Functional analysis of the Nematostella Wnt/β-catenin destruction complex provides insight into the evolution of a critical regulatory module in a major metazoan signal transduction pathway

The genesis of signaling pathways likely drove metazoan evolution, but the origins of these pathways and how they acquired signaling activity is poorly understood. Here, we studied functional evolution of the Wnt/β-catenin (cWnt) pathway destruction-complex (DC) that regulates β-catenin signaling. Bilaterian DC function requires β-catenin binding to Axin and APC proteins, and Axin-APC heterodimerization. However, bioinformatic analyses predicted that Axin and APC-like homologs in early-branching non-bilaterians lack important previously defined bilaterian β-catenin binding domains, questioning if they have functional cWnt-DCs. We demonstrate that both Axin and APC proteins in the cnidarian Nematostella vectensis (a representative of the sister taxon to bilaterians) can regulate cWnt signaling indicating an active cWnt-DC. Using in vitro analyses, we show that NvAxin binds Nvβ-catenin weakly despite lacking the conserved bilaterian Axin β-catenin-binding motif (βcatBM). Using AlphaFold3, we identified two predicted βcatBM-like sequences in NvAxin, one within the Axin-RGS domain and another towards the C-terminus. Similar analysis of placozoan, poriferan, and ctenophore Axin identified single βcatBM-like sequences located within Axin-RGS. We show that ctenophore Axin and β-catenin do not interact and changing a conserved leucine on NvAxin-βcatBM-like motifs to resemble the ctenophore sequence abolished NvAxin-Nvβ-catenin interactions. We propose that an ancestral Axin-RGS sequence acquired low-affinity β-catenin binding early in metazoan evolution, followed by motif duplication in the cnidarian-bilaterian ancestor. In bilaterians, the duplicated βcatBM evolved higher-affinity for β-catenin, while the ancestral sequence was lost. Our results demonstrate how phylogenetic insights, AI tools and functional assays can be used to reconstruct the evolution of complex signaling pathways.