Netrin -mediated signal transduction in <italic>Caenorhabditis elegans</italic>.
Lee, Jeeyong
2005
Abstract
<italic>unc-6</italic>/netrin and its receptors, <italic>unc-5</italic> and <italic>unc-40</italic>/d&barbelow;eleted in c&barbelow;olorectal c&barbelow;arcinoma (DCC), regulate axon guidance and cell migration in <italic>Caenorhabditis elegans</italic>. In the presence of <italic>unc-5, unc-6</italic>/netrin binds both <italic>unc-40</italic>/DCC and <italic>unc-5</italic>, causing repulsion. Both immunoglobulin domains of <italic>unc-5</italic> are required for the interaction with <italic>unc-6</italic>/netrin. We examined the functional consequences of removing the <italic>unc-6</italic>/netrin binding and nonbinding domains of <italic>unc-5. In vivo</italic> experiments in <italic>C. elegans </italic> indicate that both the <italic>unc-6</italic>/netrin-binding and nonbinding domains are necessary for phenotypic rescue of <italic>unc-5</italic> loss of function mutations. Over-expression of <italic>unc-5</italic> deletion constructs in wild type background cause a club-shape gonad phenotype, suggesting hyperactive receptors. Overall, we show a possible activation mechanism of <italic> unc-5</italic> receptor by <italic>unc-6</italic>/netrin ligands. Some downstream factors of <italic>unc-6</italic>/netrin signaling have been elucidated. However, many details of signal transduction still need to be clarified. When the receptors are activated by <italic>unc-6 </italic>/netrin, both <italic>unc-5</italic> and <italic>unc-40</italic>/DCC are tyrosine-phosphorylated. The activity of the receptor is compromised when some tyrosine sites of <italic>unc-5</italic> are mutated, suggesting the importance of the tyrosine phosphorylations in <italic>unc6</italic>/netrin signaling. In an effort to find the responsible tyrosine kinase(s), we tested <italic> src-1</italic> tyrosine kinase for its role in <italic>unc-6</italic>/netrin signaling pathways. We show that <italic>src-1</italic> interacts with the cytosolic domain of <italic>unc-5</italic> through its SH2 domain. This interaction also requires the intact kinase activity of <italic>src-1</italic>. Downregulation of <italic>src-1</italic> by RNA interference decreases the biological processes initiated by the <italic>unc-5</italic> protein. We also generated a chimeric protein consisting of the extracellular domain and transmembrane domain of <italic> unc-5</italic> and an intracellular domain of <italic>src-1</italic>. This fusion protein is able to partially rescue mutant phenotypes caused by <italic> unc-5</italic>, but not <italic>unc-6</italic>/netrin, <italic>unc-40</italic>/DCC and <italic>unc-34</italic>/ena. Our results support a model that <italic> src-1</italic> is required for <italic>unc-5</italic> induced axon repulsion and gonad migration signaling pathways, and localizing <italic>src-1</italic> activity to <italic>unc-5</italic> is crucial for proper signal transduction in response to <italic>unc-6</italic>/netrin.Subjects
Axon Guidance Caenorhabditis Elegans Netrin-mediated Signal Transduction Unc-5
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