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I PIK3IP1/TRIP RESTRICTS ACTIVATION of T CELLS PIK3IP1/TRIP RESTRICTS ACTIVATION OF T CELLS THROUGH INHIBITION OF THE PI3K/AKT PATHWAY by Uzodinma Nnaemeka Uche BS, Warren Wilson College, 2002 MS, The Pennsylvania State University, 2007 MS, The Pennsylvania State University, 2008 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2018 i UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This thesis was presented by Uzodinma Nnaemeka Uche It was defended on November 13, 2018 and approved by Marie C. DeFrances, Professor, Department of Pathology Adrian E. Morelli, Associate Professor, Department of Immunology Penelope A. Morel, Professor, Department of Immunology Binfeng Lu, Associate Professor, Department of Immunology Dissertation Director: Lawrence P. Kane, Professor, Department of Immunology ii Copyright © by Uzodinma Nnaemeka Uche 2018 iii PIK3IP1/TRIP RESTRICTS ACTIVATION OF T CELLS THROUGH INHIBITION OF THE PI3K/AKT PATHWAY Uzodinma Nnaemeka Uche, PhD University of Pittsburgh, 2018 Phosphatidylinositide-3-Kinases (PI3Ks) are a family of lipid kinases that play important intracellular signaling roles in cellular functions such as cell proliferation, motility, growth, intracellular trafficking, differentiation and survival. PI3K produces PIP3 which further facilitates the activation of downstream effectors such as Akt and PDK1. These effectors facilitate the cellular processes associated with PI3K activity. Conversely, because of the nature of PI3Ks roles, dysregulation of PI3K, leading to over-activity of the PI3K pathway is implicated in many cancers. PTEN, SHIP and INPP4B are negative regulators of PI3K activity that function downstream of PI3K and have been shown to act as tumor suppressors. Recently, PI3K Interacting Protein 1 (PIK3IP1) or TrIP (Transmembrane Inhibitor of PI3K), a novel negative regulator that functions upstream and proximal to PI3K, has been identified. TrIP, is a transmembrane protein and has been shown to down regulate PI3K activity leading to reduction of phosphorylation on Akt in carcinoma cell lines. Our lab was the first to show that TrIP can inhibit TCR signaling and TCR activation, in T cell lines. Here I have shown that both the extracellular kringle domain and intracellular p85-like domain are required for TrIP to inhibit PI3K and T cell activation. Interestingly, I have also found that cell surface expression of TrIP is acutely down-regulated during T cell activation. Furthermore, my results indicate that TrIP oligomerizes in order to inhibit iv PI3K signaling. Using knockout mice lacking expression of TrIP in T-cell compartments, I have been able to better define the requirements for TrIP in primary T cells upon TCR activation and in response to infection. Finally, I have developed a TrIP ecto-domain hIgG1 Fc fusion protein. This fusion protein has been used as an immunogen for the development of antibodies against TrIP in mice. This fusion protein has also been used to evaluate a possible ligand for TrIP. v TABLE OF CONTENTS PREFACE .................................................................................................................................. XII 1.0 INTRODUCTION ........................................................................................................ 1 1.1 T CELL ACTIVATION AND THE PI3K PATHWAY ................................... 1 T cell activation and signaling pathways .................................................... 1 The PI3K Superfamily .................................................................................. 3 The Class IA PI3K subfamily in T cells ...................................................... 5 TCR activation of the PI3K pathway .......................................................... 6 Regulators of the PI3K/Akt Pathway .......................................................... 8 Effects of PI3K on T cell development ...................................................... 11 Effects of PI3K on T cell differentiation ................................................... 13 The PI3K pathway in human disease ........................................................ 16 1.2 PI3K INTERACTING PROTEIN 1 (PIK3IP1): A NOVEL PI3K REGULATOR .................................................................................................................... 20 Kringle Domains ......................................................................................... 21 The p85-like Domain. ................................................................................. 23 The Importance of the Transmembrane Domain in PIK3IP1 ................ 23 2.0 STATEMENT OF THE PROBLEM ....................................................................... 25 vi 3.0 TRIP INHIBITS PI3K/AKT/MTOR PATHWAY SIGNALING AND BOTH THE EXTRACELLULAR AND INTRACELLULAR DOMAINS OF TRIP ARE REQUIRED FOR INHIBITION OF PI3K/AKT PATHWAY ..................................................................... 28 3.1 INTRODUCTION ............................................................................................. 28 3.2 METHODS AND MATERIALS ...................................................................... 29 3.3 RESULTS ........................................................................................................... 32 TrIP inhibits PI3K/Akt/mTOR pathway signaling ................................. 32 The Kringle and cytoplasmic domains are required for TrIP activity .. 34 3.4 DISCUSSION ..................................................................................................... 37 4.0 TRIP IS DOWN REGULATED UPON TCR STIMULATION THROUGH CLEAVAGE OF THE KRINGLE DOMAIN .......................................................................... 39 4.1 INTRODUCTION ............................................................................................. 39 4.2 MATERIALS AND METHODS ...................................................................... 40 4.3 RESULTS ........................................................................................................... 43 Cell-surface TrIP is down-regulated upon T cell activation ................... 43 The kringle domain is important for TrIP ligand/protein interaction and function ....................................................................................................................... 45 ADAM10/17 mediate cleavage of TrIP during T cell activation ............ 49 Mediators of TrIP cleavage are expressed on T cells .............................. 50 4.4 DISCUSSION ..................................................................................................... 53 5.0 THE P85-LIKE DOMAIN OF TRIP INTERACTS WITH P110δ PI3K ............. 55 5.1 INTRODUCTION ............................................................................................. 55 5.2 MATERIALS AND METHODS ...................................................................... 56 vii 5.3 RESULTS ........................................................................................................... 57 The p85-like domain of TrIP interacts with p110δ PI3K ........................ 57 5.4 DISCUSSION ..................................................................................................... 58 6.0 DELETION OF TRIP ON PRIMARY T CELLS LEADS TO DYSREGULATION OF T CELL ACTIVATION ...................................................................................................... 60 6.1 INTRODUCTION ............................................................................................. 60 6.2 MATERIALS AND METHODS ...................................................................... 61 6.3 RESULTS ........................................................................................................... 64 No obvious T cell developmental defects in TrIP KO mice .................... 64 Enhanced T cell activation in the absence of TrIP .................................. 66 6.4 DISCUSSION ..................................................................................................... 68 7.0 DELETION OF TRIP ENHANCES TH1 CELL INDUCTION AND INHIBITS TREG INDUCTION ................................................................................................................... 70 7.1 INTRODUCTION ............................................................................................. 70 7.2 MATERIALS AND METHODS ...................................................................... 71 7.3 RESULTS ........................................................................................................... 73 Deletion of TrIP enhances Th1 T cell polarization and inhibits Treg generation ................................................................................................................... 73 KO TrIP preference for Th1 polarization is due to enhanced PI3K activity ....................................................................................................................... 76 7.4 DISCUSSION ..................................................................................................... 78 8.0 ENHANCED ACTIVATION AND IN VIVO FUNCTION OF TRIP KO T CELLS ...................................................................................................................................... 80 viii 8.1 INTRODUCTION ............................................................................................. 80 8.2 MATERIALS AND METHODS ...................................................................... 81
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