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. 2015 Nov 3;22(5):922-35.
doi: 10.1016/j.cmet.2015.09.001. Epub 2015 Oct 1.

Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates

Nolan J Hoffman  1 , Benjamin L Parker  1 , Rima Chaudhuri  1 , Kelsey H Fisher-Wellman  2 , Maximilian Kleinert  3 , Sean J Humphrey  2 , Pengyi Yang  4 , Mira Holliday  1 , Sophie Trefely  2 , Daniel J Fazakerley  1 , Jacqueline Stöckli  1 , James G Burchfield  1 , Thomas E Jensen  5 , Raja Jothi  6 , Bente Kiens  5 , Jørgen F P Wojtaszewski  5 , Erik A Richter  5 , David E James  7
Affiliations

Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates

Nolan J Hoffman et al. Cell Metab. .

Erratum in

  • Cell Metab. 2015 Nov 3;22(5):948

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.

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Conflict of interest statement

The authors have no conflicts of interest.

Figures

Fig. 1
Fig. 1. Acute exercise-regulated phosphoproteome in human skeletal muscle
(A) Experimental design of the phosphoproteomic analysis of exercise in human muscle is shown. Muscle biopsies pre- and post-exercise from four healthy males were collected. Protein was extracted, digested with Lys-C/trypsin and peptides were isobarically labeled with iTRAQ or TMT tags. Phosphopeptides were enriched by titanium dioxide chromatography and sequential elution from immobilized metal ion affinity chromatography (SIMAC). The unbound non-phosphorylated fraction and phosphorylated fraction was further separated by hydrophilic interaction liquid chromatography (HILIC) into 12–16 fractions. Each fraction was analyzed by nano-ultra high pressure liquid chromatography coupled to tandem MS (nanoUHPLC-MS/MS) on a Q-Exactive MS operated in DDA. (B) Volcano plot showing the median phosphopeptide Log2 fold-change (post- / pre-exercise) plotted against the −Log10 P-value highlighting significantly regulated phosphopeptides (blue; P < 0.05, n=4, moderated t-test). Dotted lines indicate (+/−) 1.5-fold change (Log2 = 0.58). (C) Pearson’s correlation analysis of phosphopeptide fold-change quantification (post- / pre-exercise) between the 4 subjects is shown. (D) Summary of the quantified and regulated proteome and phosphoproteome is shown.
Fig. 2
Fig. 2. Site-specific kinase-substrate regulation in response to acute exercise
(A) Significantly regulated phosphopeptides (+/− 1.5-fold change, P < 0.05, n=4, moderated t-test) were clustered according to PhosphoSitePlus-annotated upstream kinases. Co-regulation of substrate phosphorylation sites (inner dotted circle = decreased phosphorylation; outer dotted circle = increased phosphorylation) provides insights into kinase activity in response to exercise. (B) Muscle biopsy lysates from four subjects pre- and post-exercise were immunoblotted for kinases and substrates in the phosphoproteomics data. (C) An integrative network of the exercise-regulated kinase interactome was generated using experimentally validated human protein-protein interactions and annotated kinase-substrate relationships. Direct increases (green arrows) and decreases (red arrows) in substrate phosphorylation are shown, and dotted lines represent protein-protein interactions. Colors represent kinases with exercise-regulated activity as shown in (A). Additional kinases (grey) and substrates (white) contain regulated phosphorylation sites.
Fig. 3
Fig. 3. AMPK substrate prediction using AMPK-activated phosphoproteomics
(A) Experimental design of the phosphoproteomic analysis of AICAR stimulation is depicted. 2-plex SILAC labeled L6 myotubes were treated with or without AICAR (n=4 with 2 label switching experiments). Proteins were extracted and digested with trypsin. Peptides were fractionated by strong cation exchange (SCX) chromatography and phosphopeptides enriched using titanium dioxide chromatography. An aliquot of the non-phosphorylated peptides was fractionated by HILIC. Phosphopeptide and non-phosphopeptide fractions were analysed by nanoUHPLC-MS/MS on a Q-Exactive MS operated in data-dependent acquisition followed by MaxQuant analysis. (B) Volcano plot showing median phosphopeptide Log2 fold-change (AICAR/basal) plotted against −Log10 P-value highlighting significantly regulated phosphopeptides (blue; *P < 0.05, n=4, moderated t-test). Dotted lines indicate (+/−) 1.5-fold change (Log2 = 0.58). (C) Summary of the quantified and regulated proteome and phosphoproteome is shown. (D) AMPK substrate prediction using the AICAR-regulated L6 myotube phosphoproteome. The model was trained based on the primary amino acid motif surrounding the phosphosites and magnitude of up-regulation by AICAR (AICAR-regulated sites in blue) using known AMPK substrates quantified (red).
Fig. 4
Fig. 4. AMPK substrate prediction using data-independent MS analysis of a global AMPK in vitro kinase assay
(A) Immunoblot analysis of known AMPK substrates (ACC and Raptor) in HEK293 lysates subjected to a global AMPK (α1β1γ2) in vitro kinase assay ± active AMPK and the AMPK inhibitor Compound C. (B) – (I) Targeted quantification (mean ± standard deviation, one-way ANOVA corrected for multiple testing, *P < 0.05, **P < 0.01, ***P < 0.005, n=3) of phosphopeptides from HEK293 lysates subjected to a global AMPK (α1β1γ2) in vitro kinase assay using nanoUHPLC-MS/MS on a Q-Exactive MS operated in DIA.
Fig. 5
Fig. 5. AMPK consensus motif and substrate sequence alignment
AMPK consensus motif derived from (Gwinn et al., 2008) with multiple sequence alignment of exercise-regulated phosphosites in human muscle (Human Exercise) is displayed. The alignment shows: known AMPK substrates (black circles); internally predicted AMPK substrates with validation (blue circles) in either AICAR stimulated Rat L6 myotubes (Rat AICAR) or global AMPK in vitro kinase analysis in HEK lysates (black diamonds ◆); and externally predicted AMPK substrates from (Banko et al., 2011) (orange circles).
Fig. 6
Fig. 6. AKAP1 is a mitochondrial AMPK substrate
(A) HEK293 cells expressing FLAG-tagged EV, AKAP1 WT or AKAP1 S103A were serum-starved for 2 h, followed by ± 100 μM A-769662 for 30 min. Cell lysates were immunoblotted with indicated antibodies. (B) Activated AMPK α1β1γ2 was added to immunoprecipitated AKAP1 or biotinylated ACC beads for 30 min at 33°C ± Compound C. In vitro kinase assay samples were immunoblotted. (C) L6 myotubes were serum-starved for 2 h, followed by incubation ± AMPK agonists AICAR (2 mM), A-769662 (100 μM) and DNP (200 μM) for 30 min. Lysates were immunoblotted. (D) L6 myoblasts were transfected with siScramble or siAMPKα. Cells were lysed after 72 h and immunoblotted. (E) Isolated soleus muscles from WT and AMPKα2 kinase dead (KD) mice were incubated in vitro ± 2 mM AICAR for 1 h and muscle lysates were immunoblotted. (F) WT mice were rested or subjected to treadmill exercise until exhaustion, red quadricep muscles were isolated and lysates were immunoblotted. (G) L6 myotubes were serum-starved for 2 h, followed by stimulation ± 100 μM A769662 for 30 min. Following subcellular fractionation, whole cell lysate (WCL), non-mitochondrial (Non-Mito) and mitochondrial (Mito) fractions were immunoblotted. (H) L6 myoblasts expressing pMito-LSSmOrange (Mito) and either FLAG-tagged AKAP1 WT or AKAP1 S103A were reseeded 24 h after transfection, fixed 24 h later and prepared for confocal microscopy (scale bar: 10 μm). Immunoblots are representative images from 3–6 independent experiments; quantitative AKAP1 pS103 data in panels E and F are normalized to total protein, and mean ± standard error of mean is shown from n=3–4 and n=5 mice, respectively, t-test, *P < 0.05 vs. basal WT muscle (E) or rest (F), #P < 0.05 vs. AICAR-stimulated WT muscle.
Fig. 7
Fig. 7. AKAP1 and AMPK mediated S103 phosphorylation facilitate mitochondrial respiration
(A) L6 myoblasts were transfected with siScramble or siAMPKα. Cells were lysed after 72 h and immunoblotted with indicated antibodies. (B) Immunoblotting was performed in serum-starved L6 myoblasts incubated with BSA or BSA-conjugated palmitate (Palm.; 200 μM) ± 100 μM A-769662 for 30 min. Immunoblots are representative images from 3–4 independent experiments. (C) Oxygen consumption rate (OCR) was determined in serum-starved L6 myoblasts transfected with siScramble or siAMPKα and incubated with BSA or Palm. (200 μM) ± 100 μM A-769662. (D) L6 myoblasts were transfected with siScramble or AKAP1 siRNA (siAKAP1). Cells were lysed after 72 h and immunoblotted. Respiration (JO2 in E) and OCR (F) was determined in serum-starved L6 myoblasts transfected with siScramble or siAKAP1 and incubated with BSA or Palm. (200 μM) ± 100 μM A-769662. (G) L6 myoblasts were transfected with FLAG-tagged EV control, AKAP1 WT or AKAP1 S103A mutant, incubated with BSA or Palm., and immunoblotting was performed (G), respiration (H, I) and OCR (J) were measured. (I) Respiration is shown as % increase with A-769662 stimulation in cells incubated with Palm. Quantitative OCR and JO2 data represent mean ± standard error of mean, t-test, *P < 0.05 vs. siScramble BSA or EV/WT control BSA, #P < 0.05 vs. respective siScramble or EV/WT control, n=3–6.
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Comment in

