Functional and structural analysis of xenopus muscle specific kinase

Muscle Specific Kinase (MuSK) is a receptor tyrosine kinase expressed restrictively in the lineage of mammalian skeletal muscle and localized to neuromuscular synapses. Nerve-derived factor, agrin, activates MuSK and stimulates phosphorylation and aggregation of acetylcholine receptor (AChRs) at the postsynaptic sites. Aggregation of different postsynaptic receptors, such as GABA and NMDA receptor, is also a characteristic of the postsynaptic specialization in the central nervous system (CNS). Our laboratory has recently cloned the cDNA encoding Xenopus MuSK (xMuSK). We have reported that xMuSK expression can be detected in myotomal muscle and neural tissues. In light of this evidence, it is interesting to study the potential function of MuSK in the nervous system. In this study, we have examined the role of xMuSK in neuronal development by ectopic expression of xMuSK in neuronal culture. When MuSK was overexpressed in rat pheochromocytoma (PC12) cells, expression and autophosphorylation of MuSK were induced upon NGF-induced differentiation. Northern and Western blot analysis revealed that MuSK expression was up-regulated after NGF treatment. In addition, expression of various neuronal markers such as acetylcholinesterase and neurofilaments, were induced after the introduction of exogenous xMuSK. When compared to its mammalian counterpart, xMuSK contains an extra Kringle domain just after the fourth immunoglobulin-like domain and additional tyrosine phosphorylation sites at the C-terminus. This structural specificity is absent in all known forms of mammalian MuSK. To study the possible role of specific domain and motif in xMuSK functions, mutants of MuSK were constructed. Autophosphorylation assay revealed that complete deletion of the Kringle domain did not affect the tyrosine phosphorylation of MuSK, whereas the mutation at the ectodomain tyrosine phosphorylation site inhibits the MuSK autophosphorylation. For neuronal marker expression, deletion of the Kringle domain or mouse homolog (NSK2) did not significantly increase the neuronal marker expression. Taken together, our findings suggest that xMuSK may be involved in the processes of neuronal development. This function may be mediated through the presence of Kringle domain