Src-class kinases act within the agrin/musk pathway to regulate acetylcholine receptor phosphorylation, cytoskeletal anchoring, and clustering

Abstract

Synaptogenesis at the neuromuscular junction requires agrininduced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MUSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MUSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.