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P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis

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P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis Cindy Hodakoski Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2012 Cindy Hodakoski All rights reserved ABSTRACT P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis Cindy Hodakoski PTEN, a tumor suppressor lost in multiple cancers, antagonizes PI3-kinase signaling by dephosphorylating the second messenger phosphatidylinositol (3,4,5) trisphosphate. PTEN expression and enzymatic activity is regulated through various mechanisms, including oxidation, phosphorylation, and protein-protein interactions. Our lab has recently identified a PTEN interacting protein, the Rac GEF P-REX2, which inhibits PTEN phosphatase activity in a non- competitive manner. This thesis focuses on understanding the physiological relevance of this interaction in the regulation of PI3K signaling, as well as determining the mechanism of P-REX2 mediated PTEN inhibition. The first chapter focuses on the role of P-REX2 overexpression in PI3K signaling, proliferation, and transformation. We first find that P-REX2 Rac GEF activity is dispensable for PTEN inhibition by utilizing a P-REX2 GEF dead mutant N212A. Next, we determined the effect of P-REX2 overexpression on PI3K signaling in normal mammary epithelial cells. Expression of P-REX2 or the DHPH inhibitory domain increased AKT phosphorylation, promoted cellular proliferation, and disrupted acini morphogenesis. Furthermore, P-REX2 cooperated with other oncogenes, including the PI3K E545K oncogenic mutant, c-MYC, and HER2 to promote proliferation, colony formation in soft agar, and tumor formation in mice. We also analyzed the effects of expression of P-REX2 cancer mutants, and discovered two transforming mutants, V432M and R498I that cooperated with PI3K E545K to increase anchorage independent growth and cellular proliferation. The next chapter examines the role of P-rex2 in PI3K signaling regulation in vivo. We generated Prex2 knockout mice using a gene trap method, and found that baseline signaling and proliferation in fibroblasts was not affected by P-rex2 deletion. However, insulin and IGF-1, but not PDGF or EGF stimulated PI3K signaling was reduced in Prex2-/- fibroblasts. The activity of PTEN from Prex2+/+ fibroblasts was reduced following insulin stimulation, but remained elevated in Prex2-/- cells, suggesting that insulin stimulated PTEN inhibition is dependent on P- rex2. Furthermore, P-REX2 interacted with phosphorylated insulin receptor and recruited PTEN to the membrane following insulin stimulation. Prex2-/- mice are intolerant to insulin and glucose, and have reduced PI3K signaling in the fat and liver following insulin stimulation. Furthermore, the activity of PTEN from Prex2-/- liver samples is elevated, and correlated with a decrease in cellular PIP3 levels. After uncovering an essential role for P-REX2 in PI3K signal transduction, we next examined the mechanism and regulation of P-REX2 mediated PTEN inhibition. We found that P-REX2 interacts with two different sites on PTEN. The PH domain of P-REX2 bound to the phosphatase and C2 domains of PTEN, while the inositol polyphosphate-4 phosphatase domain interacted with the PDZ-binding domain on the PTEN C-terminal tail. We discovered that the PH domain was the minimal domain that constitutively inhibited PTEN. However, the DHPH domain and full length P-REX2 required phosphorylation of the PTEN C-terminal tail for inhibition, suggesting the DH domain of P-REX2 restricts PH domain inhibition of PTEN when the C-terminal tail of PTEN is unphosphorylated. Furthermore, the PH domain of P-REX1 was not able to inhibit PTEN, and full length P-REX1 did not interact with PTEN, suggesting that there is a level of specificity involved in P-REX2 PH domain mediated phosphatase inhibition and binding. Overall, this thesis identifies P-REX2 as a dynamic inhibitor of PTEN phosphatase activity that regulates PI3K mediated cellular transformation, insulin signaling, and glucose metabolism. TABLE OF CONTENTS CHAPTER I: INTRODUCTION ..................................................................................... 1 PHOSPHOINOSITIDE SIGNALING .................................................................... 2 Substrate recognition.................................................................................. 3 Phosphoinositide regulation and disease.................................................... 5 PHOSPHOINOSITIDE-3 KINASE ........................................................................ 7 Classes of Phosphoinsotide- 3 kinases ....................................................... 7 Activation of Class IA PI3K ...................................................................... 9 AKT ....................................................................................................................... 9 Structure ................................................................................................... 10 Activation.................................................................................................. 11 Biological effects of AKT activation ...................................................... 12 PTEN .................................................................................................................. 15 Genetic alterations of PTEN in cancer .................................................... 16 PTEN structure ........................................................................................ 17 PTEN function ......................................................................................... 18 PI3K independent function of PTEN ....................................................... 22 PTEN regulation ...................................................................................... 23 P-REX2 and RHO GTPASES ............................................................................... 28 Rac GTPase function ............................................................................... 28 P-REX2 function ...................................................................................... 30 P-REX2 structure ..................................................................................... 30 Rac activation and disease ....................................................................... 31 P-REX2-PTEN interaction ....................................................................... 33 FIGURE LEGENDS ............................................................................................ 35 FIGURES ............................................................................................................. 37 CHAPTER II: Expression of Wild-type P-REX2 and P-REX2 Cancer Mutants Increases PI3K Signaling and Proliferation, and Transforms Cells in Cooperation with Various Oncogenes .................................................... 43 i ABSTRACT ......................................................................................................... 44 INTRODUCTION................................................................................................ 45 EXPERIMENTAL PROCEDURES .................................................................... 49 RESULTS ............................................................................................................ 54 DISCUSSION ...................................................................................................... 61 FIGURE LEGENDS ............................................................................................ 65 FIGURES ............................................................................................................. 68 CHAPTER III: P-REX2 Regulates Insulin Dependent PI3K Signaling and Glucose Metabolism in vivo .......................................................................................................... 74 ABSTRACT ......................................................................................................... 75 INTRODUCTION ............................................................................................... 76 EXPERIMENTAL PROCEDURES .................................................................... 79 RESULTS ............................................................................................................ 85 DISCUSSION ...................................................................................................... 93 FIGURE LEGENDS ............................................................................................ 98 FIGURES ............................................................................................................101 CHAPTER IV: Phosphorylation of the PTEN Carboxy-terminal Tail Domain Regulates P-REX2 PH Domain-mediated Inhibition of PTEN ......................... 108 ABSTRACT ....................................................................................................... 109 INTRODUCTION ............................................................................................. 110 EXPERIMENTAL PROCEDURES .................................................................. 114 RESULTS .......................................................................................................... 120 DISCUSSION ...................................................................................................
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