The goal of this research is to define the mechanisms by which protein tyrosine kinases and adaptor proteins regulate the neuromuscular junction (NMJ), a cholinergic synapse at sites of nerve-muscle contact. Proper neurochemical communication at the NMJ requires high density clustering of postsynaptic acetylcholine receptors (AChRs). This clustering occurs early in development when the presynaptic nerve secretes the glycoprotein agrin, which, in turn, activates the muscle-specific receptor tyrosine kinase (MUSK). Although this event has served as the paradigm of synapse induction for over a decade, little is known regarding the signaling network that links agrin-induced activation of MuSK to AChR clustering. We have recently identified the Abl family of nonreceptor tyrosine kinases, Abl and Arg, as critical mediators of postsynaptic assembly downstream of agrin and MUSK. We showed that Abl kinases localize to the postsynaptic membrane of the NMJ in vivo and that Abl kinase activity is required for agrin-induced AChR clustering and enhancement of MuSK tyrosine phosphorylation in myotube culture. Further, agrin stimulation of cultured myotubes increases endogenous Abl kinase activity and induces formation of a MuSK/Abl complex conducive to reciprocal tyrosine phosphorylation. Based on our novel findings, we hypothesize that Abl family kinases are required for the formation and stabilization of the NMJ and provide the developing synapse both the tyrosine kinase activity required for signal amplification and the cytoskeletal regulatory activity required for assembly and remodeling. To test this hypothesis, we propose the following specific aims: 1) determine the functional consequences of Abl-dependent MuSK phosphorylation; 2) elucidate the mechanisms by which Abl kinases regulate agrin-induced AChR clustering; 3) identify additional components of the postsynaptic MuSK/Abl signaling complex; and 4) define the in vivo role of Abl family kinases at the murine NMJ. Our results will provide a mechanistic understanding of the role of receptor and nonreceptor tyrosine kinases and adaptor proteins in the formation and stability of the NMJ. Moreover, they will shed new light on the signaling cascades affected in diseases stemming from aberrant NMJ function, such as the congenital myasthenic syndromes and myasthenia gravis, and may have broad implications for central synapse formation and intercellular communication, in general. ? ?
