Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling

Abstract

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.As the largest organ in the body, skeletal muscles comprise ϳ45% of our total body mass and play essential roles in voluntary movement, metabolic health, and maintaining quality of life (1-4). Indeed, both sedentary and active adults will lose 35-40% of their skeletal muscle mass by the age of 80, and this loss in muscle mass is associated with disability, loss of independence, an increased risk of morbidity and mortality, as well as an estimated $18.5 billion in annual healthcare costs in the United States alone (2, 5-7). Thus, the development of therapies that can maintain, restore, or even enhance muscle mass is a clinically and fiscally significant goal (8). However, to succeed in developing such therapies, we must first understand the molecular mechanisms that regulate skeletal muscle mass.Skeletal muscle is a highly plastic tissue, and it can change its mass in response to a number of environmental factors. At the most basic level, changes in muscle mass are driven by an alteration in the balance between the rate of protein synthesis and the rate of protein degradation, with a net positive balance leading to muscle growth (i.e. hypertrophy) and a net negative balance leading to muscle loss (i.e. atrophy) (9, 10). Over the last two decades, it has become apparent that a protein kinase called the mammalian/mechanistic target of rapamycin (mTO...

Resistance exercise initiates mechanistic target of rapamycin (mTOR) translocation and protein complex co-localisation in human skeletal muscle

“…To determine the specificity of antibodies towards mTOR and mTOR-associated proteins, we performed a number of validation approaches. To test the specificity of the mTOR, Rheb and TSC2 antibody, labelling experiments were performed in skeletal muscle from wild-type vs. whole-body mTOR knock-out 21 and muscle-specific, tamoxifen-inducible Rheb and TSC2 mouse 22 models (Fig. 1 ).…”

Normal Ribosomal Biogenesis but Shortened Protein Synthetic Response to Acute Eccentric Resistance Exercise in Old Skeletal Muscle

“…A second potential mechanism to explain the accelerated return to baseline MPS in Old muscle post-exercise is decreased exercise-induced sensitization to feeding (Burd et al, 2011). The early MPS response is driven by the loading stimulus and is independent of insulin/IGF-1 receptor signaling (Spangenburg et al, 2008; Jacobs et al, 2017). This mechanical response appears to be intact in Old since we observed no anabolic resistance in the activation of mTORC1.…”

“…However, at 48 h, it is possible that the prolonged increase in MPS is mediated through the potentiation of the feeding response. Unlike the exercise-induced activation of mTORC1 and protein synthesis, the feeding response requires insulin/IGF-1 signaling (Spangenburg et al, 2008; Hamilton et al, 2010; Jacobs et al, 2017). Muscle from both our 30 month old animals and samples from the NIA tissue bank showed that total IRS1 protein content is significantly lower in Old muscle.…”

Mechanical Regulation of Protein Translation in the Cardiovascular System

“…It is well established that mTOR signaling can be activated by amino acids, stress, oxygen, energy status and growth factors such as insulin (Laplante and Sabatini, 2012). There is also evidence indicating mechanical force can stimulate mTOR signaling (Kraiss et al, 2000;Guo et al, 2007;Hornberger, 2011;Philip et al, 2011;Jacobs et al, 2017;Vion et al, 2017) and this will be discussed in more detail below.…”