Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth

Abstract

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.

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

“…For immunoprecipitation, whole-cell lysate (WCL) was incubated with antibodies for ϳ2 h or overnight at 4°C, incubated Flow cytometry to measure cell size and DNA content. Flp-In HEK293 cells that stably express vector control or AU1-mTOR alleles were cultured in DMEM-FBS-hygromycin (100 g/ml) in the absence or presence of rapamycin (20 ng/ml) and/or hydroxyurea (0.5 mM) for 96 h. For cell size and DNA content analysis, cells were harvested with PBS-EDTA (0.1%), fixed in 80% ethanol, and stained with propidium iodide (PI)-RNase A solution, as described previously (1). For cell size analysis, the mean forward scatter height (FSC-H) of ϳ3,000 single, unclumped G 1 -phase cells was determined by gating on PI fluorescence using a BD Biosciences FACSCalibur with CellQuest software.…”

“…Affinity-purified antipeptide antibodies to mTOR (amino acids 221 to 237; rat), P-mTOR-S1261 (amino acids 1256 to 1266; rat), S6K1 (Cterminal amino acids 485 to 502 of the 70-kDa isoform; rat), and P-S6 (amino acids 232 to 249) were generated as described previously (1). Phospho-specific antibodies against mTOR peptides phosphorylated at S2159 (catalog no.…”

“…AU1-mTOR was immunoprecipitated overnight, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie blue R-250, excised from the gel, digested with trypsin, and analyzed by tandem mass spectrometry (MS/MS). Immunoisolated AU1-mTOR was prepared for analysis by MS/MS as described previously (1).…”

“…Insulin/ PI3K signaling leads to Akt-and mTOR-mediated phosphorylation of PRAS40, which relieves the inhibitory effect of PRAS40 on mTORC1 (20,50,57,67,69). Insulin/PI3K signaling also increases mTOR S1261 and mTOR-mediated raptor S863 phosphorylation, events that promote mTORC1 function (1,21,71). In addition to phosphorylating PRAS40 and raptor, activated mTOR also phosphorylates deptor, leading to its degradation and thus relieving its mTOR-inhibitory action (52).…”

mTOR Signaling in Lymphangioleiomyomatosis

“…19,20 Phosphorylation of S1261 in cells exposed to insulin is required for the activation of mTOR, its autophosphorylation at S2481, and its regulation of cell size. 21 mTOR also contains amino acid residues that mediate its subcellular localization. mTOR nuclear trafficking requires two leucine residues in its HEAT repeat region (Fig.…”