The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1.
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 (Ser(183)). 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 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) 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, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) 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 (Ser(183)) 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.
Oshiro
Noriko
N
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Takahashi
Rinako
R
Yoshino
Ken-ichi
K
Tanimura
Keiko
K
Nakashima
Akio
A
Eguchi
Satoshi
S
Miyamoto
Takafumi
T
Hara
Kenta
K
Takehana
Kenji
K
Avruch
Joseph
J
Kikkawa
Ushio
U
Yonezawa
Kazuyoshi
K
D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes; D020125:Mutation, Missense; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C104151:EIF4EBP2 protein, human; D039642:Eukaryotic Initiation Factors; D046912:Multiprotein Complexes; D009479:Neuropeptides; D010750:Phosphoproteins; D011506:Proteins; C490211:RHEB protein, human; C463659:RPTOR protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins