Nck Adaptor Proteins Link Nephrin at the Podocyte Slit Diaphragm to the Hippo Regulator WTIP by Ava Keyvani Chahi A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Ava Keyvani Chahi, December, 2014 ABSTRACT NCK ADAPTOR PROTEINS LINK NEPHRIN AT THE PODOCYTE SLIT DIAPHRAGM TO THE HIPPO REGULATOR WTIP Ava Keyvani Chahi Advisor: University of Guelph, 2014 Dr. Nina Jones Podocytes are specialized epithelial cells that contribute to the kidney blood filtration barrier. Their unique cytoskeletal architecture and other complex biological signals are largely maintained by a modified adherens junction known as the slit diaphragm (SD). A major component of the SD is the transmembrane protein nephrin, which upon tyrosine phosphorylation of the intracellular domain regulates actin remodeling and cell survival. Nck adaptor proteins are a critical component of the filtration barrier and connect nephrin to actin-remodeling proteins by binding phosphotyrosine residues and proline-rich motifs. Herein we identify a novel Nck binding partner, Wilm’s tumor interacting protein (WTIP). WTIP is a transcription regulator in podocytes, though Nck is not nuclear localized under conditions known to induce WTIP nuclear accumulation. However, we demonstrate that WTIP is recruited to nephrin, upon nephrin tyrosine phosphorylation by Src Family Kinases, in an Nck-dependent manner. WTIP is an evolutionarily conserved negative regulator of the Hippo kinase pathway, which inhibits the transcription factor Yap. Yap activity promotes podocyte survival. We show in mice that cannot recruit Nck to nephrin, and by extension WTIP, that Yap protein levels are downregulated. Similarly, Yap downregulation is demonstrated in wildtype mice subJected the nephrotoxic serum model of podocyte injury, which is known to induce robust decrease in nephrin tyrosine phosphorylation. Overall, through identification of a novel Nck binding partner, WTIP, we have identified a novel mechanism through which Nephrin tyrosine phosphorylation is linked to the Hippo signaling cascade, which may in turn promote podocyte survival. In dedication to the memory of my friend, Jason Kuchinsky ACKNOWLEDGEMENTS I am eternally grateful to my supervisor Dr. Nina Jones. Nina, thank you for giving me the freedom to disagree and the freedom to explore. Working with Nina and the members of the Jones Laboratory has made me develop immensely as a person and as a researcher. I also owe Dr. Terry Van Raay a debt of gratitude for really going above and beyond his role as a committee member. Dr. Van Raay is a wonderful teacher and no matter how busy he was he made his door open to offer me advice. I would also like to thank past and present members of the Jones Lab and our surrounding supervisors and colleagues in the Van Raay, Bendall and Mosser Labs; especially Jahdiel (Jaddy) Larraguibel, Rasmeet (Ras) Dhaliwal, Rachael (Katie) MacKenzie who were all awesome lab mates and housemates and without them I likely would not have found affordable housing during this degree. Others who have provided support and laughs include Agata Zienowicz, Kevin Bosse, Colin Stringer, Cameron Harris, Christopher James (Jim) Cooper, Olivia Catherine Anderson, Rebecca Rumney and so many more! The most important lab member that I need to thank is Dr. Una Adamicic-Bistrivoda, the former post-doctoral fellow in our lab, for mentorship, training and making sure I never became a “skirt-holder”. I also need to acknowledge those I collaborate with most frequently, Claire Elyse Martin and Dr. Laura A. New, who were both instrumental in designing tools used in this thesis. Lastly, but far from least, I need to thank my friend (Dr.–to-be) Melanie K.B. Wills who has been a role model for me and is always a source of scientific support, motivation and hysteric laughter. I need to thank all of my wonderful friends and family for enduring the hardships of university with me- and hopefully for enjoying it with me. I can’t list everyone but I have to iv thank Thomas Bracken Coyne for being a constant source of motivation and optimism; as well as for the countless number of times he listened while I rambled about my research. I also need to thank all of the feline sensations on the Internet that have made me laugh until I cry and were my companions during many late-nights of western blotting. Also, my research’s financial supporters: the University of Guelph College of Biological Sciences, Ontario Graduate Scholarship, and the Kidney Foundation of Canada all deserve acknowledgement. Lastly, thank you to Maman, Baba and my spunky sister, Aram. Truly, my parents have made every sacrifice a person could make for their children and I accredit them for any and every one