REGULATION OF CANONICAL WNT SIGNALING BY UBIQUITYLATION By Alison Jean Hanson Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology May, 2012 Nashville, Tennessee Approved: Susan R. Wente Robert J. Coffey Jennifer A. Pietenpol Laura A. Lee Ethan Lee For my family ii ACKNOWLEDGEMENTS I would first like to thank my mentor, Dr. Ethan Lee, for his unparalleled enthusiasm and optimism. There is not a piece of data that he doesn’t love and while this can be confusing at first, one comes to understand its importance as myriad failures mount and he is still exclaiming, “It worked!” Few scientists have as much passion and as many ideas as Ethan. I can only hope I have absorbed a fraction of these qualities as I move forward. I would also like to thank Ethan for allowing me to be a very independent investigator, which at times was frustrating (for both of us I’m sure!), but was ultimately the best training I could have received. I would also like to thank Dr. Laurie Lee for her attention to detail and uncanny ability to focus against all odds. Laurie and Ethan have put their heart and souls into fostering a fun, creative, and welcoming lab environment for which I am eternally grateful. Without their co-mentorship this work would not have been possible. I would also like to thank all members of the Lee labs past and present for helping to create an once-in-a-lifetime work environment. I am positive I will never experience anything like it again. I would like to specifically thank the first generation of Ethan Lee lab members Curtis Thorne, Chris Cselenyi, and Kristin Jernigan who took me under their scientific wings and taught me how to perform Western blots and discussed the meaning of life and science with equal gusto. I most definitely would not be the scientist I am today without their early guidance and inspiration. I would also like to thank Emilios iii Tahinci who bestowed his knowledge of Xenopus embryology upon me more times than I’d like to admit. I am grateful to each one of my thesis committee members: Dr. Robert Coffey, Dr. Susan Wente, Dr. Jennifer Pietenpol, and Dr. Laurie Lee for somehow managing to find the time to provide excellent guidance and support during my graduate school training. Dr. William Tansey and Dr. Vivian Siegel generously read my manuscript and provided very valuable feedback for which I am extremely grateful. I would not be here today without the support of Dr. Terry Dermody and the rest of the Vanderbilt MSTP leadership team who have facilitated all aspects of my education and training. Elaine Caine and Kim Kane and the rest of the administrative support team of the Cell and Developmental Biology department have similarly performed miracles numerous times in order to get me to this point. Thank you all. Financially, this work was supported by NIH grants T32 GM07347 (Medical Scientist Training Program) and a NRSA F30 fellowship from the NIEHS. Lastly, I would like to thank my family for their encouragement and support and for instilling in me a work ethic and drive without which this entire endeavor would not be possible. iv TABLE OF CONTENTS Page DEDICATION ........................................................................................................ ii ACKNOWLEDGEMENTS .................................................................................... iii LIST OF TABLES .............................................................................................. viii LIST OF FIGURES ............................................................................................ ..ix LIST OF ABBREVIATIONS ................................................................................ xi Chapter I. INTRODUCTION TO WNT SIGNALING AND THE UBIQUITIN SYSTEM .......... 1 Introduction ................................................................................................... 1 Signal Transduction: How Cells Communicate ........................................... 2 Historical Perspective: Wnt Signaling ............................................................ 5 Current Model of Wnt Signaling ..................................................................... 8 Historical Perspective: The Ubiquitin System .............................................. 17 Current Model of The Ubiquitin System ....................................................... 19 Regulation of Wnt Signaling by The Ubiquitin System ................................. 23 II. MATERIAL AND METHODS ............................................................................... 28 Drosophila dsRNA Generation and S2 Cell RNAi Screen ............................ 28 Plasmids and Purified Proteins .................................................................... 29 Cell Lines and Transfections ........................................................................ 30 Reporter Assays ........................................................................................... 31 Ubiquitylation Assays ................................................................................... 32 Gel Filtration ................................................................................................. 32 Antibodies ..................................................................................................... 33 Immunoblots, Immunoprecipitations, and GST Pull-Downs ......................... 33 Xenopus laevis Studies ................................................................................ 34 Immunofluorescence .................................................................................... 35 Real-Time RT-PCR ...................................................................................... 35 III. RNAi SCREEN TO IDENTIFY NOVEL E3 UBIQUITIN LIGASES AND DE-UBIQUITYLASES INVOLVED IN WNT SIGNALING ................................. 37 Introduction .................................................................................................. 37 v Results ......................................................................................................... 39 Results of the Drosophila E3 ligase RNAi screen .................................. 39 Results of the Drosophila DUB RNAi screen ......................................... 50 Discussion .................................................................................................... 53 IV. XIAP MONO-UBIQUITYLATES GROUCHO/TLE TO PROMOTE CANONICAL WNT SIGNALING ....................................................................... 56 Introduction ................................................................................................... 56 Results ......................................................................................................... 58 Drosophila RNAi screen identifies DIAP1 as a critical component of the Wingless signaling pathway ............................................................. 58 XIAP is required for Wnt signaling in cultured mammalian cells ............ 61 XIAP is required for Wnt signaling in Xenopus embryos ....................... 67 XIAP binds and ubiquitylates Groucho/TLE ........................................... 70 Ubiquitylation of Groucho/TLE does not affect its turnover, localization, or capacity to tetramerize ....................................................................... 81 Ubiquitylation of Groucho/TLE by XIAP disrupts its binding to TCF/Lef .............................................................................................. 87 Summary ...................................................................................................... 92 V. THE DUB USP47 IS REQUIRED FOR WNT SIGNALING ................................ 94 Introduction ................................................................................................... 