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Identification of the downstream molecules of agrin/Dok‐7 signaling in muscle

The development of the neuromuscular junction depends on signaling processes that involve protein phosphorylation. Motor neuron releases agrin to activate muscle protein Dok‐7, a key tyrosine kinase essential for the formation of a mature and functional neuromuscular junction. However, the signaling cascade downstream of Dok‐7 remains poorly understood. In this study, we combined the clustered regularly interspaced short palindromic repeats/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok‐7. We found tyrosine phosphorylation level of 36 proteins increased specifically by agrin stimulation. In Dok‐7 mutant myotubes, however, 13 of the 36 proteins failed to be enhanced by agrin stimulation, suggesting that these 13 proteins are Dok‐7‐dependent tyrosine‐phosphorylated proteins, could work as downstream molecules of agrin/Dok‐7 signaling. We validated one of the proteins, Anxa3, by in vitro and in vivo assays. Knocking down of Anxa3 in the cultured myotubes inhibited agrin‐induced AChR clustering, whereas reduction of Anxa3 in mouse muscles induced abnormal postsynaptic development. Collectively, our phosphoproteomics analysis provides novel insights into the complicated signaling network downstream of agrin/Dok‐7.