Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies.
The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.
Mothe-Satney
I
I
Departments of Pharmacology and Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Brunn
G J
GJ
McMahon
L P
LP
Capaldo
C T
CT
Abraham
R T
RT
Lawrence
J C
JC
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D011494:Protein Kinases; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D000906:Antibodies; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D007328:Insulin; D010750:Phosphoproteins; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus