Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells

SIMPLE SUMMARY: The full activation of AKT, which is necessary for cell physiological changes, is achieved through the phosphorylation of Thr308 and Ser473 in human AKT. Here, we have addressed how AKT activation at early endosomes occurs during growth factor stimulation and how mTORC2 is recruited into endosomes and associated with AKT. The explanation comes from the discovery of three important events: (1) the physical association of mSIN and Rictor, critical components for mTORC2 assembly and activity, with early endosomes; (2) the control of the recruitment of mSIN to endosomes by PtdIns(3,4)P(2); and (3) the PtdIns(3,4)P(2)-mediated endosomal AKT activation through phosphorylation at Ser473 to control a subset of AKT substrates. ABSTRACT: The serine/threonine kinase AKT is a major effector during phosphatidylinositol 3-kinase (PI3K)-driven cell signal transduction in response to extracellular stimuli. AKT activation mechanisms have been extensively studied; however, the mechanism underlying target of rapamycin complex 2 (mTORC2) phosphorylation of AKT at Ser473 in the cellular endomembrane system remains to be elucidated. Here, we demonstrate that endocytosis is required for AKT activation through phosphorylation at Ser473 via mTORC2 using platelet-derived growth factor-stimulated U87MG glioma cells. mTORC2 components are localized to early endosomes during growth factor activation, and the association of mTORC2 with early endosomes is responsible for the local activation of AKT, which is critical for specific signal transduction through glycogen synthase kinase-3 beta and forkhead box O1/O3 phosphorylation. Furthermore, endosomal phosphoinositide, represented by PtdIns(3,4)P(2), provides a binding platform for mTORC2 to phosphorylate AKT Ser473 in endosomes through mammalian Sty1/Spc1-interacting protein (mSIN), a pleckstrin homology domain-containing protein, and is dispensable for AKT phosphorylation at Thr308. This PtdIns(3,4)P(2)-mediated endosomal AKT activation provides a means to integrate PI3K activated by diverse stimuli to mTORC2 assembly. These early endosomal events induced by endocytosis, together with the previously identified AKT activation by PtdIns(3,4,5)P(3), contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.