Proximity labeling of endogenous RICTOR identifies mTOR complex 2 regulation by ADP ribosylation factor ARF1.
Proximity labeling of endogenous RICTOR identifies mTOR complex 2 regulation by ADP ribosylation factor ARF1.
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DOI：
10.1016/j.jbc.2022.102379
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发表时间：
2022-10
影响因子：
4.8
通讯作者：
Guertin, David A.
中科院分区：
文献类型：
--
作者：
Luciano, Amelia K.;Korobkina, Ekaterina D.;Lyons, Scott P.;Haley, John A.;Fluharty, Shelagh M.;Jung, Su Myung;Kettenbach, Arminja N.;Guertin, David A.
雷帕霉素（mTOR）复合物2 （mTORC2）调控代谢、细胞增殖和细胞存活。mTORC2活性受到生长因子的刺激，它磷酸化AGC激酶AKT、SGK和PKC的疏水基序位点。然而，与mTORC2相互作用以控制其活性和定位的蛋白质仍然不明确。为了鉴定活细胞中mTORC2相互作用蛋白，我们用修饰的BirA生物素连接酶BioID2标记内源性RICTOR （mTORC2的重要亚基），并进行了活细胞近距离标记。我们鉴定了215种rictor -近端蛋白，包括已知mTORC2通路相互作用的蛋白，以及135种以前未与mTORC2信号联系的蛋白（63%），包括核蛋白和细胞质蛋白。我们的成像和细胞分离实验表明，近30%的RICTOR在细胞核中，暗示了潜在的核功能。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物，从而深入了解mtorc2依赖性胰岛素信号动力学。最后，我们确定了内源性ADP核糖基化因子1 (ARF1) GTPase是mtorc2相互作用的蛋白。通过功能获得和功能丧失研究，我们提供了功能证据，证明ARF1可能负调控mTORC2。综上所述，我们提出了一种研究内源性mTORC2的新方法，这是活细胞中RICTOR/mTORC2蛋白相互作用的资源，以及ARF1 GTPase调控mTORC2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.
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