Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle

Abstract

The present study examined the effects of an acute bout of treadmill exercise on signalling through the extracellular signal-regulated kinase (ERK)1/2 and mammalian target of rapamycin (mTOR) pathways to regulatory mechanisms involved in mRNA translation in mouse gastrocnemius muscle. Briefly, C57BL/6 male mice were run at 26 m min(-1) on a treadmill for periods of 10, 20 or 30 min, then the gastrocnemius was rapidly removed and analysed for phosphorylation and/or association of protein components of signalling pathways and mRNA translation regulatory mechanisms. Repression of global mRNA translation was suggested by disaggregation of polysomes into free ribosomes, which occurred by 10 min and was sustained throughout the time course. Exercise repressed the mTOR signalling pathway, as shown by dephosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1), enhanced association of the regulatory-associated protein of mTOR with mTOR, and increased assembly of the tuberin-hamartin complex. In contrast, exercise caused no change in phosphorylation of either Akt/PKB or tuberin. Upstream of mTOR, exercise was associated with an increase in cAMP, protein kinase A activity, and AMP-activated protein kinase phosphorylation. Simultaneously, exercise caused a rapid and sustained activation of the MEK1/2-ERK1/2-p90RSK pathway, resulting in increased phosphorylation of downstream targets including eIF4E and the ribosomal protein (rp)S6 on S235/S236. Overall, the data are consistent with exercise-induced repression of mTOR signalling and global rates of mRNA translation, accompanied perhaps by up-regulated translation of selected mRNAs through regulatory mechanisms such as eIF4E and rpS6 phosphorylation, mediated by activation of the ERK1/2 pathway.

Figure 1. Effect of acute treadmill exercise on polysome aggregation in mouse gastrocnemius muscle

C57BL/6 male mice were exercised on a motorized treadmill at 26 m min⁻¹, 10% grade for 10 min (A), 20 min (B) or 30 min (C). Immediately following the exercise bout, the gastrocnemius muscle was removed, frozen between aluminium blocks pre-cooled in liquid nitrogen, and stored at −80°C. The frozen muscle was powdered under liquid nitrogen, then homogenized in lysis buffer containing cycloheximide, SUPERasin and detergent. The homogenate was clarified by centrifugation at 3000 g for 15 min, layered onto a 20–47% sucrose gradient, then centrifuged at 40 000 g for 5 h 20 min. Following centrifugation, the gradient was displaced upward through a spectrophotometer and the absorbance at 254 nm was continuously recorded. A representative profile from each condition is presented. The peaks corresponding to 40S and 60S ribosomal subunits, 80S monomers, and polysomes are denoted on the figure (n = 6–9 per group).

Figure 2. Effect of acute treadmill exercise on phosphorylation of 4E-BP1 and S6K1 in mouse gastrocnemius muscle

C57BL/6 male mice were exercised as described in the legend to Fig. 1. Immediately following the exercise bout, the gastrocnemius muscle was removed, homogenized and clarified by centrifugation at 1000 g for 3 min. The resulting muscle homogenate was combined with an equal amount of 2 × SDS sample buffer. Equal amounts of protein from each sample were assessed for the phosphorylation of 4E-BP1 on T37/T46 (A), and the hyperphosphorylation status and phosphorylation on T389 of S6K1 (B) by Western blot analysis. Representative Western blots are shown. *P < 0.05 versus control conditions (n = 6–9 per group)

Figure 3. Effect of acute treadmill exercise on the association of raptor with mTOR and hamarin with tuberin

C57BL/6 male mice were exercised as described in the legend to Fig. 1. Immediately following the exercise bout, the gastrocnemius muscle was removed and homogenized. A, mTOR was immunoprecipitated as described in Methods and the immunoprecipitates (IP) were assessed for raptor and mTOR content by immunoblot (WB) analysis, and the ratio of raptor to mTOR was calculated. The results are expressed as a percentage of control. B, tuberin was immunoprecipitated as described in Methods and the immunoprecipitates were assessed for hamartin and tuberin content by immunoblot analysis, and the ratio of hamartin to tuberin was calculated. The results are expressed as a percentage of control. *P < 0.05 versus control conditions (n = 6 per group).

