Regulation of phosphoinositide metabolism, Akt phosphorylation, and glucose transport by PTEN (lhosphatase and tensin homolog deleted on chromosome 10) in 3T3-L1 adipocytes

To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinosi tide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wildtype PTEN and its phosphatase-dead mutant (C124S) with or w ithout an N-terminal myristoylation tag were overexpressed in Sf-9 cells an d 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110 alpha catalytic subuni t of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the acc umulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylin ositol 3,4,5-trisphosphate induced by p110 alpha. In contrast, overexpressi on of the C124S mutants apparently increased these accumulations. In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedl y suppressed by overexpression of wild-type PTEN with the N-terminal myrist oylation tag, but not by that without the tag. On the contrary, the C124S m utants of PTEN enhanced insulin-induced accumulations of phosphatidylinosit ol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interesti ngly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumul ation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpressio n of these PTEN proteins. Finally, insulin-induced increases in glucose tra nsport activity were significantly inhibited by the overexpression of myris toylated wild-type PTEN, but were not enhanced by expression of the C124S m utant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phos phatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosph ate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser4 74 of Akt2 are regulated differently, and the former is regulated very sens itively by the function of PTEN. 4) The phosphorylation level of Ser474, bu t not that of Thr309, in Akt2 correlates well with insulin-stimulated gluco se transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous P TEN may not play a major role in the regulation of glucose transport activi ty in 3T3-L1 adipocytes.