Netrin-mediated signal transduction in Caenorhabditis elegans.

摘要

unc-6/netrin and its receptors, unc-5 and unc-40/d&barbelow;eleted in c&barbelow;olorectal c&barbelow;arcinoma (DCC), regulate axon guidance and cell migration in Caenorhabditis elegans. In the presence of unc-5, unc-6/netrin binds both unc-40/DCC and unc-5, causing repulsion. Both immunoglobulin domains of unc-5 are required for the interaction with unc-6/netrin. We examined the functional consequences of removing the unc-6/netrin binding and nonbinding domains of unc-5. In vivo experiments in C. elegans indicate that both the unc-6/netrin-binding and nonbinding domains are necessary for phenotypic rescue of unc-5 loss of function mutations. Over-expression of unc-5 deletion constructs in wild type background cause a club-shape gonad phenotype, suggesting hyperactive receptors. Overall, we show a possible activation mechanism of unc-5 receptor by unc-6/netrin ligands.; Some downstream factors of unc-6/netrin signaling have been elucidated. However, many details of signal transduction still need to be clarified. When the receptors are activated by unc-6 /netrin, both unc-5 and unc-40/DCC are tyrosine-phosphorylated. The activity of the receptor is compromised when some tyrosine sites of unc-5 are mutated, suggesting the importance of the tyrosine phosphorylations in unc6/netrin signaling. In an effort to find the responsible tyrosine kinase(s), we tested src-1 tyrosine kinase for its role in unc-6/netrin signaling pathways. We show that src-1 interacts with the cytosolic domain of unc-5 through its SH2 domain. This interaction also requires the intact kinase activity of src-1. Downregulation of src-1 by RNA interference decreases the biological processes initiated by the unc-5 protein. We also generated a chimeric protein consisting of the extracellular domain and transmembrane domain of unc-5 and an intracellular domain of src-1. This fusion protein is able to partially rescue mutant phenotypes caused by unc-5, but not unc-6/netrin, unc-40/DCC and unc-34/ena. Our results support a model that src-1 is required for unc-5 induced axon repulsion and gonad migration signaling pathways, and localizing src-1 activity to unc-5 is crucial for proper signal transduction in response to unc-6/netrin.