Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor.
Mammalian target of rapamycin (mTOR) is the central element of a signaling pathway involved in the control of mRNA translation and cell growth. The actions of mTOR are mediated in part through the phosphorylation of the eukaryotic initiation factor 4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eukaryotic initiation factor 4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The Arg-Ala-Ile-Pro (RAIP motif) and Phe-Glu-Met-Asp-Ile (tor signaling motif) sequences found in the NH2- and COOH-terminal regions of PHAS-I, respectively, are required for the efficient phosphorylation of PHAS-I in cells. Here we show that mutations in either motif markedly decreased the phosphorylation of recombinant PHAS-I by mTOR in vitro. Wild-type PHAS-I, but none of the mutant proteins, was coimmunoprecipitated with hemagglutinin-tagged raptor, an mTOR-associated protein, after extracts of cells overexpressing raptor had been supplemented with recombinant PHAS-I proteins. Moreover, raptor overexpression enhanced the phosphorylation of wild-type PHAS-I by mTOR but not the phosphorylation of the mutant proteins. The results not only provide direct evidence that both the RAIP and tor signaling motifs are important for the phosphorylation by mTOR, possibly by allowing PHAS-I binding to raptor, but also support the view that mTOR phosphorylates PHAS-I in cells.
Choi
Kin Man
KM
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908, USA.
McMahon
Lloyd P
LP
Lawrence
John C
JC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000076223:Regulatory-Associated Protein of mTOR; D058570:TOR Serine-Threonine Kinases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C499526:Eif4ebp1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011506:Proteins; C463659:RPTOR protein, human; D011994:Recombinant Proteins; D000076223:Regulatory-Associated Protein of mTOR; C572458:Rptor protein, rat; D011494:Protein Kinases; C546842:MTOR protein, human; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases