Patched-1 Intracellular Domains Interact with E3 Ubiquitin Ligases Smurf2 and Itch by Aaliya Tamachi A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine & Pathobiology University of Toronto © Copyright by Aaliya Tamachi 2015 Patched-1 Intracellular Domains Interact with E3 Ubiquitin Ligases Smurf2 and Itch Aaliya Tamachi Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto 2015 Abstract Patched-1 (Ptch1) is the primary receptor of the Hedgehog signalling pathway and mediates both canonical and non-canonical signalling. Previously our lab identified a group of proteins that interact with the C-terminus of murine Ptch1 that possessed SH3- and WW- binding domains, chief among them being C2-WW-HECT E3 ubiquitin ligases. Using a molecular-based approach, we showed that E3 ligases Smurf2 and Itch bind through sequences within the large intracellular middle loop and C-terminus of murine Ptch1 (mPtch1). We further demonstrated that mPtch1 is ubiquitinated in the presence of Smurf2 and Itch and this requires the middle loop and C-terminus. We also showed that mPtch1 can oligomerize and that the stability of mPtch1 is affected when the middle loop and C-terminus are deleted. Together, these data reveal a role for E3 ubiquitin ligases in mediating protein turnover of mPtch1 and show that the intracellular domains of mPtch1 interact with distinct protein factors. ii Acknowledgements I would like to start by thanking my supervisor, Prof. Paul A. Hamel, who gave me the opportunity to pursue graduate studies in his lab, as well as all of the support, patience and guidance he has given me over the years I have been here. I would also like to give my gratitude to Prof. Liliana Attisano and Prof. Michael Ohh for being part of my advisory committee and for their invaluable insights, beneficial suggestions and time taken to assist me in this project. In addition, I would like to thank Dr. Harry Elsholtz for sitting in as my committee chair and participating in my thesis defence, as well as for his friendly support over my graduate career. I would also like to thank the members of the Hamel lab: Andrew Fleet, Jennifer Lee and my 'M-Dawg' Melissa Hicuburundi, as well as former lab members Dr. Hong Chang, Dr. Laurent Balenci, Malcolm Harvey and my 'lab sister' Nadia Okolowsky. I cannot thank each of them enough for their assistance in completing my project. Their wonderful friendships and kind words of support and encouragement will always stay with me wherever I go in my life. Last, but certainly not least, I would like to thank my family who has always been there for all the ups and downs. Mum, Dad, Farah and Alikhan – I am forever indebted to all of you for your endless love and support. iii Table of Contents ABSTRACT...............................................................................................................................ii ACKNOWLEDGEMENTS......................................................................................................iii TABLE OF CONTENTS..........................................................................................................iv LIST OF FIGURES & TABLES..............................................................................................vi ABBREVIATION KEY..........................................................................................................viii INTRODUCTION.....................................................................................................................1 1 SIGNIFICANCE OF HEDGEHOG SIGNALLING..................................................1 2 CANONICAL HEDGEHOG SIGNALLING.............................................................2 3 PTCH STRUCTURE AND FUNCTION...................................................................5 3.1 Molecular Characterization..........................................................................5 3.2 Ptch1 Turnover.............................................................................................9 4 UBIQUITINATION..................................................................................................11 4.1 Process of Ubiquitination...........................................................................11 4.2 E3 Ligases: An Important Enzyme in Ubiquitin Conjugation...................13 4.3 Types of Ubiquitination.............................................................................15 RATIONALE...........................................................................................................................20 HYPOTHESIS & OBJECTIVES............................................................................................21 MATERIALS & METHODS...................................................................................................22 Cell Culture..................................................................................................................22 Cloning & Expression Constructs................................................................................22 Western Blotting & Immunoprecipitation....................................................................23 In Vitro Ubiquitination Assay.......................................................................................25 Protein Stability Assay.................................................................................................25 RESULTS.................................................................................................................................26 High-throughput LUMIER screen identifies murine Ptch1 C-Terminus binding protein factors..............................................................................................................26 E3 ubiquitin ligases bind to mPtch1 via two independent binding sites......................27 Smurf2 and Itch ubiquitin ligase expression correlate with mPtch1 ubiquitination...............................................................................................................34 iv mPtch1 can oligomerize in vitro..................................................................................40 Full-length mPtch1 is less stable than ΔML and ΔMLΔC mutants.............................42 DISCUSSION..........................................................................................................................44 Murine Ptch1 interacts with both Smurf2 and Itch......................................................44 mPtch1 is ubiquitinated in presence of Smurf2 and Itch expression...........................49 Reciprocal α-ubiquitin IP leads to investigation of mPtch1 oligomerization..............56 Deletion of domains alters stability of mPtch1............................................................58 CONCLUSIONS.....................................................................................................................61 REFERENCES........................................................................................................................63 v List of Figures & Tables FIGURE 1. CANONICAL Hh SIGNALLING PATHWAY.......................................................3 FIGURE 2. 3D SCHEMATIC OF WILD TYPE mPTCH1.......................................................6 FIGURE 3. LUMIER SCREEN USING mPTCH1 C-TERMINUS IDENTIFIES NOVEL PROTEIN INTERACTING FACTORS..................................................................................27 FIGURE 4. CONSTRUCT MAPS OF WILD TYPE mPTCH1 AND ITS MUTANT DERIVATIVES........................................................................................................................28 FIGURE 5. POLY-PROLINE SEQUENCES IN THE INTRACELLUAR MIDDLE LOOP AND C-TERMINUS OF PTCH1 ARE CONSERVED ACROSS VARIOUS SPECIES..................................................................................................................................29 FIGURE 6. FULL-LENGTH FLAG-TAGGED SMURF2 BINDS TO mPTCH1 VIA SEQUENCE IN THE INTRACELLULAR MIDDLE LOOP AND THE C-TERMINUS.....31 FIGURE 7. FULL-LENGTH ITCH ALSO BINDS TO mPTCH1 VIA SEQUENCE IN THE INTRACELLULAR MIDDLE LOOP AND THE C-TERMINUS.........................................33 FIGURE 8. ENDOGENOUS PTCH1 CAN BIND TO ENDOGENOUS SMURF2 IN HEK- 293 CELLS..............................................................................................................................34 FIGURE 9. mPTCH1 IS UBIQUITINATED IN HEK-293 CELLS WITHOUT OVEREXPRESSION OF E3 LIGASES.................................................................................36 FIGURE 10. mPTCH1 IS UBIQUITINATED IN THE PRESENCE OF SMURF2 EXPRESSION.........................................................................................................................37 FIGURE 11: mPTCH1 IS UBIQUITINATED IN THE PRESENCE OF ITCH EXPRESSION.........................................................................................................................39 FIGURE 12: mPTCH1 CAN FORM HOLO-OLIGOMERS IN VITRO.................................41 FIGURE 13: FULL-LENGTH mPTCH1 IS LESS STABLE THAN ΔML AND ΔMLΔC mPTCH1 MUTANTS..............................................................................................................43 TABLE 1: COMPILATION OF BINDING AND UBIQUITINATION RESULTS FOR EXPERIMENTS FEATURING CO-EXPRESSION OF SMURF2 OR ITCH.......................50 vi FIGURE 14: AMINO ACID SEQUENCE OF THE INTRACELLULAR MIDDLE LOOP OF mPTCH1 AND MUTANT MIDDLE LOOP CONSTRUCTS..........................................51