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, 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.
Brunn
G J
GJ
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Fadden
P
P
Haystead
T A
TA
Lawrence
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011090:Polyenes / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D020123:Sirolimus; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011090:Polyenes; D013912:Threonine; D012694:Serine; D011392:Proline; D020123:Sirolimus