MTOR Ser-2481 Autophosphorylation Monitors mTORC-specific Catalytic Activity and Clarifies Rapamycin Mechanism of Action

Diane C. Fingar,Hugo A. Acosta-Jaquez,Bilgen Ekim,Ghada A. Soliman,Elaine A. Dunlop,Nicole E. Maj,Andrew R. Tee

doi:10.1074/jbc.m109.096222

Diane C. Fingar, Hugo A. Acosta-Jaquez + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.096222

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Abstract

The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin- and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1- and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1- and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.

Highlights

The evolutionarily conserved mammalian target of rapamycin4 protein kinase functions as an environmental sensor, integrating signals from diverse cellular stimuli to control cellular physiology [1,2,3]. mTOR signals in at least two distinct multiprotein complexes distinguished by their partner proteins and differing sensitivities to rapamycin, a clinically employed immunosuppressive drug and allosteric mTOR inhibitor [4, 5]
Rapamycin Inhibits mTOR complex 1 (mTORC1) but Not mTOR complex 2 (mTORC2)-associated mTOR Ser-2481 Autophosphorylation in Response to Insulin/ PI3K Signaling—While immunoblotting whole cell lysates derived from 3T3-L1 adipocytes, we were surprised to observe that pretreatment of cells with rapamycin, an mTORC1-specific inhibitor, reduced insulin-stimulated mTOR Ser-2481 autophosphorylation (Ser(P)-2481)
Moving downstream rapamycin binding domain (42, 43, 64 – 67), the molecular of PI3K, we found that tuberous sclerosis complex (TSC) suppresses and Rheb promotes raptordetails of rapamycin-mediated mTOR inhibition remain associated mTOR Ser(P)-2481

Summary

Introduction

The evolutionarily conserved mammalian target of rapamycin (mTOR)4 protein kinase functions as an environmental sensor, integrating signals from diverse cellular stimuli to control cellular physiology [1,2,3]. mTOR signals in at least two distinct multiprotein complexes distinguished by their partner proteins and differing sensitivities to rapamycin, a clinically employed immunosuppressive drug and allosteric mTOR inhibitor [4, 5]. These data indicate that in HEK293 cells and 3T3-L1 adipocytes, rapamycin inhibits mTORC1-associated mTOR Ser-2481 autophosphorylation. In HEK293 cells, strong mTORC1-associated mTOR Ser(P)-2481 was observed as early as 5 min after insulin stimulation, before maximal phosphorylation of downstream substrates (e.g. S6), and was maintained for up to 2 h (supplemental Fig. S5).

Results

Conclusion