Wang Lifu, Harris Thurl E, Roth Richard A, Lawrence John C
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2007 Jul 6;282(27):20036-44. doi: 10.1074/jbc.M702376200. Epub 2007 May 17.
Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.
雷帕霉素哺乳动物靶蛋白（mTOR）在两种不同的信号复合物mTORC1和mTORC2中发挥作用。响应胰岛素和营养物质时，由mTOR、 Raptor（mTOR调节相关蛋白）和mLST8组成的mTORC1被激活，并使真核起始因子4E结合蛋白（4EBP）和p70 S6激酶磷酸化，以促进蛋白质合成和细胞生长。此前我们发现，响应胰岛素时mTOR激酶的激活与4EBP1与Raptor的结合增加有关。在此我们确定富含脯氨酸的Akt底物40（PRAS40）为mTORC1的结合伴侣。在PRAS40中鉴定出一个假定的TOR信号基序FVMDE，它被证明是与Raptor相互作用所必需的。胰岛素刺激显著降低了mTORC1结合的PRAS40水平。重组PRAS40在体内和体外均抑制mTORC1激酶活性，且这种抑制作用取决于PRAS40与Raptor的结合。此外，通过短发夹RNA降低PRAS40表达可增强4E - BP1与Raptor的结合，并且重组PRAS40与4E - BP1竞争与Raptor的结合。因此，我们提出PRAS40通过作为底物结合的直接抑制剂来调节mTORC1激酶活性。
