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Papers In Press, published online ahead of print March 28, 2003
Pharmacology Dept., University of Virginia, Charlottesville, VA 22908-0735
Corresponding Author: jcl3p{at}virginia.edu
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 eIF4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eIF4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The ArgAlaIlePro (RAIP motif) and PheGluMetAspIle (TOS 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 HA-tagged raptor, an mTOR-associated protein, after extracts of cells over-expressing raptor had been supplemented with recombinant PHAS-I proteins. Moreover, raptor over-expression 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 TOS 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.
J. Biol. Chem, 10.1074/jbc.M301142200
Submitted on February 3, 2003
Revised on March 24, 2003
Accepted on March 28, 2003
Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mTOR and recognition by raptor
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