Characterization of the phosphoproteins and protein kinase activity in mTOR immunoprecipitates.
The mammalian target of rapamycin, mTOR, has been shown to be an upstream regulator of translational effectors. In the present study, in order to detect potential molecules involved in the mTOR signaling, an in vitro phosphorylation assay using mTOR immunoprecipitates from HEK293 cells was carried out. In addition to the autophosphorylation of mTOR, 32P incorporation into 80-kDa (pp80) and 175-kDa (pp175) bands was observed in mTOR immunoprecipitates. The protein kinase activity toward the recombinant eIF-4E binding protein 1 (4E-BP1) was also detected as previously described. When mTOR immunoprecipitates from HEK293 cells were prepared in the presence of a detergent, Nonidet P-40, the 4E-BP1 kinase activity and 32P incorporation into pp175 dramatically diminished, while the phosphorylation of mTOR and 32P incorporation into pp80 did not change. These results raised a possibility that mTOR may associate with protein cofactors, some of which may be involved in the regulation of kinase activities associated with mTOR.
Nishiuma
T
T
Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Kobe, Nada-ku, 657-8501, Japan.
Hara
K
K
Tsujishita
Y
Y
Kaneko
K
K
Shii
K
K
Yonezawa
K
K
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D006224:Cricetinae; D039642:Eukaryotic Initiation Factors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008970:Molecular Weight; D017830:Octoxynol; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C499527:Eif4ebp1 protein, mouse; C499526:Eif4ebp1 protein, rat; D039642:Eukaryotic Initiation Factors; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; C546843:mTOR protein, mouse; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin