The INSR/AKT/mTOR pathway regulates the pace of myogenesis in a syndecan-3-dependent manner

•A phosphoproteomic survey of muscle stem cell progeny reveals that syndecans-3 regulates tyrosine kinase receptor-mediated signal transduction at multiple levels, including insulin/INSR signaling as one of the most enriched pathways.•The presence of syndecan-3 in muscle stem cells during the early stages of myogenesis inhibits insulin/INSR signaling via AKT/mTOR leading to muscle stem cell progeny expansion.•Syndecan-3 downregulation during the late stages of myogenesis releases the block on AKT/mTOR activation by insulin/INSR, leading to muscle stem cell progeny differentiation.•Syndecan-3 acts as a timekeeper of myogenesis, by functioning as a molecular switch for insulin signaling in muscle stem cell progeny. Muscle stem cells (MuSCs) are indispensable for muscle regeneration. A multitude of extracellular stimuli direct MuSC fate decisions from quiescent progenitors to differentiated myocytes. The activity of these signals is modulated by coreceptors such as syndecan-3 (SDC3). We investigated the global landscape of SDC3-mediated regulation of myogenesis using a phosphoproteomics approach which revealed, with the precision level of individual phosphosites, the large-scale extent of SDC3-mediated regulation of signal transduction in MuSCs. We then focused on INSR/AKT/mTOR as a key pathway regulated by SDC3 during myogenesis and mechanistically dissected SDC3-mediated inhibition of insulin receptor signaling in MuSCs. SDC3 interacts with INSR ultimately limiting signal transduction via AKT/mTOR. Both knockdown of INSR and inhibition of AKT restore Sdc3−/− MuSC differentiation to wild type levels. Since SDC3 is rapidly downregulated at the onset of differentiation, our study suggests that SDC3 acts a timekeeper to restrain proliferating MuSC response and prevent premature differentiation.