title: 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.
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Brittany L
authors.initials: BL
authors.lastname: Jacobs
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Rachel M
authors.initials: RM
authors.lastname: McNally
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Kook-Joo
authors.initials: KJ
authors.lastname: Kim
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Rocky
authors.initials: R
authors.lastname: Blanco
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Privett
authors.affiliation: From the Department of Comparative Biosciences and.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: You
authors.affiliation: From the Department of Comparative Biosciences and troy.hornberger@wisc.edu.|the School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, 53706.
authors.forename: Troy A
authors.initials: TA
authors.lastname: Hornberger
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D005260:Female; D006720:Homozygote; D007328:Insulin / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D000076205:Ras Homolog Enriched in Brain Protein; D015398:Signal Transduction; D020123:Sirolimus / Q000737:chemistry; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D013629:Tamoxifen / Q000737:chemistry; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D007328:Insulin; D009479:Neuropeptides; D000076205:Ras Homolog Enriched in Brain Protein; C490213:Rheb protein, mouse; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins; D020123:Sirolimus
keywords: cell growth; exercise; mechanotransduction; protein synthesis; skeletal muscle