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J. Biol. Chem., Vol. 282, Issue 28, 20329-20339, July 13, 2007

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The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1^*

Noriko Oshiro, Rinako Takahashi, Ken-ichi Yoshino, Keiko Tanimura, Akio Nakashima, Satoshi Eguchi, Takafumi Miyamoto, Kenta Hara, Kenji Takehana^¶, Joseph Avruch^||1, Ushio Kikkawa, and Kazuyoshi Yonezawa²

From the Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, the Department of Internal and Geriatric Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan, the ^¶Pharmaceutical Research Laboratories, Ajinomoto Co. Inc., Kawasaki 210-8681, Japan, and the ^||Department of Molecular Biology and the Diabetes Unit, Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser¹⁸³). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe¹²⁹ to Ala). Immediately carboxyl-terminal to Phe¹²⁹ are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser¹⁸³, to Asp. PRAS40 (Ser¹⁸³) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser¹⁸³) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser¹⁸³) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.

Received for publication, March 27, 2007 , and in revised form, May 17, 2007.

^* This work was supported in part by research grants from the Scientific Research Funds of the Ministry of Education, Culture, Sports, Science and Technology of Japan and CREST, Japan Science and Technology Agency. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

² Deceased on July 8, 2005.

¹ Supported by National Institutes of Health Grants DK17776 and CA73818. To whom correspondence should be addressed: Diabetes Research Laboratory, Dept. of Molecular Biology, Massachusetts General Hospital, 185 Cambridge St., Boston, MA 02114. Tel.: 617-726-6909; Fax: 617-726-5649; E-mail: avruch{at}molbio.mgh.harvard.edu.

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