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 AMPK-dependent 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.
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Nolan J
authors.initials: NJ
authors.lastname: Hoffman
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Rima
authors.initials: R
authors.lastname: Chaudhuri
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.
authors.forename: Kelsey H
authors.initials: KH
authors.lastname: Fisher-Wellman
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; University of Copenhagen, August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen 2100, Denmark.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Kleinert
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Humphrey
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; Systems Biology Section, Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Pengyi
authors.initials: P
authors.lastname: Yang
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Mira
authors.initials: M
authors.lastname: Holliday
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.
authors.forename: Sophie
authors.initials: S
authors.lastname: Trefely
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Fazakerley
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Stöckli
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: James G
authors.initials: JG
authors.lastname: Burchfield
authors.affiliation: University of Copenhagen, August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen 2100, Denmark.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Jensen
authors.affiliation: Systems Biology Section, Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Raja
authors.initials: R
authors.lastname: Jothi
authors.affiliation: University of Copenhagen, August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen 2100, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Kiens
authors.affiliation: University of Copenhagen, August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen 2100, Denmark.
authors.forename: Jørgen F P
authors.initials: JF
authors.lastname: Wojtaszewski
authors.affiliation: University of Copenhagen, August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen 2100, Denmark.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Richter
authors.affiliation: Charles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia; School of Medicine, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address: david.james@sydney.edu.au.
authors.forename: David E
authors.initials: DE
authors.lastname: James
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics* / Q000378:metabolism; D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D004734:Energy Metabolism; D015444:Exercise / Q000502:physiology*; D006801:Humans; D000069550:Machine Learning; D008297:Male; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010805:Physical Conditioning, Animal; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D010750:Phosphoproteins; D055372:AMP-Activated Protein Kinases