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

Abstract

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 p70(S6k), and it also prevented the EC-induced dissociation of Raptor from p70(S6k). 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.

Keywords: EC; Exercise; G-protein β-subunit-like protein; GFP; GβL; Hypertrophy; JNK; MAPK; Mechanotransduction; PI3K; PRAS40; Raptor; Skeletal muscle; TA; c-jun n-terminal kinase; eccentric contraction; green fluorescent protein; lethal with sec13 protein 8; mLST8; mTOR; mTOR complex 1; mTOR complex 2; mTORC1; mTORC2; mammalian [or mechanistic] target of rapamycin; mitogen-activated protein kinase; p38; p38 mitogen-activated protein kinase; p70(S6k); phosphotidylinositol-3-kinase; proline-rich Akt substrate of 40kDa; regulatory associated protein of mTOR; ribosomal S6 kinase 1; tibialis anterior muscle.