Application of the [γ-32P] ATP kinase assay to study anabolic signaling in human skeletal muscle
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Updated: May 3, 2026
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Chris McGlory1, Amanda White, Caroline Treins
1Health & Exercise Sciences Research Group University of Stirling, Stirling, United Kingdom;
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.
Insights
A new kinase assay (KA) reveals increased p70S6K1 activity in human skeletal muscle after resistance exercise and protein intake, despite unchanged phosphorylation levels. This method offers a better way to study muscle protein synthesis signaling.
Area of Science:
- Muscle physiology and metabolism
- Molecular signaling pathways
- Biochemical assay development
Background:
- The AMP-dependant protein kinase (AMPK)-mechanistic target of rapamycin in complex 1 (mTORC1)-ribosomal protein S6 kinase 1 of 70 kDa (p70S6K1) pathway is vital for muscle protein synthesis (MPS).
- Western blot (WB) is commonly used but may not fully capture complex posttranslational modifications in mTORC1 signaling.
- Existing methods may not accurately reflect dynamic metabolic signaling in skeletal muscle.
Purpose of the Study:
- To develop and validate a quantitative in vitro [γ-(32)P] ATP kinase assay (KA) for p70S6K1 activity in human skeletal muscle.
- To assess p70S6K1 signaling responses to resistance exercise (RE) and protein feeding using the developed KA.
- To compare KA results with traditional WB markers of muscle signaling.
Main Methods:
- Development and validation of a quantitative in vitro [γ-(32)P] ATP kinase assay (KA) for p70S6K1.
- Application of the KA to muscle biopsies from six men undergoing unilateral resistance exercise (RE) and consuming 20 g of protein.
- Measurement of p70S6K1 activity via KA and assessment of various phosphorylated proteins (p70S6K1(thr389), ACC(ser79), 4EBP1(thr37/46)) and total protein kinase B activity using Western blot (WB).
Main Results:
- The KA demonstrated significantly increased p70S6K1 activity in human skeletal muscle 1 and 3 hours post-RE and protein consumption compared to pre-RE levels.
- Contrary to KA findings, phosphorylated p70S6K1(thr389) levels did not show a significant increase.
- AMPK activity was suppressed at 3h post-RE, while phosphorylated ACC(ser79) and total protein kinase B activity remained unchanged; 4EBP1(thr37/46) phosphorylation was the only other significant WB marker increase.
Conclusions:
- The quantitative kinase assay (KA) provides a valuable tool for assessing p70S6K1 activity in skeletal muscle.
- KA revealed an increase in p70S6K1 activity following resistance exercise and protein feeding, which was not detected by traditional phosphorylation-based Western blot methods.
- These findings highlight the utility of the KA for studying skeletal muscle plasticity and signaling responses to stimuli.