of my accomplishments. v DECLARATION OF WORK PERFORMED All of the work in this thesis was performed by Ava Keyvani Chahi, with the following exceptions. Isolation and generation of wildtype, Nck1 knockout and Nck1 and 2 knockout mouse podocyte cells was performed by Claire Elyse Martin. Subcloning of WTIPΔPRR from pEGFP-C1 to pCDNA3.1-HA was designed and organized by Ava Keyvani Chahi and performed by Olivia Catherine Anderson. All mouse organization and handling was done by Dr. Laura A. New and the University of Guelph Central Animal Facility. The β-actin forward and reverse primers were previously designed by Steve Hawley. Also, the Western Blot for Figure 16A was partially provided by Claire Elyse Martin. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………………….iv DECLARATION OF WORK PERFORMED………………………………………………...vi TABLE OF CONTENTS……………………………………………………………………....vii LIST OF TABLES………………………………………………………………………….........x LIST OF FIGURES……………………………………………………………………………..xi LIST OF ABBREVIATIONS……………………………………………………………….....xii CHAPTER 1: INTRODUCTION……………………………………………………………….1 SIGNIFICANCE OF KIDNEY RESEARCH……………………………………………………….......2 KIDNEY ANATOMY………………………………………………………………………………..3 GLOMERULAR FILTRATION BARRIER……………………………………………………………5 PODOCYTES……………………………………………………………………………………….6 PODOCYTE CYTOARCHITECTURE………………………………………………………………...6 PODOCYTE EFFACEMENT…………………………………………………………………………9 PODOCYTE ADHESION STRUCTURES……………………………………………………………10 NEPHRIN: A MAJOR COMPONENT OF THE SLIT DIAPHRAGM……………………………………12 NCK ADAPTOR PROTEINS……………………………………………………………………….15 NCK AND NEPHRIN INTERACTION………………………………………………………………16 WILM’S TUMOR-1 INTERACTING PROTEIN (WTIP)……………………………………………19 WTIP IS A REGULATOR OF THE HIPPO KINASE PATHWAY…………………………………….21 SUMMARY AND RATIONALE……………………………………………………………………..24 CHAPTER 2: MATERIALS AND METHODS……………………………………………...26 TISSUE CULTURE………………………………………………………………………………...27 vii LPS TREATMENT………………………………………………………………………………..28 TRANSFECTION…………………………………………………………………………………..28 VECTORS……………………………………..…………………………………………………..28 CLONING OF GFP- AND HA- TAGGED WTIPΔPRR……………………………………………28 ANTIBODIES……………………………………………………………………………………...30 CD16 STIMULATION AND PP2 TREATMENT…………………………………………………….30 CELL LYSIS………………………………………………………………………………………31 CELL FRACTIONATION…………………………………………………………………………..31 IMMUNOBLOTTING………………………………………………………………………………32 IMMUNOPRECIPITATION………………………………………………………………………...33 GST PULLDOWN ASSAY…………………………………………………………………………33 RNA EXTRACTION AND CDNA PREPARATION…………………………………………………34 SEMI-QUANTITATIVE REAL TIME PCR………………………………………………………...34 Y3F NEPHRIN MICE AND NTS INJECTION………………………………………………………..35 GLOMERULI ENRICHMENT……………………………………………………………………...35 CHAPTER 3: RESULTS………………………………………………………………………37 NCK AND WTIP INTERACT……………………………………………………………………...38 MAPPING OF THE WTIP-NCK INTERACTION TO THE FIRST AND THIRD NCK SH3 DOMAINS...39 MAPPING OF THE WTIP-NCK INTERACTION TO THE PRR OF WTIP…………………………44 NCK SUBCELLULAR LOCALIZATION DOES NOT EXHIBIT SIMILAR TRENDS AS WTIP AND NCK EXPRESSION DOES NOT INFLUENCE TRANSCRIPT LEVELS OF WT1 TARGET GENES……………46 WTIP IS RECRUITED TO CD16-NEPHIN IN AN NCK-DEPENDENT MANNER…………………….49 viii WTIP RECRUITMENT TO CD16-NEPHRIN IS DEPENDENT ON NEPHRIN PHOSPHORYLATION BY SFK AND ON NCK’S PHOSPHO-TYROSINE- AND PROLINE- BINDING FUNCTIONS……………….50 TOTAL YAP LEVELS IN MOUSE GLOMERULI ARE INFLUENCED BY NEPHRIN PHOSPHORYLATION……………………………………………………………………………...53 CHAPTER 4: DISCUSSION…………………………………………………………………..55 CHAPTER 5: CONCLUSIONS……………………………………………………………….67 REFERENCES………………………………………………………………………………….69 ix LIST OF TABLES Table 1: Primer sequences (from 5’-3’)………………………………………………………....36 Table 2: Summary of Nck interaction partners and reinterpretation of mapping data……….…58 x LIST OF FIGURES Figure 1: Schematic of a kidney and nephron……………………………………………………4 Figure 2: Schematic of glomerulus and glomerular filtration barrier…………………………….6 Figure 3: Podocyte Structure……………………………………………………………………..8 Figure 4: Podocyte Effacement………………………………………………………………….10 Figure 5: Schematic of adjacent foot processes connected by Slit Diaphragm………………....12 Figure 6: Schematic of Nck protein domains…………………………………………………...16 Figure 7: Schematic of WTIP protein domains and motifs……………………………………..20 Figure 8: Schematic of the Hippo kinase pathway in mammals………………………………...23 Figure 9: Summary of the molecular signals in healthy and injured podocytes relevant to the research in this thesis…………………………………………………………………………….25 Figure 10: Nck and WTIP interaction…………………………………………………………...39 Figure 11: Nck and WTIP interact via Nck SH3 domains……………………………………...41 Figure 12: Mapping of Nck and WTIP interaction to the first and third Nck SH3 domains……43 Figure 13: Mapping of the Nck