REGULATION OF CANONICAL WNT SIGNALING BY UBIQUITYLATION By Alison Jean Hanson ...

184 Pages·2012·37.19 MB·English

by  Ali Hanson

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

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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
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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.

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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
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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
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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

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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

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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
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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

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