Identification of a novel xPAK1 partner important for proper FGF signaling in Xenopus laevis

Abstract

The protein complex of Axin, APC and GSK-3 acts as a primary negative regulator of theWnt signaling pathway. Axin is a key scaffolding protein of the -catenin degradation complex and contains a -catenin binding domain, a GSK binding domain, an RGS domain and a DIX domain. Our study focuses on the role of the Axin-RGS domain. Typically, RGS domains contain a conserved asparagine (Asn), critical for its ability to bind heterotrimeric G-proteins. Interestingly, we find that the Axin-RGS domain encodes a glutamine (Gln) at the equivalent position, suggesting a novel function for Axin-RGS. Because Wnt signaling is thought to involve activation of G-proteincoupled Frizzled receptors, we tested whether the Axin-RGS domain functions in this context. We generated a point mutation converting the original Gln to Asn, predicted to increase affinity for GT and another converting Gln to Ala, predicted to abolish binding activity. Wild type and mutant Axin were overexpressed in zebrafish (Danio rerio) and frog (Xenopus laevis) embryos. We also tested for the ability of the mutant forms to rescue antisense knockdown of Axin protein. We found that overexpression of Axin-RGS domain mutants inhibits endogenous Wnt signaling and showed a differential ability to restore normal development in fish and frog embryos, albeit with differing efficiencies. Our results suggest that the interaction of Axin with G-proteins, via its RGS domain, may play a key role in the regulation of Wnt signaling during axial development.