Abstract
The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-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 phosphorylation of PHAS-I in cells.
Highlights
The mammalian target of rapamycin, mTOR1 (1) ( known as FRAP (2) or RAFT1 (3)), is a homolog of the Tor1p and Tor2p proteins that are required for cell cycle progression in Saccharomyces cerevisiae (4)
Insulin promotes the phosphorylation of PHAS-I at five Ser/Thr residues, each of which is located on the NH2-terminal side of an adjacent Pro residue (19)
The apparent Mr of the mTOR estimated by SDS-PAGE was 240,000, which is similar to the Mr of 289,000 predicted from the nucleotide sequence of the mTOR cDNA
Summary
The mammalian target of rapamycin, mTOR1 (1) ( known as FRAP (2) or RAFT1 (3)), is a homolog of the Tor1p and Tor2p proteins that are required for cell cycle progression in Saccharomyces cerevisiae (4). We describe an antibody that activates mTOR and identify the sites in PHAS-I that are phosphorylated by mTOR. Beads with immunoprecipitated or affinity-purified mTOR were suspended in 50 l of a reaction mixture that contained solution B plus 200 nM microcystinLR, 10 mM MnCl2, 100 M [␥-32P]ATP (10 Ci) (NEN Life Science Products), 1 g [His6]PHAS-I, and 50 mM -glycerophosphate, pH 7.4.
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