A role for Raptor phosphorylation in the mechanical activation of mTOR signaling

• Published in: Cellular signalling Vol. 26; no. 2; pp. 313 - 322
• Main Authors: Frey, John W., Jacobs, Brittany L., Goodman, Craig A., Hornberger, Troy A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.02.2014
• Subjects: Activation; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Bacterial Agents - pharmacology; Exercise; Hypertrophy; In Vitro Techniques; Mass Spectrometry; Mechanical activation; Mechanotransduction; Mice; Mice, Inbred C57BL; Molecular Sequence Data; mTOR; Muscle, Skeletal - metabolism; Muscles; Pathways; Phosphopeptides - analysis; Phosphoproteins - metabolism; Phosphorylation; Phosphorylation - drug effects; Protein Binding; Raptor; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Signal Transduction; Sirolimus - pharmacology; Skeletal muscle; Stimulation; Stimuli; Stress, Mechanical; TOR Serine-Threonine Kinases - metabolism; Upstream; GβL; p70S6k; JNK; MAPK; TA; Raptor; Skeletal muscle; GFP; Exercise; p38; PI3K; PRAS40; mTOR; Mechanotransduction; mLST8; mTORC2; mTORC1; EC; Hypertrophy; tibialis anterior muscle; mTOR complex 2; mTOR complex 1; phosphotidylinositol-3-kinase; mitogen-activated protein kinase; G-protein β-subunit-like protein; regulatory associated protein of mTOR; p70(S6k); mammalian [or mechanistic] target of rapamycin; lethal with sec13 protein 8; green fluorescent protein; eccentric contraction; proline-rich Akt substrate of 40kDa; c-jun n-terminal kinase; p38 mitogen-activated protein kinase; ribosomal S6 kinase 1
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2013.11.009

The activation of mTOR signaling is necessary for mechanically-induced changes in skeletal muscle mass, but the mechanisms that regulate the mechanical activation of mTOR signaling remain poorly defined. In this study, we set out to determine if changes in the phosphorylation of Raptor contribute to the mechanical activation of mTOR. To accomplish this goal, mouse skeletal muscles were subjected to mechanical stimulation via a bout of eccentric contractions (EC). Using mass spectrometry and Western blot analysis, we found that ECs induced an increase in Raptor S696, T706, and S863 phosphorylation, and this effect was not inhibited by rapamycin. This observation suggested that changes in Raptor phosphorylation might be an upstream event in the pathway through which mechanical stimuli activate mTOR. To test this, we employed a phospho-defective mutant of Raptor (S696A/T706A/S863A) and found that the EC-induced activation of mTOR signaling was significantly blunted in muscles expressing this mutant. Furthermore, mutation of the three phosphorylation sites altered the interactions of Raptor with PRAS40 and p70S6k, and it also prevented the EC-induced dissociation of Raptor from p70S6k. Combined, these results suggest that changes in the phosphorylation of Raptor play an important role in the pathway through which mechanical stimuli activate mTOR signaling. •Mechanical stimulation induces multisite phosphorylation of Raptor•The S696, T706, and S863 residues are the primary sites of regulation•Changes in Raptor phosphorylation alter its interaction PRAS40 and p70S6k•Raptor phosphorylation contributes to the mechanical activation of mTOR signaling.