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Protein Tyrosine Phosphatase Sigma (Ptpσ) Targets Apical Junction Complex Proteins in the Intestine and Modulates

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Protein Tyrosine Phosphatase Sigma (Ptpσ) Targets Apical Junction Complex Proteins in the Intestine and Modulates Protein tyrosine phosphatase sigma (PTPσ) targets apical junction complex proteins in the intestine and modulates epithelial permeability. by Ryan Murchie A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Ryan Murchie 2013 Protein tyrosine phosphatase sigma (PTPσ) targets apical junction complex proteins in the intestine and modulates epithelial permeability. Ryan Murchie Master of Science Department of Biochemistry University of Toronto 2012 Abstract Protein tyrosine phosphatase sigma (PTPσ), encoded by PTPRS, was shown previously by us to contain SNP polymorphisms that can confer susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype and show increased susceptibility to acute models of murine colitis. The function of PTPσ in the intestine is uncharacterized. Here, I show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in trans-epithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. We identified several putative PTPσ intestinal substrates; among these were several proteins that form and regulate the apical junction complex, including ezrin. My results show that ezrin binds to and undergoes tyrosine de-phosphorylation by PTPσ in vitro, suggesting it is a direct substrate for this PTP. The results suggest a role for PTPσ as a positive regulator of epithelial barrier integrity in the intestine. The proteins identified in the screen, including ezrin, suggest that PTPσ may modulate epithelial cell adhesion through the targeting of AJC-associated proteins, a process impaired in IBD. ii Acknowledgements First and foremost, I would like to thank my supervisors Dr. Daniela Rotin and Dr. Aleixo Muise for all of your guidance over the course of my project. Your encouragement and support were essential to the success my endeavours. I would also like to thank my supervisory committee members Dr. John Brumell and Dr. Christine Bear for their helpful guidance and suggestions during my committee meetings. To all the members of the Rotin and Muise labs, thank you for making my time in the lab a memorable one. Your constant support always made the lab a welcoming place to work. Thank you to Chong, Chen, Angie and Hui for all your invaluable technical support throughout my time in the lab without which I would not have been successful. Special thanks to Melanie Gareau from Dr. Phil Sherman’s lab for training on the Ussing Chamber experiments and to Ramzi Fattouh from Dr. John Brumell’s lab for additional training. Lastly, thank you to my friends and family for all of your support over the years! iii Table of Contents Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abbreviations ............................................................................................................................... viii Chapter 1: Introduction ................................................................................................................... 1 1) Tyrosine Phosphorylation ....................................................................................... 2 2) Protein Tyrosine Phosphatases ............................................................................... 2 3) Protein Tyrosine Phosphatase Sigma (PTPσ) ......................................................... 8 4) PTPσ(-/-) Mouse Model ........................................................................................ 11 5) Inflammatory Bowel Disease ................................................................................ 12 6) Protein Tyrosine Phosphatases in IBD ................................................................. 13 7) PTPσ in IBD ......................................................................................................... 14 8) Role of Intestinal Barrier Defence in IBD ............................................................ 19 9) Apical Junction Complex and Epithelial Barrier Function ................................... 20 10) PTPσ and the Apical Junction Complex ............................................................... 20 11) Rationale and Goals .............................................................................................. 26 Chapter 2: Materials & Methods................................................................................................... 27 1) Animal Experimentation ....................................................................................... 28 2) Constructs ............................................................................................................. 28 3) Tissue Preparation ................................................................................................. 28 4) Tandem immunoprecipitation of phosphotyrosine-mass spectrometry (TIPY- MS) ....................................................................................................................... 29 5) MS/MS Data Analysis .......................................................................................... 29 6) In vitro substrate trapping assay ........................................................................... 30 7) Tissue culture ........................................................................................................ 31 8) para-Nitrophenyl phosphate (pNPP) phosphatase activity assay .......................... 31 9) In vitro dephosphorylation assay .......................................................................... 31 10) Ussing Chamber Studies ....................................................................................... 32 11) Macromolecular Permeability ............................................................................... 33 12) FITC-dextran assay ............................................................................................... 33 13) Dextran Sodium Sulfate (DSS) Model for IBD .................................................... 33 14) Immunohistochemistry ......................................................................................... 34 15) Transmission Electron Microscopy ...................................................................... 34 Chapter 3: Results ......................................................................................................................... 36 1) PTPσ(-/-) mice exhibit defects in intestinal barrier integrity ................................ 37 2) PTPRS is expressed in the crypts regions of the mouse intestine ......................... 40 3) Tyrosine phosphorylation is enriched in the crypts of PTPσ(-/-) mouse colon and small bowel .................................................................................................... 40 iv 4) Identification of Villin and Ezrin as PTPσ binding partners by mass spectrometry .......................................................................................................... 45 5) Ezrin is a substrate of PTPσ .................................................................................. 49 6) E-cadherin and β-catenin colocalization and β-catenin localization to the nucleus is unaffected in the PTPσ(-/-) mouse small bowel and colon .................. 54 7) Ezrin localization is altered in PTPσ(-/-) mouse small bowel after DSS treatment ............................................................................................................... 58 8) Intestinal morphology is not disrupted in the small bowel and colon of neonatal PTPσ (-/-) mice ....................................................................................... 61 9) Lysozyme immunostaining is reduced in the Paneth cells of the PTPσ(-/-) mouse small bowel ................................................................................................ 64 10) Ki-67 immunostaining, a marker for cell proliferation, appears unchanged in PTPσ(-/-) mouse small bowel and colon .............................................................. 64 Chapter 4: Discussion ................................................................................................................... 70 Role of PTPσ in intestinal epithelia .............................................................................................. 71 1) Evidence of defects in intestinal barrier integrity ................................................. 71 2) Role for E-cadherin and β-catenin ........................................................................ 72 3) Ezrin as a colonic PTPσ substrate ......................................................................... 73 Proposed Mechanism: PTPσ regulation of adherens junction proteins ........................................ 74 Implications for PTPσ in Paneth cell function .............................................................................. 78 PTPσ in IBD pathogenesis ...........................................................................................................
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