Investigating Βpix As a Novel Upstream Regulator of the Hippo Pathway
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Investigating βPix as a novel upstream regulator of the Hippo pathway by Ki Myung Song A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Ki Myung Song 2015 Investigating βPix as a novel upstream regulator of the Hippo pathway Ki Myung Song Master of Science Department of Biochemistry University of Toronto 2015 Abstract The Hippo pathway regulates cell growth and organ size and dysregulation of the pathway leads to cancer. In mammals, the core Hippo pathway consists of Mst/Lats kinases, which phosphorylate and inhibit transcriptional co-activators, Yap and Taz, by promoting cytoplasmic sequestration. Here, we identify the guanine nucleotide exchange factor (GEF), Arhgef7, or βPix, as a positive Hippo pathway regulator. Upon upstream Hippo signals emanating from cell-cell contact and actin cytoskeletal rearrangements, βPix functions to regulate Yap/Taz localization and activity in a GEF-independent manner. βPix interacts with Yap via the C-terminal KER domain and this physical interaction plays a key role in βPix-mediated Yap/Taz regulation. In normal mammary epithelial cells, Lats kinases are required for βPix function while Mst kinases are not required for Hippo signalling. In breast cancer cells, ectopic expression of βPIX is sufficient to re-couple the Hippo kinase cassette to Yap/Taz, suggesting a possible role as a tumour suppressor. ii Acknowledgments I would like to express my deepest gratitude to my supervisor, Professor Liliana Attisano, for her guidance, invaluable insights and engagement throughout the learning process of my Master’s thesis. This dissertation would not have been possible without her unprecedented supervision and constant support. I would also like to extend my appreciation to my committee members, Professor Andrew Wilde and Professor Michael Ohh. I am sincerely grateful for their helpful advice and genuine remarks during the course of my study. I want to express my warmest thanks to all members of the Attisano Lab, past and present, for offering suggestions and assistance on numerous occasions, sharing their knowledge and techniques, and encouraging me with kind words and hearts. I would also like to thank Dr. Jeff Wrana and all members of the Wrana lab for sharing their expertise and intuitions. Last but not least, I want to wholeheartedly thank my loved ones, including my parents, siblings and friends. I have stumbled and fallen many times, but I was able to get up with their love and support from both local and overseas. I love you. iii Table of Contents Abstract…………………………………………………………………………………………………….…ii Acknowledgements........................................................................................................................iii List of Tables………………………………………………...……………………………..………………vii List of Figures…..……….………………………………………………………………………………..viii Chapter 1: Introduction……………………………………………………………………..……1 1.1 Core components of the Hippo pathway……..……………………..…………………1 1.1.1 Mst1 and Mst2 kinases…………………………………………………….3 1.1.2 Lats1 and Lats2 kinases…………………………………………………...5 1.1.3 Yap and Taz……………………………………………………………….6 1.2 Upstream regulators of the Hippo pathway…………………………..……………….8 1.2.1 Apical-basal polarity…………………..………………………………….....8 1.2.2 Planar cell polarity (PCP)………………………………………………….12 1.2.3 Mechanotransduction ………………………...……………………………13 1.2.4 G-protein-coupled receptor (GPCR) signalling ...…………………………15 1.3 Biological implications of the Hippo pathway…...………….………………………15 1.4 αPix and βPix ……………………………………...…………………..…………….18 1.4.1 Structure of Pix…………………………………………………………….18 1.4.2 Function of Pix……………………………………………………………..19 1.4.3 βPix-Git complex…………………………………………………………..21 iv 1.5 Thesis overview………………….…………………………………………………..22 Chapter 2: Materials and Methods……………………………….……………………….…….24 2.1 Cell culture and transfection...……………………….………………………………24 2.2 Plasmids and chemicals...………………………………….………………………...24 2.3 Immunoblotting (IB) and Immunoprecipitation (IP).…...…….……………………..25 2.4 Quantitation …………………………………………………….……………………25 2.5 Tead reporter assay ….…………...………..………………………………………...26 2.6 Immunofluorescence microscopy……...…………………………………………….26 2.7 Quantitative Real-Time PCR …...…...…………………………..…………………..27 Chapter 3: Results………………………………………………………….……………………32 3.1 LUMIER validation and characterization of βPix-Yap interaction……………….....32 3.1.1 βPix interacts with Taz and Yap….…………….………………………….32 3.1.2 Domain mapping of Pix and Yap.…………….…………..………………..34 3.1.2.1 KER of βPix is required for Yap interaction…...………………..34 3.1.2.2 Corresponding KER of αPix is required for Yap interaction…….38 3.2 Functional analysis of Pix in the Hippo pathway ....……………….