FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions

The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases, These kinases medi ate cellular responses to stresses such as DNA damage and nutrient deprivat ion in a variety of eukaryotes from yeast to humans. FRAP regulates G(1) ce ll cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulato rs. Although FRAP is known to be phosphorylated in vivo and to phosphorylat e several proteins (including itself) in vitro, FRAP's phosphorylation site s and substrate specificity are unknown. We report here the identification of a FRAP autophosphorylation site. This site, Ser-2481, is located in a hy drophobic region near the conserved carboxyl-terminal FRAP tail. We demonst rate that the COOH-terminal tail is required for FRAP kinase activity and f or signaling to the translational regulator p70(s6k) (ribosomal subunit S6 kinase), Phosphorylation of wild-type but not kinase-inactive FRAP occurs a t Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells. FRAP autophosphorylation is blocked com pletely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphory lation of eIF4E-binding protein (4E-BP1) and p70(s6k). Ser-2481 phosphoryla tion increases slightly upon c-Akt/ PHB activation and dramatically upon ca lyculin A treatment of T-cells, These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70(s6k) occurs through a mechanism other t han inhibition of intrinsic FRAP kinase activity.