Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

Nolan J Hoffman; Benjamin L Parker; Rima Chaudhuri; Kelsey H. Fisher-Wellman; Maximilian Kleinert; Sean J Humphrey; Pengyi Yang; Mira Holliday; Sophie Trefely; Daniel J Fazakerley; Jacqueline Stöckli; James G Burchfield; Thomas Elbenhardt Jensen; Raja Jothi; Bente Kiens; Jørgen Wojtaszewski; Erik Richter; David E James

doi:10.1016/j.cmet.2015.09.001

Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

Nolan J Hoffman, Benjamin L Parker, Rima Chaudhuri, Kelsey H. Fisher-Wellman, Maximilian Kleinert, Sean J Humphrey, Pengyi Yang, Mira Holliday, Sophie Trefely, Daniel J Fazakerley, Jacqueline Stöckli, James G Burchfield, Thomas Elbenhardt Jensen, Raja Jothi, Bente Kiens, Jørgen Wojtaszewski, Erik Richter, David E James

The August Krogh Section for Molecular Physiology

181 Citations (Scopus)

Abstract

Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPKdependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

Original language	English
Journal	Cell Metabolism
Volume	22
Issue number	5
Pages (from-to)	922-935
Number of pages	14
ISSN	1550-4131
DOIs	https://doi.org/10.1016/j.cmet.2015.09.001
Publication status	Published - 3 Nov 2015

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Hoffman, N. J., Parker, B. L., Chaudhuri, R., Fisher-Wellman, K. H., Kleinert, M., Humphrey, S. J., Yang, P., Holliday, M., Trefely, S., Fazakerley, D. J., Stöckli, J., Burchfield, J. G., Jensen, T. E., Jothi, R., Kiens, B., Wojtaszewski, J., Richter, E., & James, D. E. (2015). Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metabolism, 22(5), 922-935. https://doi.org/10.1016/j.cmet.2015.09.001

Hoffman, NJ, Parker, BL, Chaudhuri, R, Fisher-Wellman, KH, Kleinert, M, Humphrey, SJ, Yang, P, Holliday, M, Trefely, S, Fazakerley, DJ, Stöckli, J, Burchfield, JG, Jensen, TE, Jothi, R, Kiens, B , Wojtaszewski, J , Richter, E & James, DE 2015, 'Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates', Cell Metabolism, vol. 22, no. 5, pp. 922-935. https://doi.org/10.1016/j.cmet.2015.09.001

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title = "Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates",

abstract = "Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPKdependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.",

author = "Hoffman, {Nolan J} and Parker, {Benjamin L} and Rima Chaudhuri and Fisher-Wellman, {Kelsey H.} and Maximilian Kleinert and Humphrey, {Sean J} and Pengyi Yang and Mira Holliday and Sophie Trefely and Fazakerley, {Daniel J} and Jacqueline St{\"o}ckli and Burchfield, {James G} and Jensen, {Thomas Elbenhardt} and Raja Jothi and Bente Kiens and J{\o}rgen Wojtaszewski and Erik Richter and James, {David E}",

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T1 - Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

AU - Hoffman, Nolan J

AU - Parker, Benjamin L

AU - Chaudhuri, Rima

AU - Fisher-Wellman, Kelsey H.

AU - Kleinert, Maximilian

AU - Humphrey, Sean J

AU - Yang, Pengyi

AU - Holliday, Mira

AU - Trefely, Sophie

AU - Fazakerley, Daniel J

AU - Stöckli, Jacqueline

AU - Burchfield, James G

AU - Jensen, Thomas Elbenhardt

AU - Jothi, Raja

AU - Kiens, Bente

AU - Wojtaszewski, Jørgen

AU - Richter, Erik

AU - James, David E

N1 - CURIS 2015 NEXS 351

PY - 2015/11/3

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N2 - Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPKdependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

AB - Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPKdependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

U2 - 10.1016/j.cmet.2015.09.001

DO - 10.1016/j.cmet.2015.09.001

M3 - Journal article

C2 - 26437602

SN - 1550-4131

VL - 22

SP - 922

EP - 935

JO - Cell Metabolism

JF - Cell Metabolism

IS - 5

ER -

Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

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