  • Metabolism: One step forward for exercise.
    Hawley JA, Krook A. Hawley JA, et al. Nat Rev Endocrinol. 2016 Jan;12(1):7-8. doi: 10.1038/nrendo.2015.201. Epub 2015 Nov 27. Nat Rev Endocrinol. 2016. PMID: 26610411 No abstract available.

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References

    1. Banko MR, Allen JJ, Schaffer BE, Wilker EW, Tsou P, White JL, Villen J, Wang B, Kim SR, Sakamoto K, et al. Chemical genetic screen for AMPKalpha2 substrates uncovers a network of proteins involved in mitosis. Molecular cell. 2011;44:878–892. - PMC - PubMed
    1. Bennetzen MV, Cox J, Mann M, Andersen JS. PhosphoSiteAnalyzer: a bioinformatic platform for deciphering phospho proteomes using kinase predictions retrieved from NetworKIN. Journal of proteome research. 2012;11:3480–3486. - PubMed
    1. Bo H, Zhang Y, Ji LL. Redefining the role of mitochondria in exercise: a dynamic remodeling. Annals of the New York Academy of Sciences. 2010;1201:121–128. - PubMed
    1. Bridges D, MacDonald JA, Wadzinski B, Moorhead GB. Identification and characterization of D-AKAP1 as a major adipocyte PKA and PP1 binding protein. Biochemical and biophysical research communications. 2006;346:351–357. - PubMed
    1. Chaudhuri R, Sadrieh A, Hoffman NJ, Parker BL, Humphrey SJ, Stöckli JS, Hill A, James DE, Yang JY. PhosphOrtholog: A web-based tool for cross-species mapping of orthologous protein post-translational modifications. BMC Genomics 2015 - PMC - PubMed

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