PRAS40 Is a Target for Mammalian Target of Rapamycin Complex 1 and Is Required for Signaling Downstream of This Complex

Abstract

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid-and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.

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 vs cells transfected with NT siRNA; † p <0.05 for the effect of insulin-treated (+) vs untreated (−) cells. Rapa, rapamycin cells, neither knockdown nor overexpression of PRAS40 affected insulin-mediated Akt phosphorylation [22]. Yet, in line with our findings with hSkMC, knockdown of PRAS40 resulted in the degradation of IRS1 in 3T3L1 adipocytes, HepG2 cells and C2C12 myoblasts [21,32], and reduction of insulin-mediated Akt phosphorylation in 3T3L1 adipocytes and HepG2 cells [21].…”

“…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]. However, this proposed function for PRAS40 is in contrast with our findings on hSkMC and multiple other studies [16,22,33,34]. In HEK293E cells, the dissociation of PRAS40 from raptor was found to increase 4E-BP1 binding to raptor, but not to affect the basal or insulin-stimulated phosphorylation of p70S6K and 4E-BP1 [33].…”

“…*** 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].…”

“…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].…”

“…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].…”