94 Results ......................................................................................................... 95 Drosophila RNAi screen identifies the DUB Ubp64E as a novel Wingless signaling component .............................................................. 95 USP47 is required for Wnt signaling in cultured mammalian cells ......... 100 USP47 is localized in the cytoplasm ...................................................... 101 USP47 is expressed throughout Xenopus development ....................... 103 USP47 affects primary body axis formation in Xenopus embryos ......... 105 USP47 interacts with β-TRCP and Smad4, but does not affect their stability ................................................................................................... 108 USP47 interacts with XIAP, but does not affect TLE3 or XIAP stability ...................................................................................... 113 Summary ...................................................................................................... 115 VI. DISCUSSION AND FUTURE DIRECTIONS .................................................... 117 Introduction ................................................................................................... 117 Part I ............................................................................................................. 117 Discussion ................................................................................................ 117 Future Directions ...................................................................................... 123 Is XIAP required for Wnt signaling in the absence of Groucho/TLE? .................................................................................. 124 What are the sites of XIAP-mediated ubiquitylation on Groucho/TLE? ............................................................................. 124 vi What are the concentrations of β-catenin and Groucho/TLE in the nucleus? ........................................................................................... 126 How and when is XIAP recruited to the TCF/Lef transcriptional complex? ................................................................... 127 Does loss of XIAP inhibit tumor formation? ...................................... 128 What is the DUB that opposes XIAP-mediated Groucho/TLE ubiquitylation? .................................................................................. 129 Part II ............................................................................................................ 130 Discussion ................................................................................................ 130 USP47 and β-TRCP ......................................................................... 130 USP47 and XIAP .............................................................................. 132 USP47 and transcription .................................................................. 134 Future Directions ...................................................................................... 136 Is USP47 required for Wnt signaling in mammalian cells? ............... 136 Is USP47 required for Wnt signaling in Xenopus embryos? ............ 137 Does USP47 bind endogenous Smad4 and XIAP? ......................... 138 Does USP47 affect the localization or ubiquitylation of β-TRCP, β- catenin, XIAP, or Groucho/TLE? ...................................................... 138 Is USP47 involved in Wnt-mediated transcriptional events in the nucleus? ........................................................................................... 139 What is the USP47 substrate in the Wnt pathway? .......................... 140 Significance .............................................................................................. 141 BIBLIOGRAPHY ..................................................................................................... 148 vii LIST OF TABLES Table Page 3.1 List of the predicted E3 ligases in the Drosophila genome used for the RNAi screen .......................................................................................................... 50 3.2 List of the predicted DUBs in the Drosophila genome used for the RNAi screen. ......................................................................................................... 53 viii LIST OF FIGURES Figure Page 1.1. Schematic of Canonical Wnt Signaling .......................................................... 9 1.2. Schematic of the ubiquitin system ................................................................ 21 3.1. Schematic of Drosophila RNAi screen to identify E3 ligases and DUBs involved in regulating Wg/Wnt signlaling ..................................................... 39 3.2. Results of Drosophila E3 ligase RNAi screen for Wg/Wnt signaling regulators. ..................................................................................................... 47 3.3. Results of Drosophila DUB RNAi screen for Wg/Wnt signaling regulators .. 52 4.1. Drosophila S2 cell RNAi screen identifies the E3 ligase DIAP1 as a positive regulator of Wingless signaling .................................................................... 60 4.2. XIAP is required for Wnt signaling in cultured mammalian cells .................. 63 4.3. Knockdown of XIAP does not inhibit CMV-Luciferase or Notch signaling .... 64 4.4. XIAP loss or knockdown has no effect on β-catenin levels or localization ... 65 4.5. XIAP is required for Wnt signaling in vivo. ................................................... 69 4.6. XIAP binds and ubiquitylates Groucho/TLE ................................................. 73 4.7. TLE3 promotes XIAP nuclear localization .................................................... 73 4.8 Domain structure of the IAP family ............................................................... 74 4.9. Overexpression of Smac does not inhibit XIAP-mediated Groucho/TLE ubiquitylation. ............................................................................................... 78 4.10. Overexpression of cIAP1 or cIAP2 fails to promote Groucho/TLE3 ubiquitylation ................................................................................................ 79 ix 4.11. Ubiquitylation of Groucho/TLE by XIAP does not affect its stability, nuclear localization, or capacity to tetramerize ........................................................... 83 4.12. XIAP ubiquitylates all human TLE isoforms and Drosophila Groucho ....... 86 4.13. Ubiquitylation of Groucho/TLE by XIAP disrupts TCF/Lef binding.. ........... 89 4.14. Wnt signaling does not increase TLE3 ubiquitylation ................................. 90 4.15. Model of XIAP-mediated regulation of Groucho/TLE in the Wnt pathway ........................................................................................ 92 5.1. Drosophila RNAi screen identifies the DUB Ubp64E as a novel Wingless signaling component ................................................................................... 98 5.2. USP47 is required for Wnt signaling in cultured mammalian cells ............... 99 5.3. USP47 is located in the cytoplasm ............................................................. 102 5.4. USP47 is dynamically expressed throughout Xenopus development ........ 105 5.5. Loss and gain of USP47 perturbs axis formation in Xenopus embryos ..... 108 5.6. USP47 interacts with β-TRCP and Smad4 ................................................ 110 5.7. USP47 interacts with XIAP. ........................................................................ 112 5.8. USP47 and XIAP do not affect the stability of each other .......................... 114 6.1. Model of XIAP-mediated regulation of Groucho/TLE in the Wnt pathway. 118 6.2. Genetic changes associated with colorectal tumorigenesis. ...................... 143 x