The in vitro and in vivo phosphotyrosine map of activated MuSK

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in n euromuscular synapse formation. Activation of MuSK is induced by agrin lead ing to clustering of several proteins, including acetylcholine receptors, a t synaptic sites. In a first step to elucidate the signal transduction casc ade following MuSK activation and leading to clustering of synaptic protein s, we sought to identify the tyrosine residues that are phosphorylated in a ctivated MuSK. We mapped the tyrosine residues that are phosphorylated in v itro and in vivo using methods that provide high sensitivity and do not req uire radioactive tracers. We expressed MuSK in insect cells by using a bacu lovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In additio n, we isolated MuSK from Torpedo electric organ and used nanoelectrospray t andem mass spectrometry and parent ion scanning to identify the tyrosine re sidues that are phosphorylated in activated, endogenous MuSK in vivo. We fo und that six of the nineteen intracellular tyrosine residues in MuSK are ph osphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyro sines within the activation loop (Y750, Y754, and Y755), a tyrosine near th e beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C -terminal lobe of the kinase domain. Our biochemical data are consistent wi th results from functional experiments and establish a good correlation bet ween tyrosine residues that are phosphorylated in activated MuSK and tyrosi nes that are required for MuSK signaling.