FKBP12-Rapamycin-associated Protein (FRAP) Autophosphorylates at Serine 2481 under Translationally Repressive Conditions

• Published in: The Journal of biological chemistry Vol. 275; no. 10; pp. 7416 - 7423
• Main Authors: Peterson, Randall T., Beal, Peter A., Comb, Michael J., Schreiber, Stuart L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.03.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Akt protein; Amino Acid Sequence; Androstadienes - pharmacology; Animals; FKBP12-rapamycin associated protein; Humans; Immunization; Initation factor eIF-4E-binding protein; Jurkat Cells; Molecular Sequence Data; phosphatidylinositol kinase-related kinase; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - immunology; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein Biosynthesis; Protein Kinases; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Rabbits; rapamycin; Serine - metabolism; Sirolimus - pharmacology; TOR Serine-Threonine Kinases; Wortmannin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.10.7416

The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases. These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation in a variety of eukaryotes from yeast to humans. FRAP regulates G1cell cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulators. Although FRAP is known to be phosphorylatedin vivo and to phosphorylate several proteins (including itself) in vitro, FRAP's phosphorylation sites and substrate specificity are unknown. We report here the identification of a FRAP autophosphorylation site. This site, Ser-2481, is located in a hydrophobic region near the conserved carboxyl-terminal FRAP tail. We demonstrate that the COOH-terminal tail is required for FRAP kinase activity and for signaling to the translational regulator p70s6k (ribosomal subunit S6 kinase). Phosphorylation of wild-type but not kinase-inactive FRAP occurs at Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells. FRAP autophosphorylation is blocked completely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphorylation of eIF4E-binding protein (4E-BP1) and p70s6k. Ser-2481 phosphorylation increases slightly upon c-Akt/PKB activation and dramatically upon calyculin A treatment of T-cells. These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70s6k occurs through a mechanism other than inhibition of intrinsic FRAP kinase activity.