Src Family Kinase for Wnt5 Signaling

Abstract

Although most members of the Wnt [Wingless (Wg) in flies] family of ligands signal through seven-transmembrane receptors of the Frizzled family, Wnt5 binds to receptor tyrosine kinase-like proteins called RYKs [ROR in mammals and Derailed (Drl) in flies]. RYKs lack conserved residues that are important for kinase activity and so are likely not catalytically active kinases. Wouda et al . identified the Src family tyrosine kinase (SFK) Src64B as a protein that interacted with Drl in a directed yeast two-hybrid screen. Genetic interactions supported a role for Src64B and the related protein Src42A in Wnt5-mediated axonal repulsion of neurons that cross the midline during embryonic development of the Drosophila nervous system. Flies deficient in both Src64B and Src42A showed the most penetrant phenotype of abnormal commissural axonal projections, a phenotype similar to the Wnt5 -null flies. The Wnt5A overexpression phenotype was suppressed in flies heterozygous for a null allele of Src64B , whereas the phenotype was not suppressed in flies heterozygous for Src42A . Thus, although the two SFKs are partially redundant, Src64B appears to be the primary SFK that participates in Wnt5 signaling. Flies overexpressing Src64B and mildly overexpressing Drl showed higher numbers of neurons crossing the midline than did flies in which either was expressed alone. (No effect of overexpression of Src64B was observed in the absence of overexpressed Drl, which suggests that Drl abundance may be limiting.) Transcripts for Src64B and Drl were detected in the same neurons. Drl and Src64B or the mammalian homologs (ROR and Src) were coimmunoprecipitated from transfected cultured cells. The interaction in the transfected cells did not require Wnt5. In a mammalian two-hybrid assay, only catalytically active Src64B interacted with a fusion protein containing the Drl intracellular domain. Even in the absence of Wnt5, the interaction between Drl and Src64B in the transfected cells resulted in tyrosine phosphorylation of Src64B, which is required for its activity, and tyrosine phosphorylation of Drl. Cells transfected with Wnt5, Drl, and Src64B did not activate a reporter gene responsive to the transcription factor known as TCF/LEF, which is an indicator of canonical Wnt pathway activation. How Wnt5 influences the activity of the Drl-Src64B complex remains unclear, and the authors speculate that Wnt5 may direct the receptor-bound SFK to specific substrates. R. R. Wouda, M. R. K. S. Bansraj, A. W. M. de Johng, J. N. Noordermeer, L. G. Fradkin, Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system. Development 135 , 2277-2287 (2008). [Abstract] [Full Text]