Abstract
The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.
Highlights
MTORC2, the rapamycin-insensitive form, contains mammalian target of rapamycin (mTOR), rictor, SIN1, mLST8, and PRR5 [1, 2] and functions as a PDK2 to phosphorylate Akt/protein kinase B at Ser-473 and regulates the actin cytoskeleton [1, 2]
HEK293 cells. mTOR complexes were isolated with antibodies toward FLAG-tagged mTOR, and mTOR kinase assays were performed with PRAS40 as substrate
D, phosphorylation of the indicated PRAS40 mutants in kinase assays with mTORC1 and Akt1 immune complexes were assessed by 32P incorporation
Summary
MTORC2, the rapamycin-insensitive form, contains mTOR, rictor, SIN1, mLST8, and PRR5 [1, 2] and functions as a PDK2 (phosphoinositide-dependent protein kinase 2) to phosphorylate Akt/protein kinase B at Ser-473 and regulates the actin cytoskeleton [1, 2]. PRAS40 wild type and mutant proteins were phosphorylated by mTORC1 in vitro, and after SDS-PAGE, 32P-labeled PRAS40 was digested by trypsin and subjected to phosphopeptide mapping analysis. To confirm that these identified residues are the major sites phosphorylated by mTORC1 in vitro, PRAS40 mutants S212A, S221A, and the combined S183A/S212A/S221A mutations were used in an mTORC1 kinase assay.
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