Acosta-Jaquez Hugo A, Keller Jennifer A, Foster Kathryn G, Ekim Bilgen, Soliman Ghada A, Feener Edward P, Ballif Bryan A, Fingar Diane C
Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA.
Mol Cell Biol. 2009 Aug;29(15):4308-24. doi: 10.1128/MCB.01665-08. Epub 2009 Jun 1.
The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.
雷帕霉素靶蛋白（mTOR）复合物1（mTORC1）作为一种对雷帕霉素敏感的环境传感器，可响应生长因子和营养物质促进细胞生物合成过程。尽管多种生理刺激可调节mTORC1信号传导，但mTORC1调节的直接生化机制仍不清楚。实际上，虽然已报道了三个mTOR磷酸化位点，但位点特异性mTOR磷酸化的功能作用尚未得到证实。在这里，我们通过串联质谱鉴定了一个新的mTOR磷酸化位点（S1261），并证明胰岛素-磷脂酰肌醇3-激酶信号传导在mTORC1和mTORC2中均促进mTOR S1261磷酸化。在这里，我们聚焦于mTORC1，并表明TSC/Rheb信号传导以氨基酸依赖性、雷帕霉素不敏感且自磷酸化非依赖性的方式促进mTOR S1261磷酸化。我们的数据揭示了mTOR S1261磷酸化在mTORC1作用中的功能作用，因为S1261磷酸化促进mTORC1介导的底物磷酸化（例如，p70核糖体蛋白S6激酶1 [S6K1]和真核起始因子4E结合蛋白1）以及细胞生长至细胞大小增加。此外，Rheb驱动的mTOR S2481自磷酸化和S6K1磷酸化需要S1261磷酸化。这些数据提供了首个证据，即位点特异性mTOR磷酸化调节mTORC1功能，并提出了一个模型，即胰岛素刺激的mTOR S1261磷酸化促进mTORC1自激酶活性、底物磷酸化和细胞生长。
