The mTOR (Mammalian Target of Rapamycin) Kinase Maintains Integrity of mTOR Complex 2

Chien-Hung Chen,Dos D Sarbassov

doi:10.1074/jbc.m111.282590

Chien-Hung Chen, Dos D Sarbassov

Open Access

https://doi.org/10.1074/jbc.m111.282590

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Abstract

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.

Highlights

MTORC2 is a major regulatory kinase of Akt, and its regulation remains poorly characterized
This complex dissociates into two heterodimers rictor/SIN1 and mTOR/ mLST8 when cells are lysed under stringent buffer containing Triton X-100 detergent
These observations led to an assumption that two heterodimers rictor/SIN1 and mTOR/mLST8 are formed at the initial stage of the mTORC2 assembly, and these heterodimers form a low affinity Triton X-100-sensitive mTORC2 complex

Summary

Introduction

MTORC2 is a major regulatory kinase of Akt, and its regulation remains poorly characterized. To prevent a potential effect of autophosphorylation of Akt, we carried the kinase assay with the kinaseinactive form of GST-Akt. We found that mTORC2 assembled with the wild-type mTOR shows a high kinase activity toward the inactive Akt kinase, indicating that autophosphorylation of Akt does not occur in the mTORC2-dependent phosphorylation of Akt. In a follow-up analysis we found that the kinase-dead form of mTOR did not interfere with expression and binding of its binding partner mLST8, but it caused a substantial decrease in expression of SIN1 and rictor that subsequently translated to a low assembly of mTORC2 (Fig. 2A, middle and bottom).

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