title: 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-(32)P] 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 p70S6K1(thr389) was not significantly elevated. AMPK activity was suppressed from pre-RE at 3 h post-RE, whereas phosphorylated ACC(ser79) was unchanged. Total protein kinase B activity also was unchanged after RE from pre-RE levels. Of the other markers we assessed by WB, 4EBP1(thr37/46) 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.
authors.affiliation: Health & Exercise Sciences Research Group University of Stirling, Stirling, United Kingdom;
authors.forename: Chris
authors.initials: C
authors.lastname: McGlory
authors.forename: Amanda
authors.initials: A
authors.lastname: White
authors.forename: Caroline
authors.initials: C
authors.lastname: Treins
authors.forename: Barry
authors.initials: B
authors.lastname: Drust
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Close
authors.forename: Don P M
authors.initials: DP
authors.lastname: Maclaren
authors.forename: Iain T
authors.initials: IT
authors.lastname: Campbell
authors.forename: Andrew
authors.initials: A
authors.lastname: Philp
authors.forename: Simon
authors.initials: S
authors.lastname: Schenk
authors.forename: James P
authors.initials: JP
authors.lastname: Morton
authors.forename: D Lee
authors.initials: DL
authors.lastname: Hamilton
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D025181:ATP Synthetase Complexes; D000328:Adult; D000818:Animals; D015153:Blotting, Western; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000502:physiology*; D010710:Phosphates / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019275:Radiopharmaceuticals; D055070:Resistance Training; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D046509:Tissue Culture Techniques; D055815:Young Adult
chemical_list: D010710:Phosphates; D019275:Radiopharmaceuticals; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D055372:AMP-Activated Protein Kinases; D025181:ATP Synthetase Complexes
keywords: AMPK; mTORC1; p70S6K1; resistance exercise