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Characterization of Novel Substrates of the Tankyrase and RNF146 Destruction Complex and Mechanisms of Its Regulation

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Characterization of Novel Substrates of the Tankyrase and RNF146 Destruction Complex and Mechanisms of Its Regulation Characterization of Novel Substrates of the Tankyrase and RNF146 Destruction Complex and Mechanisms of its Regulation by Arun Chandrakumar A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Medical Biophysics University of Toronto © Copyright by Arun Chandrakumar 2020 Characterization of Novel Substrates of the Tankyrase and RNF146 Destruction Complex and Mechanisms of its Regulation Arun Chandrakumar Doctor of Philosophy Medical Biophysics University of Toronto 2020 Abstract Poly-ADP-ribose is a post-translational modification that was first described over 50 years ago as a polymer derived from nicotinamide adenine dinucleotide or NAD. Since then, a family of 17 enzymes responsible for generating Poly-ADP-ribosylation or PARylation post translational modification has been identified and characterized. A unique member of this family of enzymes, called Poly-ADP-ribose Polymerases (PARPs), are Tankyrases. There are two mammalian Tankyrases, Tankyrase 1 and 2, whose domain organization include ankyrin repeat clusters which mediate enzyme-substrate interactions and a SAM domain which regulates oligomerization of Tankyrase into large macromolecular complexes. Tankyrases bind to proteins through a ‘RxxxxG’ peptide motif which facilities these proteins to undergo PARylation. A subset of Tankyrase substrates are recognized by an E3 ligase, RNF146, which facilities protein degradation through PARylation dependent ubiquitylation. The studies summarized in this thesis have focused on the identification of new proteins targets involved in the Tankyrase:RNF146 degradation pathway and investigates how Tankyrase PARylation is regulated through FIH dependent hydroxylation. I have identified SH3BP5 and SH3BP5L as new Tankyrase substrates that are targets of RNF146. I have shown that both substrates are guanine exchange factors for the small GTPase Rab11a. I have defined the minimal catalytic core of SH3BP5/L and shown it to be composed of a novel two α-helix GEF domain. My work has demonstrated that SH3BP5 and SH3BP5L are both required for optimal activation of Rab11a in epithelial cells during lumenogenesis and that their activities are repressed by Tankyrase-mediated PARylation. RNF146 regulates Rab11a ii activity by controlling Tankyrase protein abundance, marking the first time that RNF146 has been described as antagonistic towards Tankyrase function. I have demonstrated that FIH-mediated hydroxylation of Tankyrase affects the ability of substrates to bind to the ankyrin repeat clusters and inhibits autoPARylation and substrate PARylation yet does not affect protein degradation. Since PARylation dependant degradation is unaffected, it suggests that hydroxylation could affect the degradation independent functions of Tankyrase. iii Acknowledgments Foremost, I am extremely grateful for the opportunity given to me to pursue graduate work and realize my dream of becoming a scientist by my supervisor Dr. Robert Rottapel. Thank you for taking a chance on a 21-year-old who did not have a clue what he wanted to do in life but knew he enjoyed lab research. Thank you for your constant encouragement through this up and down journey and always being available whenever I really needed your help. I will always appreciate the support you have given me and I’m sure you will continue to provide as I go into the next phase of my career. Many thanks to my committee members, Dr. Raught and Dr. Wouters, for their helpful feedback and encouragement during my graduate work. Secondly, I have been so fortunate to have amazing, helpful, hilarious, and enjoyable lab colleagues. There have been so many people that have come and gone through the lab over the years and someway every individual has had an impact on me that has made working in the Rottapel lab an enjoyable experience. Thank you to my collaborators who have contributed critical pieces of data that made completion of this thesis possible. Dr. Etienne Coyaud, you were such a pleasure to work with and cannot thank you enough for your help and advice and willingness to let me bother you so many times. Dr. Chris Marshall, I learned so much from you over the past couple years and it has been truly a pleasure to work with you. Can’t thank you enough for all the time you spent collecting data, brainstorming and working through road-bumps. Also, a big thank you to Dr. Sebastian Guettler and Dr. David Bryant for their advice and providing valuable reagents. A big thank you to my family and friends for your support and encouragement for the last seven years as I navigated through this challenging yet satisfying journey. Lastly, huge thanks to the Toronto Raptors for putting the cherry on top of 2019, one of the best years of my life. WHAT IT DOOOO BAYYBEEEE!!!!!! iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations ................................................................................................................... xiv Chapter 1 ..........................................................................................................................................1 Introduction .................................................................................................................................1 1.1 Post translational modification ............................................................................................1 1.2 Poly-ADP-Ribose ................................................................................................................1 1.3 Poly-ADP-Ribose Polymerases (PARPs) ............................................................................2 1.3.1 Revised classification system based on the PARP domain .....................................2 1.3.2 PARP biology defined by PAR binding domains ....................................................3 1.4 Tankyrases ...........................................................................................................................4 1.4.1 Tankyrase family .....................................................................................................4 1.4.2 Tankyrases bind to substrates through a specific amino-acid motif ........................5 1.4.3 Tankyrases oligomerize and form higher order assemblies .....................................8 1.4.4 Role of Tankyrases at human telomeres ..................................................................8 1.4.5 Tankyrase in mitosis ................................................................................................9 1.4.6 Tankyrase controls glucose metabolism ..................................................................9 1.4.7 Role of Tankyrase in WNT signaling and cancer ..................................................10 1.4.8 Tankyrase promotes tumour growth independently of WNT signaling ................10 1.4.9 Tankyrase regulates bone metabolism and dysregulation of 3BP2 is the underlying cause of Cherubism .............................................................................11 1.4.10 Tankyrase promotes YAP signaling and cell polarity ...........................................11 1.5 Regulation of Tankyrase ....................................................................................................12 v 1.5.1 Factors that regulate catalytic activity ...................................................................12 1.5.2 Protein-protein interactions regulate Tankyrase stability ......................................12 1.5.3 Tankyrase stability is regulated by PAR dependent Ubiquitylation (PARdU) ......13 1.6 RNF146 ..............................................................................................................................14 1.6.1 RNF146 regulates DNA damage and PAR-mediated cell death ...........................15 1.6.2 RNF146 regulates bone metabolism ......................................................................15 1.7 SH3BP5..............................................................................................................................16 1.7.1 Role of SH3BP5 in stress kinase signaling ............................................................16 1.7.2 SH3BP5 is a guanine nucleotide exchange factor for Rab11 ................................18 1.7.3 SH3BP5 in cancer ..................................................................................................20 1.8 Rab11 GTPases ..................................................................................................................20 1.8.1 Rab11 family ..........................................................................................................20 1.8.2 Rab11 function at recycling endosomes ................................................................21 1.8.3 Rab11 in epithelial polarity ....................................................................................21 1.8.4 Rab11 in disease ....................................................................................................22
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