Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.
Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
J Biol Chem. 2022 Oct;298(10):102379. doi: 10.1016/j.jbc.2022.102379. Epub 2022 Aug 13.
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.
雷帕霉素靶蛋白复合物 2（mTORC2）调节代谢、细胞增殖和细胞存活。mTORC2 的活性受生长因子刺激，它磷酸化 AGC 激酶 AKT、SGK 和 PKC 的疏水性基序位点。然而，与 mTORC2 相互作用以控制其活性和定位的蛋白质仍然定义不明确。为了在活细胞中鉴定 mTORC2 相互作用蛋白，我们用改良的 BirA 生物素连接酶 BioID2 标记内源性 RICTOR（mTORC2 的必需亚基），并进行活细胞邻近标记。我们鉴定了 215 个 RICTOR 近端蛋白，包括具有已知 mTORC2 途径相互作用的蛋白，以及 135 个（63%）以前与 mTORC2 信号无关的蛋白，包括核蛋白和细胞质蛋白。我们的成像和细胞分级实验表明，近 30%的 RICTOR 位于细胞核内，暗示其可能具有核功能。我们还鉴定了 29 个含有 RICTOR 依赖性、胰岛素刺激磷酸化位点的相互作用蛋白，从而深入了解 mTORC2 依赖性胰岛素信号动态。最后，我们鉴定了内源性 ADP 核糖基化因子 1（ARF1）GTPase 作为 mTORC2 相互作用蛋白。通过功能获得和功能丧失研究，我们提供了功能证据表明 ARF1 可能负调节 mTORC2。总之，我们提出了一种研究内源性 mTORC2 的新方法，提供了活细胞中 RICTOR/mTORC2 蛋白相互作用的资源，以及 ARF1 GTPase 调节 mTORC2 的潜在机制。
