{"id":{"repo_id":"columbia-diss","oai_identifier":"oai:academiccommons.columbia.edu:10.7916/D86T0TRZ"},"canonical_url":"https://search.dev.ndltd.org/etd/columbia-diss/oai:academiccommons.columbia.edu:10.7916/D86T0TRZ","repository":{"repo_id":"columbia-diss","name":"Columbia University","base_url":"https://academiccommons.columbia.edu/oai"},"display":{"title":"P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Hodakoski, Cindy Marie"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012","date_published":"2012","updated_at":"2026-07-24T01:44:22Z","subjects":["Biology","Biochemistry"],"languages":["English"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.7916/D86T0TRZ","outbound_label":"DOI","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Hodakoski, Cindy Marie"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2012"]},{"key":"dc:type","label":"Dc Type","values":["Theses"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology","Biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://doi.org/10.7916/D86T0TRZ"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:title","label":"Title","values":["P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis"]}]}],"canonical_facts":{"dc:creator":["Hodakoski, Cindy Marie"],"dc:date":["2012"],"dc: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."],"dc:identifier":["https://doi.org/10.7916/D86T0TRZ"],"dc:language":["English"],"dc:subject":["Biology","Biochemistry"],"dc:title":["P-REX2 PH Domain Inhibition of PTEN Regulates Transformation, Insulin Signaling, and Glucose Homeostasis"],"dc:type":["Theses"]},"updated_at":"2026-07-24T01:44:22Z"}