雷帕霉素(mTOR)复合物2(mTORC2)调节代谢的机制靶点,细胞增殖,细胞存活。mTORC2活性受生长因子刺激,它磷酸化AGC激酶AKT的疏水基序位点,SGK,PKC。然而,与mTORC2相互作用以控制其活性和定位的蛋白质仍然不明确。为了鉴定活细胞中的mTORC2相互作用蛋白,我们标记了内源性RICTOR,一个必需的mTORC2亚基,用修饰的BirA生物素连接酶BioID2进行活细胞邻近标记。我们鉴定了215个RICTOR近端蛋白,包括已知mTORC2通路相互作用的蛋白质,135种蛋白质(63%)以前与mTORC2信号没有联系,包括细胞核和细胞质蛋白。我们的成像和细胞分级分离实验表明,近30%的RICTOR在细胞核中,暗示潜在的核功能。我们还确定了29个含有RICTOR依赖性的相互作用者,胰岛素刺激的磷酸化位点,从而深入了解mTORC2依赖性胰岛素信号传导动力学。最后,我们将内源性ADP核糖基化因子1(ARF1)GTPase鉴定为mTORC2相互作用蛋白.通过功能增益和功能损失研究,我们提供了ARF1可能负向调节mTORC2的功能证据。总之,我们提出了一种研究内源性mTORC2的新方法,mTORC2是活细胞中RICTOR/mTORC2蛋白相互作用的资源,以及ARF1GTPase对mTORC2调控的潜在机制。
Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase
BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.