Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation
Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation
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DOI：
10.1074/jbc.m109.029637
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发表时间：
2010-01-01
影响因子：
4.8
通讯作者：
Fingar, Diane C.
中科院分区：
文献类型：
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作者：
Foster, Kathryn G.;Acosta-Jaquez, Hugo A.;Fingar, Diane C.
雷帕霉素敏感的mTOR复合物1（mTORC 1）促进蛋白质合成，细胞生长和细胞增殖，以响应生长因子和营养提示。为了阐明mTORC 1调控的机制，我们研究了mTOR相互作用伴侣raptor的磷酸化。我们已经确定了六个猛禽磷酸化位点，位于两个集中定位的集群（集群1，Ser（696）/Thr（706）和集群2，Ser（855）/Ser（859）/Ser（863）/Ser（877）），使用串联质谱法，并为这些网站的每一个产生磷酸特异性抗体。在这里，我们主要集中在猛禽Ser（863）磷酸化。我们报道胰岛素通过经典的PI 3 K/TSC/Rheb途径以雷帕霉素敏感的方式促进mTORC 1相关的raptor Ser（863）磷酸化。mTORC 1通过其他刺激激活（例如，G.氨基酸、表皮生长因子/MAPK信号传导和细胞能量）也促进raptor Ser（863）磷酸化。Rheb过表达增加了猛禽Ser（863）以及其他五个鉴定位点（e. G. Ser（859）、Ser（855）、Ser（877）、Ser（696）和Thr（706））。引人注目的是，raptor Ser（863）磷酸化是raptor Ser（859）和Ser（855）磷酸化绝对必需的。这些数据表明，mTORC 1激活导致raptor多位点磷酸化，raptor Ser（863）磷酸化作为调节分级raptor磷酸化的主生化开关发挥作用（例如，G.在Ser（859）和Ser（855）上）。重要的是，mTORC 1含有磷酸化位点缺陷的猛禽表现出对底物4 EBP 1的体外激酶活性降低，多位点猛禽6A突变体比单位点猛禽S863 A更强烈地缺陷。总之，这些数据表明，复杂的猛禽磷酸化功能作为一个生化变阻器，调节mTORC 1信号根据环境的线索。
The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser(696)/Thr(706) and cluster 2, Ser(855)/Ser(859)/Ser(863)/Ser(877)) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser(863) phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser(863) via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e. g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser(863) phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser(863) as well as on the five other identified sites (e. g. Ser(859), Ser(855), Ser(877), Ser(696), and Thr(706)). Strikingly, raptor Ser(863) phosphorylation is absolutely required for raptor Ser(859) and Ser(855) phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser(863) phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e. g. on Ser(859) and Ser(855)). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.