Exploring the Late Endosome/ Lysosomal System: A Defined Role for Rheb and TSC2 in the Mechanical Activation of mTOR Signaling

It is well recognized that the activation of signaling by the mechanistic target of rapamycin (mTOR) is necessary for mechanically-induced changes in skeletal muscle mass; however, the mechanism(s) through which mechanical stimuli activate mTOR signaling remain poorly defined. Nevertheless, advancements are being made, and recent studies have indicated that the late endosome/lysosomal system (LEL) may serve as an integration center for mTOR signaling. Indeed, this study and others have shown that mTOR and its direct activator, Ras homolog enriched in brain (Rheb), are enriched at the late endosome / lysosomal system (LEL). Moreover, we have determined that tuberin (TSC2), an inhibitor of Rheb, is enriched at the LEL under basal conditions, and that it becomes hyper-phosphorylated and translocates away from the LEL in response to mechanical stimulation. These findings suggest that mechanically-induced mTOR signaling may be regulated by the dissociation of TSC2 from the LEL, resulting in Rheb attaining an active state and, consequently, activation of mTOR signaling. Although an intriguing hypothesis, the necessity of Rheb and TSC2 for the mechanical activation of mTOR signaling is currently unknown. Thus, to address this question, we generated inducible and skeletal muscle-specific knockout mice for Rheb (iRhebKO) and TSC2 (iTSC2KO), and subjected these mice to a bout of eccentric contractions (EC) as a source of mechanical stimulation. Our results demonstrated that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both iRhebKO and iTSC2KO mice. Next, we explored the importance of changes in TSC2 phosphorylation. Our mass spectrometric analyses revealed 6 phospho-residues on TSC2 that are upregulated in response to ECs, and introducing phospho-defective mutations into these residues nearly abolished the ability of ECs, but not insulin, to activate mTOR signaling. Taken together, the results from this study firmly establish Rheb and TSC2 as critical regulators of mechanically-induced mTOR signaling and provide indirect evidence that the LEL is important to this process. Moreover, our results highlight the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway and significantly contribute to the EC-induced activation of mTOR signaling.