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

Abstract

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.The mammalian target of rapamycin (mTOR) 3 is the founding member of the PI-3 kinase-related family of protein (Ser/ Thr) kinases (PIKKs) and controls many important aspects of the cellular response to nutrient sufficiency and growth factors (1). The mTOR polypeptide is now known to function in two distinct, independently regulated hetero-oligomeric complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes contain mTOR and the polypeptide mLST8/G␤L; mTORC1 in addition contains raptor (an ortholog of Saccharomyces cerevisiae KOG1), which binds directly to the known mTORC1 substrates S6K1 and 4E-BP1 and is indispensable for their phosphorylation by mTOR in vivo and in vitro. mTORC2 lacks raptor but contains the polypeptides rictor (an ortholog to ScAVO3) and mSin1 (an ortholog of ScAVO1). mTORC2 is one of the activating kinases for Akt, previously called PDK2, and also regulates the actin cytoskeleton through as yet unidentified effectors. Although rapamycin, in complex with FKBP12, binds directly to mTOR in a segment just amino-terminal to the catalytic domain, only mTORC1 binds the FKBP12-rapamycin complex, and thus only mTORC1 is directly susceptible to inhibition by rapamycin.Rapamycin is among the most selective kinase inhibitors k...

Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells

“…*** p<0.001, ** p<0.01 and * p<0.05 for the comparisons indicated in the bar graphs. Rapa, rapamycin; PF, PF-4708671 Accordingly, overexpression of PRAS40 impaired the insulin-mediated phosphorylation of the mTORC1 substrates p70S6K and 4E-BP1 [19,20,22,23]. These findings led to the suggestion that the dissociation of phosphorylated PRAS40 from raptor could promote downstream mTORC1 signalling by increasing substrate binding to raptor [16,17].…”

“…Yet studies of the function of this protein within mTORC1 have yielded conflicting results [18][19][20][21][22][23]. Silencing and overexpression studies, mostly in immortalised cultured cell lines, have ascribed both inhibitory and stimulatory functions to PRAS40 in the regulation of mTORC1 activity [18][19][20][21][22][23]. Importantly, a recent study conducted in Drosophila melanogaster shed light on these seemingly contrasting findings, demonstrating that D. melanogaster dPRAS40 acts as an inhibitor of TORC1 signalling in a tissue-specific way that depends on post-translational modification of the protein [24].…”

“…PRAS40 is a component of mTORC1 [16,17]. Yet studies of the function of this protein within mTORC1 have yielded conflicting results [18][19][20][21][22][23]. Silencing and overexpression studies, mostly in immortalised cultured cell lines, have ascribed both inhibitory and stimulatory functions to PRAS40 in the regulation of mTORC1 activity [18][19][20][21][22][23].…”

Not all substrates are treated equally: Implications for mTOR, rapamycin-resistance, and cancer therapy

“…This motif, termed the TOR signaling (TOS) motif, is necessary for substrate-Raptor interaction, and the abrogation of this interaction correlates with an inability for mTORC1 to signal to the mutated substrate. [15][16][17][18][19] The TOS motif is present in the known mTORC1 substrates S6K1, 4E-BP1 and PRAS40. [17][18][19] However, it remains to be determined if the TOS motif is required for all mTORC1 substrates to be phosphorylated.…”

“…Although little is known about the details of mTORC1's proteomic composition, proteins such as mLST8 and PRAS40 have been shown to be critical regulators of mTORC1's activity. 18,19,[28][29][30][31] Whether a protein like PRAS40 dissociates from mTORC1 or an activator associates with mTORC1 after long-term rapamycin treatment remains to be investigated. Such a model would be consistent with the requirement of newly synthesized proteins for the re-emerging phosphorylation.…”