Résumé :
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Communication n° 643 The muscle-specific receptor tyrosine kinase (MuSK) is part of an agrinreceptor complex, that orchestrates the differentiation of the neuromuscular junction (NMJ). Activation of MuSK stimulates tyrosine phosphorylation and drives clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane. MuSK also regulates synaptic gene transcription in subsynaptic nuclei.To gain insight into the function of the MuSK complex, we have developped a proteomic approach to identify new MuSK partners. Mass spectrometry analysis of MuSK crosslink products from postsynaptic membranes of the Torpedo electrocytes identified the adaptor protein 14-3-3 gamma. The 14-3-3 proteins constitute a family of regulatory proteins involved in cell signaling. The 14-3-3 gamma protein was localized at the adult rat NMJ, and cotransfection experiments in COS-7 cells showed that 14-3-3 gamma codistributed with MuSK at the plasma membrane. Futhermore, 14-3-3 gamma copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. 14-3-3 gamma did not colocalize with agrin-elicited acetylcholine receptor (AChR) aggregates in cultured myotubes, suggesting that it is not involved in AChR clustering. Expression of 14-3-3 gamma in cultured myotubes specifically repressed the transcription of several synaptic reporter genes (epsilon- and delta-AChR, Utrophin (promoter A), leaving the MCK unchanged. This down-regulation is caused by the inactivation of Raf-1 in the MAPKinase pathway. This repression was potentiated by MuSK expression. Moreover, the expression of 14-3-3 gamma in muscle fibers in vivo caused both the repression of AChR, utrophin, rapsyn transcription and morphological perturbations of the NMJ. Our results indicate that, apart from its well documented role in AChR clustering, the MuSK complex might also be involved in the regulation of synaptic gene expression in skeletal muscle cells. Supported by:CNRS, Universités Paris 6 and 7, AFM.
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