A role for Raptor phosphorylation in the mechanical activation of mTOR signaling.
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.
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA.
John W
JW
Frey
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA.
Brittany L
BL
Jacobs
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA.
Craig A
CA
Goodman
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, 2015 Linden Dr., Madison, WI 53706, USA. Electronic address: thornb1@svm.vetmed.wisc.edu.
Troy A
TA
Hornberger
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D000076223:Regulatory-Associated Protein of mTOR; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013314:Stress, Mechanical; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000900:Anti-Bacterial Agents; D010748:Phosphopeptides; D010750:Phosphoproteins; D000076223:Regulatory-Associated Protein of mTOR; C540162:Rptor protein, mouse; C508191:proline-rich Akt substrate, 40 kDa protein, mouse; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus
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