Structure of S6 Kinase 1 Determines whether Raptor-mTOR or Rictor-mTOR Phosphorylates Its Hydrophobic Motif Site

Siraj M Ali,David M Sabatini

doi:10.1074/jbc.c500125200

Siraj M Ali, David M Sabatini

Open Access

https://doi.org/10.1074/jbc.c500125200

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Abstract

The mTOR protein kinase is the target of the immunosuppressive and anti-cancer drug rapamycin and is increasingly recognized as a key regulator of cell growth in mammals. S6 kinase 1 (S6K1) is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. Nutrients and growth factors activate S6K1 by inducing the phosphorylation of threonine 389 in the hydrophobic motif of S6K1. As phosphorylation of Thr(389) is rapamycin sensitive and mTOR can phosphorylate the same site in vitro, it has been suggested that mTOR is the physiological Thr(389) kinase. This proposal is not supported, however, by the existence of mutants of S6K1 that are phosphorylated in vivo on Thr(389) in a rapamycin-resistant fashion. Here, we demonstrate that the raptor-mTOR complex phosphorylates the rapamycin-sensitive forms of S6K1, while the distinct rictor-mTOR complex phosphorylates the rapamycin-resistant mutants of S6K1. Phosphorylation of Thr(389) by rictor-mTOR is independent of the TOR signaling motif and depends on removal of the carboxyl terminal domain of S6K1. Because many members of the AGC family of kinases lack an analogous domain, rictor-mTOR may phosphorylate the hydrophobic motifs of other kinases.

Highlights

From the Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts 02142
Using RNAi to suppress the expression of mTOR and its associated proteins we find that mTOR is the threonine 389 (Thr389) kinase of wild-type and rapamycin-resistant S6 kinase 1 (S6K1)
To determine whether mTOR is essential for the phosphorylation of rapamycin-resistant mutants of S6K1, we inhibited the expression of mTOR and its associated proteins with RNAi mediated by lentivirally transduced shRNAs [15, 17]

Summary

Introduction

These results are consistent with raptor-mTOR being the in vivo regulator and likely hydrophobic motif site kinase for S6K1 and indicate that rictor-mTOR is not involved in S6K1 signaling. Having validated our RNAi reagents we asked whether the raptor-mTOR complex regulates Thr389 phosphorylation of the rapamycin-resistant mutants of S6K1. As a rictor knockdown does not affect the phosphorylation of wild-type S6K1 (Fig. 1B), rictor-mediated regulation of S6K1 must require deletion of the region COOH-terminal to the kinase domain and/or destruction of the TOS motif.

Results

Conclusion