Application of the [γ-32P] ATP kinase assay to study anabolic signaling in human skeletal muscle

Abstract

AMPK (AMP-dependant protein kinase)-mTORC1 (mechanistic target of rapamycin in complex 1)-p70S6K1 (ribosomal protein S6 kinase 1 of 70 kDa) signaling plays a crucial role in muscle protein synthesis (MPS). Understanding this pathway has been advanced by the application of the Western blot (WB) technique. However, because many components of the mTORC1 pathway undergo numerous, multisite posttranslational modifications, solely studying the phosphorylation changes of mTORC1 and its substrates may not adequately represent the true metabolic signaling processes. The aim of this study was to develop and apply a quantitative in vitro [γ-32P] ATP kinase assay (KA) for p70S6K1 to assess kinase activity in human skeletal muscle to resistance exercise (RE) and protein feeding. In an initial series of experiments the assay was validated in tissue culture and in p70S6K1-knockout tissues. Following these experiments, the methodology was applied to assess p70S6K1 signaling responses to a physiologically relevant stimulus. Six men performed unilateral RE followed by the consumption of 20 g of protein. Muscle biopsies were obtained at pre-RE, and 1 and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity as assessed by the KA was significantly increased from pre-RE at 1 and 3 h post-RE. However, phosphorylated p70S6K1thr389 was not significantly elevated. AMPK activity was suppressed from pre-RE at 3 h post-RE, whereas phosphorylated ACCser79 was unchanged. Total protein kinase B activity also was unchanged after RE from pre-RE levels. Of the other markers we assessed by WB, 4EBP1thr37/46 phosphorylation was the only significant responder, being elevated at 3 h post-RE from pre-RE. These data highlight the utility of the KA to study skeletal muscle plasticity.