………………..41 3.2.1 Analysis of βPix function via Tead reporter assay ……..……....…………41 3.2.2 αPix is not involved in the Hippo pathway in mouse mammary epithelial cells……………………………………………………………………………….43 3.3 Analysis of βPix GEF activity in Hippo pathway regulation………..………………43 3.3.1 βPix functions independent of GEF activity on Cdc42/Rac1 ……..………43 v 3.4 βPix and core Hippo kinases………….………….......................................................50 3.4.1 βPix and Lats kinases…...………………………………………………….50 3.4.1.1 βPix functions upstream of Lats kinases ………..……………….50 3.4.1.2 βPix does not affect phosphorylation status of Lats kinases at Thr1079/1041…………………………………………………………….50 3.4.2 βPix and Mst kinases...…….………………..……………………………..54 3.4.2.1 βPix does not interact with Mst kinases………………………….54 3.4.2.2 Increased expression of Mst by overexpressed βPIX……………57 3.4.2.3 Mst kinases bypass the canonical Hippo pathway……………….57 3.5 βPix as a re-coupler of the Hippo signal in metastatic cancer cell…………...……...62 Chapter 4: Discussion and the future direction…………….……………………..……………69 References……………………………………………………………………….………………76 vi List of Tables Table 1: List of siRNAs………………………………………………………………………….28 Table 2: Sequence of qPCR Primers (mouse)……..……………………………………………31 vii List of Figures Figure 1.1: The core Hippo pathway……………………………………………………………...2 Figure 1.2: Domain organization of core components of the Hippo pathway…………………….4 Figure 1.3: Domain organization of the homologous βPix and αPix………………………….…20 Figure 3.1: βPix interacts with Taz/Yap ………………………………………………………...33 Figure 3.2: A schematic representation of the wild-type and deletion cDNA constructs of βPIX and αPIX………………………………………………………………………………………...35 Figure 3.3: The KER of βPix is required for interaction with Yap………………………………36 Figure 3.4: The KER of αPix is required for interaction with Yap……………………………....37 Figure 3.5: The LZ domain is required for βPix dimerization…………………………………...39 Figure 3.6: The sequence alignment of human β/αPIX, showing amino acid sequence of a possible region of interaction with Yap………………………………………………………...40 Figure 3.7: βPix knockdown promotes accumulation of Yap/Taz in the nucleus……………….42 Figure 3.8: βPix GEF activity is dispensable for regulation of Yap/Taz ………..……………....45 Figure 3.9: βPix regulates Yap/Taz localization in response to cell density in a GEF-independent manner …………………………………………………………………………………………46 Figure 3.10: βPix regulates Yap/Taz localization in response to actin cytoskeleton rearrangement in a GEF-independent manner ………………………………………………………………….48 Figure 3.11: βPix functions upstream of Lats kinases…………………………………...………51 Figure 3.12: Phosphorylated Lats1/2 kinases at Thr1079/1041 localize to cell periphery………52 Figure 3.13: Knockdown of βPix does not affect the phosphorylation status at Thr1079/1041 on Lats1/2 kinases…………………………………………………………………………………...53 viii Figure 3.14: Overexpression of βPIX does not affect the phosphorylation status at Thr1079/1041 on Lats1/2 kinases………………………………………………………………………………..55 Figure 3.15: βPix does not interact with Mst kinases …………………………………………...56 Figure 3.16: Increased expression of Mst kinases by βPix is not physiologically relevant .….....58 Figure 3.17: Mst kinases bypass the canonical Hippo pathway in NMuMG cells ………….......59 Figure 3.18: Mst kinases bypass the canonical Hippo pathway in EpH4 cells.…………….........63 Figure 3.19: LATS1/2 kinases are required for βPIX-mediated YAP/TAZ inactivation in MDA- MB-231 cells……………………………………………………………………………………..66 Figure 4.1: Model of the regulatory mechanism of βPix in the Hippo pathway………………...73 ix 1 Chapter 1 Introduction In developmental biology, the fundamental biological processes of cell proliferation, differentiation and death have been extensively explored. Proliferation ensures growth during development and proper differentiation of multiple cell types assures their functionality, while appropriate apoptosis functions to replace defective cells. Coordination of these vital processes is critical for a wide range of physiological phenomena such as the exquisite control of organ size. The mechanisms underlying the regulation of organ size had remained under a veil until the emergence of the Hippo signalling pathway approximately two decades ago. Not only has the Hippo pathway been recognized as a master regulator of organ size, this complex signalling network plays critical roles in diverse cellular processes such as tissue homeostasis and regeneration, cell fate and stem cell self-renewal (Tapon et al. 2002, Harvey et al. 2003, Jia et al. 2003, Udan et al. 2003, Wu et al. 2003; Zhao et al. 2008). Dysfunction of the Hippo pathway is also implicated in pathological conditions including tumourigenesis and organ degeneration (O’Neill et al. 2004; Jonasson et al. 2007; Cordenonsi et al. 2011; Nishio et al.