Netrins are a family of well-characterized guidance cues that are conserved across different species and are homologous to laminin beta2. They promote axon outgrowth and guide neuronal growth cones in the developing nervous system. The intracellular signaling mechanism of netrin is unclear. In preliminary studies, we found that DCC (deleted in colorectal cancer), a netrin receptor, interacts directly with the focal adhesion kinase (FAK), a tyrosine kinase implicated in integrin signaling. Netrin-1 stimulation results in increased DCC-FAK interaction and FAK tyrosine phosphorylation at multiple residues including tyrosines 397 and 861. Furthermore, netrin-1 induces DCC/neogenin tyrosine phosphorylation in a manner dependent on FAK and an Src family kinase. The tyrosine phosphorylated DCC, on the other hand, binds to Fyn, an Src family kinase enriched in the brain. Functionally, treatment of rat cortical explants with PP2, an inhibitor of Src kinases, prevents netrin-1 from inducing neurite outgrowth. Expression of dominant negative FAK mutant blocks netrin-1 induced attractive turning in Xenopus spinal neurons. Based on these preliminary results, we hypothesize that FAK is important in mediating and/or regulating netrin functions. To test this hypothesis, we will: (1) determine the role of DCC tyrosine phosphorylation in netrin-induced axon outgrowth and turning, (2) determine the role of FAK in netrin-induced axon outgrowth and turning, and (3) investigate the effects of laminin on netrin-induced axon outgrowth and turning. The proposed research will provide not only key pieces of the signaling pathway by which netrin-DCC regulate axon outgrowth and growth cone guidance, but also insight into how signaling networks between netrin-DCC and extracellular matrix-integrins are organized. Such knowledge is essential for understating the mechanisms involved in axon path-finding in neural development and birth defects with malformation of the brain and spinal cord.
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