THE MAMMALIAN TARGET OF RAPAMYCIN PHOSPHORYLATES SITES HAVING A (SER THR)-PRO MOTIF AND IS ACTIVATED BY ANTIBODIES TO A REGION NEAR ITS COOH TERMINUS/

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, w as phosphorylated rapidly and stoichiometrically when incubated with [ gamma-P-32]ATP and the mammalian target of rapamycin (mTOR) that had b een immunoprecipitated with an antibody, mTAb1, directed against a reg ion near the COOH terminus of mTOR. PHAS-I was phosphorylated more slo wly by mTOR obtained either by immunoprecipitation with other antibodi es or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phospho rylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr(36), Thr(45), Ser(64) , Thr(69), and Ser(82) in PHAS-I. All five of these sites fit a (Ser/T hr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the p hosphorylation of PHAS-I in cells.