Figure 4. Effect of acute treadmill exercise on PKB phosphorylation on S473 and tuberin phosphorylation on T1462

C57BL/6 male mice were exercised as described in the legend to Fig. 1. Immediately following the exercise bout, the gastrocnemius muscle was removed, frozen between aluminium blocks pre-cooled in liquid nitrogen, and stored at −80°C until analysed. A, PKB phosphorylation on S473 was measured by protein immunoblot analysis of muscles homogenized in 2 × SDS sample buffer as described in Methods. Results were normalized for PKB content and are expressed as a percentage of control condition. B, phosphorylation of tuberin on T1462 was measured in tuberin immunoprecipitates as described in Methods. Values were normalized for tuberin content and are expressed as a percentage of the control value. *P < 0.05 versus control (n = 6 per group).

Figure 5. Effect of acute treadmill exercise on AMPK phosphorylation, cAMP concentration and PKA activity in mouse gastrocnemius muscle

C57BL/6 male mice were exercised as described in the legend to Fig. 1. A, immediately following the exercise bout, the gastrocnemius muscle was removed, homogenized and clarified by centrifugation at 1000 g for 3 min. The resulting muscle homogenate was combined with an equal volume of 2 × SDS sample buffer and equal amounts of protein from each sample were assessed for the phosphorylation of AMPK on T172 by Western blot analysis. The results are expressed as a percentage of the control conditions. Representative Western blots are shown (CON; control). For analysis of cAMP concentration (B) and PKA activity (C), gastrocnemius muscle was quickly removed and frozen between aluminium blocks pre-cooled in liquid nitrogen and then stored at −80°C until analysed. *P < 0.05 versus control (n = 6–9 per group).

Figure 6. Effect of acute treadmill exercise on phosphorylation of MEK1/2 on S217/S221, ERK1/2 on T202/Y204, p90RSK on S380, and eIF4E on S209 in mouse gastrocnemius muscle

C57BL/6 male mice were exercised as described in the legend to Fig. 1. Immediately following the exercise bout, the gastrocnemius muscle was removed, homogenized, and clarified by centrifugation at 1000 g for 3 min. The resulting muscle homogenate was combined with an equal volume of 2 × SDS sample buffer and equal amounts of protein from each sample were assessed for the phosphorylation of MEK1/2 on S217/S221 (A), ERK1/2 on T202/Y204 (B), p90RSK on S380 (C), and eIF4E on S209 (D) by Western blot analysis. Results are expressed as a percentage of the control. Representative Western blots are shown. *P < 0.05 versus control (n = 6–9 per group).

Figure 7. Effect of acute treadmill exercise on phosphorylation of ribosomal protein (rp) S6 in mouse gastrocnemius muscle

C57BL/6 male mice were exercised as described in the legend to Fig. 1. Immediately following the exercise bout, the gastrocnemius muscle was removed, homogenized, and clarified by centrifugation at 1000 g for 3 min. The resulting muscle homogenate was combined with an equal volume of 2 × SDS sample buffer and equal amounts of protein from each sample were assessed for the phosphorylation of rpS6 on S235/S236 and S240/S244 by Western blot analysis. Results are expressed as a percentage of the control. Representative Western blots are shown. *P < 0.05 versus control (n = 6–9 per group).

Figure 8. Signal transduction pathways through which endurance exercise regulates mRNA translation in skeletal muscle

Endurance exercise promotes the activation of multiple signalling pathways in skeletal muscle including the extracellular signal-regulated kinase (ERK)1/2 and protein kinase A (PKA) pathways. Both ERK1/2 and the downstream effector, p90 ribosomal protein S6 kinase (p90RSK), phosphorylate and inhibit the mTOR repressor complex consisting of tuberin and hamartin. In contrast, PKA phosphorylates the AMP-activated protein kinase (AMPK), which subsequently phosphorylates and activates the tuberin–hamartin complex. Thus, activation of ERK1/2 and p90RSK activate, whereas protein kinase A/AMPK inhibit mTOR signalling to eIF4E binding protein (4E-BP1) and promotes the ribosomal protein S6 kinase (S6K1). Simultaneously, ERK1/2 phosphorylation of eIF4E. A continuous line represents direct signalling whereas a dashed line represents indirect signalling involving intermediate effectors that are not shown for simplicity. A line with an arrowhead represents activation whereas a line ending in a perpendicular line represents inhibition.