Dataset Information

MTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.

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.

SUBMITTER: Soliman GA

PROVIDER: S-EPMC2832937 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.

Soliman Ghada A GA Acosta-Jaquez Hugo A HA Dunlop Elaine A EA Ekim Bilgen B Maj Nicole E NE Tee Andrew R AR Fingar Diane C DC

The Journal of biological chemistry 20091218 11

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 c ...[more]

PMID: 20022946

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Project description:BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.

Dataset Information

MTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.

Publications

mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.

Similar Datasets

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