Exercise improves health through adaptive metabolic and mechanical remodelling, mediated by a network of kinases in response to homeostatic stress. The phosphoproteome of acutely exercised human skeletal muscle provides a blueprint of kinase regulation to promote health. However, of this phosphoproteome, only 10% of the >1000 regulated phosphosites have a known upstream kinase. Consequently, an in vitro model of exercise is needed to further characterise the role of known and novel kinases. We therefore performed a phosphoproteomic screen of 10 candidate exercise-like treatments in rat L6 myotubes quantifying 20,249 unique Class I phosphopeptides, of which 37.8% were regulated in at least one treatment. Of the regulated phosphosites in exercised human skeletal muscle that mapped to the in vitro exercise mimetics, 74% were regulated by at least one treatment. We predicted the combination of isoproterenol and thapsigargin to most closely recapitulate the exercise phosphoproteome. As exercise involves multiple stimuli, we measured the phosphoproteome of this combination to determine the extent of interactions between exercise-like treatments. Synergy between the treatments produced a phosphoproteome closer to exercise than either treatment or their expected combination, revealing the importance of interactions between stimuli.
Our phosphoproteomic data was also used to predict the involvement of >20 kinases not previously implicated in exercise signalling that may be attractive drug targets. One of these targets was PAK2, previously implicated in cytoskeletal dynamics, however its function in muscle is incompletely understood. CRISPR/Cas9 knockout of PAK2 in L6 myotubes revealed roles as a negative regulator of glucose uptake via an increase in plasma-membrane localised GLUT4. Phosphoproteomic screening identified several novel PAK2 substrates enriched in vesicle trafficking and endosomal recycling. Our data suggests that PAK2 inhibition is a potential novel strategy to promote glucose uptake into muscle cells relevant for the treatment of hyperglycemia and type-2 diabetes.