P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis
By: Cindy Marie Hodakoski
Publisher: [publisher not identified]
Published: 2012
Place of publication: [New York, N.Y.?]
Language: eng
Description: PTEN, a tumor suppressor lost in multiple cancers, antagonizes PI3-kinase signaling by dephosphorylating the second messenger phosphatidylinositol (3,4,5) trisphophate. 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 over expression 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.
Notes: Department: Cellular, Molecular, Structural, and Genetic Studies.

Thesis advisor: Ramon E. Parsons.

Thesis (Ph.D.)--Columbia University, 2012.