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J. Biol. Chem., Vol. 278, Issue 22, 19667-19673, May 30, 2003

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Two Motifs in the Translational Repressor PHAS-I Required for Efficient Phosphorylation by Mammalian Target of Rapamycin and for Recognition by Raptor^*

Kin Man Choi , Lloyd P. McMahon  and John C. Lawrence, Jr.   ¶

From the Departments of Pharmacology and Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908

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.

Received for publication, February 3, 2003 , and in revised form, March 24, 2003.

^* This work was supported by National Institutes of Health Grants DK52753 and DK28312. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, P. O. Box 800735, University of Virginia Health System, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 434-924-1584; Fax: 434-982-3575; E-mail: jcl3p{at}virginia.edu.

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