Evidence for Direct Activation of mTORC2 Kinase Activity by Phosphatidylinositol 3,4,5-Trisphosphate
Evidence for Direct Activation of mTORC2 Kinase Activity by Phosphatidylinositol 3,4,5-Trisphosphate
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DOI：
10.1074/jbc.m110.195016
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发表时间：
2011-04-01
影响因子：
4.8
通讯作者：
Wu, Dianqing
中科院分区：
文献类型：
--
作者：
Gan, Xiaoqing;Wang, Jiyong;Wu, Dianqing
mTORC 2（mammalian target of rapamycin complex 2）通过调节一系列下游效应物（包括蛋白激酶AKT）在信号转导中起重要作用。然而，上游监管机构对其监管的特点仍然很差。虽然已知磷脂酰肌醇3，4，5-三磷酸（PtdIns（3，4，5）P-3）通过mTORC 2调节AKT Ser（473）（疏水基序（HM）位点）的磷酸化，但尚不清楚PtdIns（3，4，5）P-3是否可以直接调节mTORC 2激酶活性。在这里，我们使用两种膜对接的AKT突变蛋白，一种具有和另一种不具有普列克底物蛋白同源性（PH）结构域，作为mTORC 2的底物，以剖析PtdIns（3，4，5）P-3在培养细胞和体外激酶测定中AKT HM磷酸化中的作用。在HEK 293 T细胞中，胰岛素和组成型活性突变体的小GT3 H-Ras和PI 3 K可以诱导HM磷酸化的AKT突变体，这是由PI 3 K抑制剂LY 294002阻断。重要的是，PtdIns（3，4，5）P-3能够在体外激酶测定中通过免疫沉淀的mTOR 2复合物刺激两种AKT突变体的磷酸化。在体内和体外测定中，含有PH结构域的AKT突变体似乎是比没有PH结构域的AKT突变体更好的底物。因此，这些结果表明PtdIns（3，4，5）P-3可以通过多种机制调节mTORC 2对HM的磷酸化。其机制之一是直接刺激mTORC 2的激酶活性。
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.