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A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

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The Journal of Physiology

Published online on


The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal‐intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal‐intensity contractions (MIC) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC‐regulated phosphorylation events, a large proportion of these identifications were present on highly‐abundant myofibrillar proteins. We subsequently incorporated a centrifugation‐based fractionation step to deplete the highly‐abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5,983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase‐substrate predictions indicated that the MIC‐regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPK's and CAMK's. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z‐disc, with over 74% of the Z‐disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z‐disc kinases (striated muscle‐specific serine/threonine protein kinase (SPEG) and obscurin) was dramatically altered by MIC, and we propose how these kinases could play a fundamental role in skeletal muscle mechanotransduction. This article is protected by copyright. All rights reserved