Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men

Abstract

Protein synthesis in skeletal muscle is known to decrease during contractions but the underlying regulatory mechanisms are unknown. Here, the effect of exercise on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation, a key component in protein translation machinery, was examined. Eight healthy men exercised on a cycle ergometer at a workload eliciting approximately 67% peak pulmonary oxygen consumption (VO2 peak) with skeletal muscle biopsies taken from the vastus lateralis muscle at rest as well as after 1, 10, 30, 60 and 90 min of exercise. In response to exercise, there was a rapid (i.e. < 1 min) 5- to 7-fold increase in eEF2 phosphorylation at Thr56 that was sustained for 90 min of continuous exercise. The in vitro activity of skeletal muscle eEF2 kinase was not altered by exercise indicating that the increased activity of eEF2 kinase to eEF2 is not mediated by covalent mechanisms. In support of this, the increase in AMPK activity was temporally unrelated to eEF2 phosphorylation. However, skeletal muscle eEF2 kinase was potently activated by Ca(2)(+)-calmodulin in vitro, suggesting that the higher eEF2 phosphorylation in working skeletal muscle is mediated by allosteric activation of eEF2 kinase by Ca(2)(+) signalling via calmodulin. Given that eEF2 phosphorylation inhibits eEF2 activity and mRNA translation, these findings suggest that the inhibition of protein synthesis in contracting skeletal muscle is due to the Ca(2)(+)-induced stimulation of eEF2 kinase.

Figure 1. Skeletal muscle eEF2 kinase activity is Ca²⁺–calmodulin dependent

eEF2 kinase was immunoprecipitated from human skeletal muscle samples and activity was measured in vitro in the absence and presence of Ca²⁺–calmodulin (Ca²⁺-CaM). Data are mean ±s.e.m. from 4 samples, *P < 0.01 versus EGTA.

Figure 2. Time course of the effect of exercise on eEF2 phosphorylation and expression

Human skeletal muscle samples were subject to SDS-PAGE and immunoblotted with pT56-eEF2 (top panel) and eEF2 (bottom panel) antibodies. Data are mean ±s.e.m., n = 8; *P < 0.01 versus time 0. Representative immunoblots are shown. AU, arbitrary units.

Figure 3. Time course of the effect of exercise on eEF2 kinase activity expression and phosphorylation

eEF2K was immunoprecipitated from skeletal muscle samples and in vitro activity was measured (top panel). Human skeletal muscle samples were subject to SDS-PAGE and immunoblotted with eEF2 kinase (bottom panel) antibodies. Data are mean ±s.e.m., n = 8; *P < 0.05 versus time 0. Representative immunoblots are shown. AU, arbitrary units; OD, optical density

Figure 4. Time course of the effect of exercise on AMPK-α and ACC-β phosphorylation

Human skeletal muscle samples were subject to SDS-PAGE and immunoblotted with pT172-AMPK-α (top panel) and pS221-ACC-β (bottom panel) antibodies. Data are mean ±s.e.m., n = 8; *P < 0.01 versus time 0. Representative immunoblots are shown. OD, optical density.