Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function

Authors

  • Noriko Oshiro,

    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
    2. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
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  • Ken-ichi Yoshino,

    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
    2. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
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  • Sujuti Hidayat,

    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
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  • Chiharu Tokunaga,

    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
    2. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
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  • Kenta Hara,

    1. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
    2. Department of Geriatric Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan
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  • Satoshi Eguchi,

    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
    2. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
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  • Joseph Avruch,

    1. Department of Molecular Biology and the Diabetes Unit, Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA
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  • Kazuyoshi Yonezawa

    Corresponding author
    1. Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
    2. CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
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  • Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: yonezawa@kobe-u.ac.jp

Abstract

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that plays a crucial role in a nutrient-sensitive signalling pathway that regulates cell growth. TOR signalling is potently inhibited by rapamycin, through the direct binding of a FK506-binding protein 12 (FKBP12)/rapamycin complex to the TOR FRB domain, a segment amino terminal to the kinase catalytic domain. The molecular basis for the inhibitory action of FKBP12/rapamycin remains uncertain. Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that is essential for mTOR signalling in vivo, and whose binding to mTOR is critical for mTOR-catalysed substrate phosphorylation in vitro. Here we investigated the stability of endogenous mTOR/raptor complex in response to rapamycin in vivo, and to the direct addition of a FKBP12/rapamycin complex in vitro. Rapamycin diminished the recovery of endogenous raptor with endogenous or recombinant mTOR in vivo; this inhibition required the ability of mTOR to bind the FKBP12/rapamycin complex, but was independent of mTOR kinase activity. Rapamycin, in the presence of FKBP12, inhibited the association of raptor with mTOR directly in vitro, and concomitantly reduced the mTOR-catalysed phosphorylation of raptor-dependent, but not raptor-independent substrates; mTOR autophosphorylation was unaltered. These observations indicate that rapamycin inhibits mTOR function, at least in part, by inhibiting the interaction of raptor with mTOR; this action uncouples mTOR from its substrates, and inhibits mTOR signalling without altering mTOR's intrinsic catalytic activity.

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