mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action.
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.
Soliman
Ghada A
GA
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA.
Acosta-Jaquez
Hugo A
HA
Dunlop
Elaine A
EA
Ekim
Bilgen
B
Maj
Nicole E
NE
Tee
Andrew R
AR
Fingar
Diane C
DC
D041721:3T3-L1 Cells; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000906:Antibodies / Q000494:pharmacology; D002384:Catalysis; D002461:Cell Line, Transformed; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D046912:Multiprotein Complexes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D011506:Proteins; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D000906:Antibodies; C493245:CRTC2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D011506:Proteins; D014157:Transcription Factors; D012694:Serine; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus