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.

Mammalian Target of Rapamycin (mTOR): Conducting the Cellular Signaling Symphony

“…Prior to the identification of distinct mTOR complexes, mTOR Ser 2481 autophosphorylation was believed to be insensitive to rapamycin and amino acid withdrawal, leading to the idea that modulation of mTOR intrinsic kinase activity does not universally underlie mTOR regulation (88). More recent work indicates, however, that rapamycin and amino acid withdrawal reduce mTORC1-but not mTORC2-associated mTOR Ser 2481 autophosphorylation, consistent with the known sensitivities or lack thereof of mTORC1 and mTORC2 to these conditions (11,12). Thus, mTORC1-and mTORC2-associated mTOR Ser 2481 autophosphorylation serves as a simple biomarker that monitors intrinsic mTOR catalytic activity.…”

mTOR Kinase Domain Phosphorylation Promotes mTORC1 Signaling, Cell Growth, and Cell Cycle Progression

“…By modulating inhibitory interactions of mTOR with mTORC1 components, mTOR S2159/ T2164 phosphorylation shifts mTORC1 toward a more active conformation. We have shown previously that insulin stimulation or Rheb overexpression promotes mTOR S2481 autophosphorylation within mTORC1, which monitors mTORC1 intrinsic catalytic activity in intact cells (62). Additionally, mTOR S1261 phosphorylation is required, at least in part, for the ability of Rheb to promote mTORC1-associated mTOR S2481 autophosphorylation (1).…”

“…mTOR interacts with different partner proteins to form at least two functionally distinct signaling complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (2,4). Acute rapamycin treatment inhibits the intrinsic catalytic activity and signaling capacity of mTORC1, which contains mTOR, mLST8 (lethal with sec13 protein 8)/G␤L (G-protein, ␤-subunit-like protein), raptor, PRAS40 (proline-rich Akt substrate of 40 kDa), and deptor (DEP domain protein that interacts with mTOR) (25,27,38,39,43,52,57,62,67). Acute rapamycin treatment fails to inhibit mTORC2, which contains shared and distinct partners (2,4,22).…”

Phosphatidic Acid Activates Mammalian Target of Rapamycin Complex 1 (mTORC1) Kinase by Displacing FK506 Binding Protein 38 (FKBP38) and Exerting an Allosteric Effect

“…Kinase Activity-To evaluate a potential effect of PA on the kinase activity of mTOR in cells, we examined the phosphorylation of mTOR on Ser-2481, an autophosphorylation site that has recently been reported to monitor mTORC-specific catalytic activities (27). To avoid potential complications from exogenous PA-derived lysophosphatidic acid (28), which would initiate signaling through the membrane-bound lysophosphatidic acid receptors, we used a shortchain PA (C8-PA) for delivery into cells, which would not be converted into active lysophosphatidic acid (29,30).…”