorylation and identify two regulatory phospho-serines in the C-terminal (CT) tail, Ser-1093 and Ser-1163, that are phosphorylated by the AGC-kinase family member, RSK1. Finally, we show that 14-3-3 family members bind and regulate both the cellular localization and activity of CdGAP in a Ser-1093 and Ser-1163 phosphorylation-dependent manner. Overall, this work provides two novel CdGAP- regulatory mechanisms that can be applied in therapeutic approaches targeting this RhoGAP, particularly in breast cancers. iii | P a g e Résumé Les petites protéines G de la famille Rho sont des commutateurs moléculaires qui contrôlent divers procédés cellulaires associés notamment, à la régulation du cytosquelette et jouent par conséquent, un rôle clé dans la régulation de la motilité cellulaire. La dérégulation de leur activité peut entrainer des aberrations se manifestant en particulier, par une progression du cancer et des métastases. Ainsi, les protéines régulatrices comme les facteurs d'échange de nucléotide (RhoGEFs) et les protéines activatrices des Rho GTPases (RhoGAPs) sont essentielles pour une signalisation cellulaire normale et sont en général, affectées lors des transformations oncogéniques. CdGAP (Cdc42-GTPase activating protein) est un membre d’une sous-famille de protéines RhoGAPs bien conservée qui régule négativement les Rho GTPases Cdc42 et Rac1. Associé à un trouble du développement rare, le syndrome d'Adams-Oliver ou AOS et nécessaire pour une angiogenèse normale, CdGAP joue un rôle important dans la régulation de la migration et la prolifération cellulaires au cours du développement. Récemment, CdGAP est identifié comme une composante synergique essentielle entre les voies de signalisation de TGFβ et HER2/Neu/ErbB-2 et qui joue un rôle proto- oncogénique, en particulier dans le cancer du sein. CdGAP est régulée par des mécanismes incluant les lipides, les interactions protéine-protéine et la phosphorylation, néanmoins, ces mécanismes ne sont pas bien élucidés. Dans cette étude, nous étudions en premier l'interaction entre CdGAP et son régulateur négatif, la protéine endocytique, Intersectin. En utilisant une approche in vitro, nous identifions un nouveau motif atypique, xKx (K/R) (SKSKK) dans la région riche en résidus basiques (BR) de CdGAP interagissant directement avec le domaine SH3D d’Intersectin. Le motif est en outre, bien conservé est requis pour la régulation de l'activité CdGAP par Intersectin. Par la suite, nous identifions deux sites de phosphorylation clés dans la région C- terminale de CdGAP, Ser-1093 et Ser-1163 qui sont phosphorylés par la protéine AGC- kinase, RSK1. Nous démontrons finalement, que les protéines adaptatrices 14-3-3 lient et régulent la localisation cellulaire et l’activité de CdGAP d’une manière dépendante de la phosphorylation des résidus, Ser-1093 et Ser-1163. La présente étude identifie deux nouveaux mécanismes de régulation de CdGAP qui peuvent être exploités dans des approches thérapeutiques ciblant cette protéine, notamment dans les cancers du sein. iv | P a g e Table of Contents ACKNOWLEDGEMENTS……………………………………………………………………....ii ABSTRACT…………………………………………………………………….........................iii RÉSUMÉ………………………………………………………………………………………...iv LIST OF TABLES……………………………………………………………………..............viii LIST OF FIGURES…………………………………………………………………….............ix ABBREVIATIONS………………………………………………………………………..…......x CONTRIBUTION OF AUTHORS TO MANUSCRIPTS……………………………………xvi ORIGINAL CONTRIBUTIONS TO KNOWLEDGE…....................................................xviii SUBMITTED MANUSCRIPTS…………………………………………………………….…xix CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW…………………………….1 1.0 General introduction…………………………………………… ………………………….2 1.1 Overview of the small GTPases……………………………………….………………….2 1.1.1 Ras GTPase regulation………………………………………………………………….2 1.1.2 Roles of Ras GTPases……………………………………………………………….….4 1 .2 The Rho GTPase subfamily……………………………………………………………....7 1.2.1 Rho GTPase regulation ………………………………………………………………....7 1.2.1.1 Rho Guanine nucleotide Exchange Factors (RhoGEFs)…………………………..8 1.2.1.2 Rho Guanine nucleotide Dissociation Inhibitors (RhoGDIs)……….………………9 1.2.1.3 Posttranslational regulation……………………………………………………….…..9 1.2.2 Role of Rho GTPases in cancer………………………….……………………………..9 1.3 Rho GTPase-Activating Proteins (RhoGAPs)………………………………………….11 1.3.1 RhoGAP regulation by phosphorylation………………………………………………11 1.3.2 RhoGAP membrane localization………………………………………………………11 1.3.3 RhoGAP role in cancer…………………………………………………………………12 1.4 RhoGAP protein subfamilies……………………………………………………………..13 1.4.1 PH-domain containing RhoGAPs……………………………………………………..18 1.4.2 BAR/FCH-domain containing RhoGAPs……………………………………………..24 1.4.3 C1-domain containing RhoGAPs……………………………………………………...26 1.4.4 SEC14-domain containing RhoGAPs…………………………………………………30 v | P a g e 1.4.5 START-domain containing RhoGAPs………………………………………...………31 1.4.6 Multiple LBD-containing RhoGAPs……………………………………………………33 1.4.7 PBR-containing RhoGAPs.....................................................................................36 1.5 CdGAP regulation and function………………………………………………………….47 1.5.1 Domain organization……………………………………………………………………47 1.5.2 CdGAP regulation…………………………………………………….…………………48 1.5.2.1 Protein-protein interactions…………………………………………………………..48 1.5.2.2 Phosphorylation……………………………………………………………………….48 1.5.2.3 Lipid binding…………………………………………………………………………...49 1.5.3 Cellular functions of CdGAP…………………………………………………………...50