Postsynaptic role for Abl kinases and Disabled-1 in assembly of the neuromuscular junction

This document describes work 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). Though paradigmatic, this event is poorly understood. We combined immunohistochemical analysis of murine muscle with pharmacologic and genetic manipulation of cultured myotubes to identify the AN family of nonreceptor tyrosine kinases, Abl and Arg, as critical mediators of postsynaptic assembly downstream of agrin and MuSK. AN kinases localize to the postsynaptic membrane of the NMJ in vivo and AN kinase activity is required for agrin-induced AChR clustering and enhancement of MuSK tyrosine phosphorylation in myotube culture. Further, agrin stimulation increases endogenous Abl kinase activity and induces formation of a MuSK-Abl complex conducive to reciprocal phosphorylation. We propose that Abl family kinases provide the developing synapse both a tyrosine kinase activity required for signal amplification and a cytoskeletal regulatory capacity required for assembly and remodeling. To tackle, at once, the composition of the postsynaptic apparatus and the mechanisms of its regulation, we performed an immunohistochemical screen of murine muscle to identify Abl-associated proteins exhibiting specific localization to the NMJ. Thus, we identified the phosphotyrosine binding (PTB) domain-containing protein Disabled-1 (Dab1) as a novel component of the NMJ in vivo and an endogenous MUSK-interacting protein in myotube culture. Both depletion and overexpression of Dab1 diminish agrin-induced AChR clustering, consistent with distinct roles for Dab1 in potentiation of MuSK cell surface expression and inhibition of its activation. Thus Dab1 demonstrates the importance of intracellular adaptors to proper synaptic function and provides an unexpected mechanistic link between surface expression of a receptor and prevention of its illicit activation.