Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA.
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA.
Cell Signal. 2014 Feb;26(2):313-22. doi: 10.1016/j.cellsig.2013.11.009. Epub 2013 Nov 13.
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.
mTOR 信号的激活对于骨骼肌肉质量的机械诱导变化是必要的，但调节 mTOR 信号机械激活的机制仍未得到很好的定义。在这项研究中，我们旨在确定 Raptor 的磷酸化变化是否有助于 mTOR 的机械激活。为了实现这一目标，通过一系列离心收缩（EC）对小鼠骨骼肌进行机械刺激。使用质谱和 Western blot 分析，我们发现 EC 诱导 Raptor S696、T706 和 S863 磷酸化增加，而雷帕霉素不能抑制这种效应。这一观察结果表明，Raptor 磷酸化的变化可能是机械刺激激活 mTOR 的途径中的上游事件。为了验证这一点，我们使用了 Raptor 的磷酸化缺陷突变体（S696A/T706A/S863A），并发现表达该突变体的肌肉中 mTOR 信号的 EC 诱导激活明显减弱。此外，三个磷酸化位点的突变改变了 Raptor 与 PRAS40 和 p70(S6k) 的相互作用，并且还阻止了 Raptor 从 p70(S6k) 诱导的解离。综上所述，这些结果表明 Raptor 磷酸化的变化在机械刺激激活 mTOR 信号的途径中起着重要作用。
