Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

Abstract

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [ 32 P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/ protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition. Mammalian target of rapamycin (mTOR)2 has been demonstrated as a key element in signaling pathways controlling cell size, proliferation, and metabolism (1, 2). Two mTOR signaling complexes, mTORC1 and mTORC2, have been discovered. The rapamycin-sensitive mTORC1 consists of the catalytic subunit mTOR, the substrate-binding subunit raptor (regulatory associated protein of mTOR), mLST8 (also known as G␤L), and PRAS40 (1, 2) and controls protein translation (1, 2). mTORC2, the rapamycin-insensitive form, contains mTOR, rictor, SIN1, mLST8, and PRR5 (1, 2) and functions as a PDK2 (phosphoinositide-dependent protein kinase 2) to phosphorylate Akt/protein kinase B at Ser-473 and regulates the actin cytoskeleton (1, 2). The best characterized downstream effectors of mTORC1 are S6K1 and 4E-BP1 (also known as PHAS-I), both of which are phosphorylated by mTORC1 at multiple sites and are involved in the control of mRNA translation (1, 2). The nature of the phosphorylation sites in these two mTORC1 substrates is surprisingly different: either (S/T)P (3) or h(S/T)h (where h represents hydrophobic) (4). Thus, the surrounding amino acids appear not to be the major determinant for phosphorylation. A critical motif called the TOR signaling (TOS) motif has been discovered in the NH 2 terminus of S6K1 (FDIDL) and COOH terminus of 4E-BP1 (FEMDI) (5, 6). Mutation of the TOS motif not only decreases the rate of phosphorylation of S6K1 and 4E-BP1 by mTOR but also disrupts interaction between these substrates and raptor (5-8).PRAS40 has been recently identified as a protein associated with mTORC1 (9, 10). PRAS40 predominantly interacts with raptor, althoug...

mTOR Kinase Domain Phosphorylation Promotes mTORC1 Signaling, Cell Growth, and Cell Cycle Progression

“…mTOR S2159/T2164 phosphorylation additionally modulates the interaction of raptor with PRAS40, as expression of DE-mTOR weakens the raptor-PRAS40 interaction in the absence of serum growth factors while expression of AA-mTOR strengthens the raptor-PRAS40 interaction in both the absence and presence of insulin. Thus, similar to Akt-and mTOR-mediated phosphorylation of PRAS40 (50,57,67,69), phosphorylation on mTOR itself weakens the raptor-PRAS40 interaction to promote mTORC1 signaling. As PRAS40 contains a TOS motif and may thus inhibit mTORC1 signaling by functioning as a competitive substrate (20,50,70), mTOR S2159/T2164 phosphorylation may facilitate S6K1 or 4EBP1 docking and/or positioning by suppressing competitive PRAS40 action.…”

“…Insulin/ PI3K signaling leads to Akt-and mTOR-mediated phosphorylation of PRAS40, which relieves the inhibitory effect of PRAS40 on mTORC1 (20,50,57,67,69). Insulin/PI3K signaling also increases mTOR S1261 and mTOR-mediated raptor S863 phosphorylation, events that promote mTORC1 function (1,21,71).…”

mTOR-Dependent Cell Survival Mechanisms

“…Recent work is beginning to reveal insight into the structure of mTORC1 (Yip et al 2010). A cryo-EM structure of mTORC1 at 26 Å indicates that the complex is a dimer with interlocking mTOR-RAPTOR interactions and where PRAS40 acts as a competitive inhibitor for the binding of mTORC1 substrates to RAPTOR (Wang et al 2008;Yip et al 2010). mLST8 associates with the mTOR kinase domain, located in the carboxyl terminus (Kim et al 2003).…”

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

“…32 P radiolabeling and two-dimensional phosphopeptide mapping were performed as described previously (23).…”