Evidence for Direct Activation of mTORC2 Kinase Activity by Phosphatidylinositol 3,4,5-Trisphosphate

mTORC2 (mammalian target of rapamycin complex 2) plays important roles in signal transduction by regulating an array of downstream effectors, including protein kinase AKT. However, its regulation by upstream regulators remains poorly characterized. Although phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) is known to regulate the phosphorylation of AKT Ser(473), the hydrophobic motif (HM) site, by mTORC2, it is not clear whether PtdIns(3,4,5)P-3 can directly regulate mTORC2 kinase activity. Here, we used two membrane-docked AKT mutant proteins, one with and the other without the pleckstrin homology (PH) domain, as substrates for mTORC2 to dissect the roles of PtdIns(3,4,5)P-3 in AKT HM phosphorylation in cultured cells and in vitro kinase assays. In HEK293T cells, insulin and constitutively active mutants of small GTPase H-Ras and PI3K could induce HM phosphorylation of both AKT mutants, which was blocked by the PI3K inhibitor LY294002. Importantly, PtdIns(3,4,5)P-3 was able to stimulate the phosphorylation of both AKT mutants by immunoprecipitated mTOR2 complexes in an in vitro kinase assay. In both in vivo and in vitro assays, the AKT mutant containing the PH domain appeared to be a better substrate than the one without the PH domain. Therefore, these results suggest that PtdIns(3,4,5)P-3 can regulate HM phosphorylation by mTORC2 via multiple mechanisms. One of the mechanisms is to directly stimulate the kinase activity of mTORC2.