Phosphorylation of Raptor by p38β Participates in Arsenite-induced Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation

Xiao-Nan Wu,Xue-Kun Wang,Su-Qin Wu,Jiawei Lu,Min Zheng,Yan-Hai Wang,Huamin Zhou,Hongbing Zhang,Jiahuai Han

doi:10.1074/jbc.m111.233122

Abstract

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.

Highlights

The p38 mitogen-activated protein kinase (MAPK) signal pathway plays an important role in a variety of biological processes, including inflammation, cell differentiation, and cell death [1,2,3]
We demonstrate that a specific p38 group member, p38␤, plays an important role in arsenite-induced activation of mTOR complex 1 (mTORC1)
It was known that MK2 plays a role in anisomycin-induced mTORC1 activation [33], whereas it has no role in arseniteinduced mTORC1 activation

Summary

EXPERIMENTAL PROCEDURES

Materials—Sodium arsenite, rapamycin, insulin, and anisomycin were obtained from Sigma. The immunoprecipitates were washed three times each with lysis buffer followed by addition of 1ϫ SDS sample buffer, boiling for 10 min, resolved by SDSPAGE, and analyzed by immunoblotting. Calf Intestinal Alkaline Phosphatase (CIAP) Treatment—As described [24], anti-HA beads used for immunoprecipitating HA-Raptor deletions were washed three times in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na3VO4, 1 ␮g/ml of leupeptin, 0.1% SDS, and 1 mM PMSF) followed by two additional washes in CIAP buffer (100 mM NaCl, 50 mM Tris, pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol). Transfected cells were treated with 1 mM arsenite for 30 min as indicated in the figures and lysed in CHAPS lysis buffer followed by immunoprecipitation by HA antibodies. The reactions were terminated by 2ϫ SDS sample buffer, and subjected to SDS-PAGE and immunoblotting

RESULTS

DISCUSSION