Figure 1.
Mechanical stimulation activates mTOR signaling via an ERK-independent mechanism.
EDL muscles were held at optimal length (Lo) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
Figure 2.
Validation of mTOR-dependent signaling events that are induced by mechanical stimulation.
(A) EDL muscles from wild type mice, and (B) transgenic mice with muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR), were held at Lo and pre-incubated with 150 nM rapamycin (RAP +) or the vehicle (RAP –, DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions followed by western blot analysis for phosphorylated (P) and total p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the genotype-matched vehicle control samples (RAP–, Stretch–). Note: total 4E-BP1 gels were also run under conditions that minimize the gel mobility shift (short-run). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
Figure 3.
Mechanical stimulation induces an increase in protein synthesis via an ERK-independent mechanism.
EDL muscles were held at Lo and pre-incubated with 50 µM U0126 or the vehicle (DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions, and protein synthesis rates were measured during the final 30 min. All values are presented as the mean + SEM (n = 4−7 per group). *Significantly different from the drug-matched control group, P≤0.05.
Figure 4.
Exogenous phosphatidic acid activates mTOR signaling via an ERK-independent mechanism.
(A) EDL muscles were held at Lo and pre-labeled with [3H]-myristic acid for 2 h. The muscles were then subjected to 15 or 90 min of stretch or control conditions. Muscles were collected at the end of the 15 or 90 min interval, and the amount of 3H-labeled PA (3H PA) was measured and then expressed as a percentage of time-matched control values. The values are presented as the mean + SEM (n = 4−7 per group). • Significantly different from the time-matched control group. (B) C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min, followed by 20 min stimulation with 30 µM C8 PA, 300 µM Egg PA or the vehicle (Control, PBS). The samples were then subjected to western blot analysis for phosphorylated (P) and total ERK, p70, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples (U0126 –, PBS). All values are presented as the mean and were obtained from three independent experiments (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
Figure 5.
Phosphatidic acid directly promotes mTOR kinase activity in-vitro.
Wild type C2C12 myoblasts and C2C12 myoblasts stably expressing FLAG-tagged mTOR (FLAG-mTOR) were serum starved overnight, collected, and then the cell lysates were subjected to immunoprecipitation against the FLAG epitope. The immunoprecipitates were incubated for 15 min with either 150 µM C8 PA vesicles (50% C8 PA +50% PC) or 150 µM phosphatidylcholine (PC) vesicles (100% PC) as a control condition. mTOR kinase activity was then assayed with GST-p70 as a substrate. The resulting samples were subjected to western blot analysis, and the phospho:total ratios for GST-p70 were expressed relative to the values obtained in the PC treated FLAG-mTOR group. All values are presented as the mean and were obtained from at least three independent experiments (n = 5−8 per group). *Significantly different from the cell type-matched control group, P≤0.05.