Abstract
Simple SummaryThe 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)P2; and (3) the PtdIns(3,4)P2-mediated endosomal AKT activation through phosphorylation at Ser473 to control a subset of AKT substrates.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)P2, 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)P2-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)P3, contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.
Highlights
This article is an open access articleAKT/PKB is a central signaling effector that phosphorylates specific serine or threonine residues of core proteins that control cell physiological processes, such as cell survival, metabolism, growth, proliferation, and migration [1,2]
We demonstrate that mSIN1—an essential contributor for mechanistic target of rapamycin complex 2 (mTORC2) activity and integrity—is localized to the early endosome in a PtdIns(3,4)P2 -dependent manner and that endosomal mTORC2 is responsible for AKT activation through Ser473 phosphorylation
We investigated whether endocytosis is required for platelet-derived growth factor (PDGF)-induced AKT activation in U87MG glioma cells, which display hyperactivated AKT signaling caused by phosphatase and tensin homolog (PTEN) deficiency [37,38], using endocytosis inhibitors
Summary
This article is an open access articleAKT/PKB is a central signaling effector that phosphorylates specific serine or threonine residues of core proteins that control cell physiological processes, such as cell survival, metabolism, growth, proliferation, and migration [1,2]. AKT activity is upregulated by phosphatidylinositol 3-kinase (PI3K) signaling during the activation of receptor tyrosine kinases or G-protein-coupled receptors [3], and full AKT activation in human cells requires the phosphorylation of Thr308 in the activation loop and. AKT is phosphorylated at Thr308 by phosphoinositidedependent protein kinase 1 (PDK1) at the plasma membrane and at Ser473 by mechanistic target of rapamycin complex 2 (mTORC2) [5,6]. PDK1-mediated AKT Thr308 phosphorylation is dependent on the accumulation of PtdIns(3,4,5)P3 by PI3K at the plasma membrane, indicating that PtdIns(3,4,5)P3 molecules provide a binding platform for PDK1 and AKT through the pleckstrin homology (PH) domain of each protein. The mechanism underlying mTORC2-dependent AKT activation through phosphorylation at Ser473 has not been elucidated based on cell types or their extracellular stimulations
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