Abstract
Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mM and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.
Highlights
MTORC2 integrity is dependent on SIN1 phosphorylation
These data indicate that the basal kinase activity of mTORC2 is sensitive to glucose but not amino acid deprivation, and its low activity is associated with a decrease in abundance of SIN1 and rictor
Our results show that the cellular ATP level regulates the basal kinase activity of mTORC2 because the optimal kinase activity of this complex requires ATP concentration at the physiological range above 1.2 mM
Summary
Materials and Cell Culture—Reagents were obtained from the following sources: Dulbecco’s modified Eagle’s medium (DMEM)/F-12 from Invitrogen; fetal bovine serum (FBS) from Hyclone; Fugene 6 transfection reagent and the complete protease inhibitor mixture from Roche Applied Science; protein G-Sepharose from Pierce; insulin-like growth factor I from Peprotech; the antibodies to Xpress, Myc, and V5 tag from Invitrogen; the antibodies to phospho-Ser-260 SIN1, mTOR, rictor, Akt, phospho-Ser-473, and phospho-Thr-450 Akt from Cell Signaling Technologies; the antibodies to rictor, HRP-labeled anti-rabbit, anti-mouse, anti-goat secondary antibodies, and tubulin from Santa Cruz Biotechnology, Inc.; and the raptor antibody from Bethyl. Following a 1-h incubation with 40 l of a 50% slurry of protein G-agarose, immunoprecipitates captured by protein G-agarose were washed four times with the CHAPS-containing lysis buffer and once with rictor-mTOR kinase buffer (25 mM Hepes, pH 7.5, 100 mM potassium acetate, 2 mM MgCl2). After a 48-h transfection, cells were washed with cold PBS, lysed with 0.3% CHAPS buffer, and incubated with mild agitation for 20 min at 4 °C. After a 48-h transfection, cells were washed with cold PBS, lysed with 0.3% CHAPS buffer, and subjected to centrifugation for 15 min at 10,000 rpm. The Myc-rictor/SIN1-V5 immunoprecipitates were incubated with or without the purified soluble FLAG-mLST/ Myc-mTOR heterodimer at room temperature for 2 h in mTORC2 kinase buffer with 1 mM ATP.
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