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Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction

Abstract

Agrin signals through the muscle-specific receptor tyrosine kinase (MuSK) to cluster acetylcholine receptors (AChRs) on the postsynaptic membrane of the neuromuscular junction (NMJ). This stands as the prevailing model of synapse induction by a presynaptic factor, yet the agrin-dependent MuSK signaling cascade is largely undefined. Abl1 (previously known as Abl) and the Abl1-related gene product Abl2 (previously known as Arg) define a family of tyrosine kinases that regulate actin structure and presynaptic axon guidance. Here we show that the Abl kinases are critical mediators of postsynaptic assembly downstream of agrin and MuSK. In mouse muscle, Abl kinases were localized to the postsynaptic membrane of the developing NMJ. In cultured myotubes, Abl kinase activity was required for agrin-induced AChR clustering and enhancement of MuSK tyrosine phosphorylation. Moreover, MuSK and Abl kinases effected reciprocal tyrosine phosphorylation and formed a complex after agrin engagement. Our findings suggest that Abl kinases provide the developing synapse with the kinase activity required for signal amplification and the intrinsic cytoskeletal regulatory capacity required for assembly and remodeling.

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Figure 1: Abl kinases are expressed in mouse muscle and are localized to the developing NMJ.
Figure 2: Abl kinases concentrate postsynaptically at the NMJ.
Figure 3: A specific pharmacologic inhibitor of Abl kinase activity blocks agrin-induced AChR clustering in cultured myotubes.
Figure 4: A kinase-defective Abl1 allele blocks agrin-induced AChR clustering in C2C12 myotubes.
Figure 5: Abl kinases and MuSK effect reciprocal tyrosine phosphorylation.
Figure 6: Abl kinases are recruited to the MuSK signaling complex.

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Acknowledgements

We thank members of the Feng and Pendergast laboratories for technical assistance and critical reading of the manuscript; M.D. Ehlers for critical input; J.R. Sanes for MuSK constructs and A.J. Koleske for Abl2(SH2-SH3) antibody. This work was supported by the Duke University Medical Center Medical Scientist Training Program (A.J.F.); the Sloan Fellowship, the Klingenstein Fellowship in the Neurosciences and a research grant (5-FY01-500) from the March of Dimes Birth Defects Foundation (G.F.) and NIH grants CA70940 and GM62375 (A.M.P.).

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Correspondence to Guoping Feng or Ann Marie Pendergast.

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Supplementary Fig. 1.

Proposed function of Abl kinases in synaptic assembly downstream of agrin/MuSK. Agrin engagement of the MuSK receptor complex leads to activation of MuSK and Abl kinases via reciprocal tyrosine phosphorylation. This may result in recruitment of additional proteins to the MuSK scaffold; targeting of synaptic components and cytoskeletal effectors by Abl kinases; and mobilization of the intrinsic actin bundling activity of the Abl kinases at sites of postsynaptic assembly. (PDF 88 kb)

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Finn, A., Feng, G. & Pendergast, A. Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction. Nat Neurosci 6, 717–723 (2003). https://doi.org/10.1038/nn1071

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