Characterization of Rictor Phosphorylation Sites Reveals Direct Regulation of mTOR Complex 2 by S6K1
Characterization of Rictor Phosphorylation Sites Reveals Direct Regulation of mTOR Complex 2 by S6K1
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DOI：
10.1128/mcb.00735-09
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发表时间：
2009-11-01
影响因子：
5.3
通讯作者：
Manning, Brendan D.
中科院分区：
文献类型：
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作者：
Dibble, Christian C.;Asara, John M.;Manning, Brendan D.
哺乳动物雷帕霉素靶点（mTOR）在两个不同的复合物（mTORC1和mTORC2）中起作用，控制细胞生长、增殖、存活和代谢。虽然我们对mTORC1调控的理解已经取得了很大进展，但调控mTORC2的信号机制尚未明确。在这项研究中，我们使用液相色谱-串联质谱分析鉴定了核心mTORC2组分Rictor上的21个磷酸化位点。我们发现其中一个位点T1135经历了生长因子响应性磷酸化，该磷酸化对雷帕霉素非常敏感，并且在mTORC1下游磷酸化。我们发现Rictor-T1135被mtorc1依赖性激酶S6K1直接磷酸化。尽管该磷酸化事件不影响mTORC2的完整性或体外激酶活性，但在野生型或Rictor空细胞中表达Rictor磷酸化位点突变体（T1135A）会导致Akt在S473上的mTORC2依赖性磷酸化增加。然而，Rictor-T1135磷酸化似乎不调节mtorc2介导的对SGK1或PKC α的作用。虽然影响Akt的确切分子机制尚不清楚，但T1135的磷酸化刺激了Rictor与14-3-3蛋白的结合。我们提供的证据表明，Rictor-T1135磷酸化与其他mtorc1依赖的反馈机制（如影响IRS-1向PI3K信号传导的机制）平行作用，以调节Akt对胰岛素的反应。
The mammalian target of rapamycin (mTOR) functions within two distinct complexes (mTORC1 and mTORC2) to control cell growth, proliferation, survival, and metabolism. While there has been great progress in our understanding of mTORC1 regulation, the signaling mechanisms that regulate mTORC2 have not been defined. In this study, we use liquid chromatography-tandem mass spectrometry analyses to identify 21 phosphorylation sites on the core mTORC2 component Rictor. We find that one site, T1135, undergoes growth factor-responsive phosphorylation that is acutely sensitive to rapamycin and is phosphorylated downstream of mTORC1. We find that Rictor-T1135 is directly phosphorylated by the mTORC1-dependent kinase S6K1. Although this phosphorylation event does not affect mTORC2 integrity or in vitro kinase activity, expression of a phosphorylation site mutant of Rictor (T1135A) in either wild-type or Rictor null cells causes an increase in the mTORC2-dependent phosphorylation of Akt on S473. However, Rictor-T1135 phosphorylation does not appear to regulate mTORC2-mediated effects on SGK1 or PKC alpha. While the precise molecular mechanism affecting Akt is unknown, phosphorylation of T1135 stimulates binding of Rictor to 14-3-3 proteins. We provide evidence that Rictor-T1135 phosphorylation acts in parallel with other mTORC1-dependent feedback mechanisms, such as those affecting IRS-1 signaling to PI3K, to regulate the response of Akt to insulin.