Reconstructing the evolutionary origin of β-catenin recognition in the Wnt destruction complex.

Emergence of Wnt/β-catenin signaling was a major metazoan innovation, yet how its core molecular interactions evolved remains unresolved. Here, we investigated evolution of the Wnt/β-catenin destruction complex (BDC), a conserved regulatory module that represses Wnt/β-catenin signaling by targeting cytoplasmic β-catenin for degradation. In bilaterians, BDC activity depends on interactions between Axin, adenomatous polyposis coli (APC), and β-catenin. However, bioinformatic analyses suggested that early-diverging metazoans lack the canonical β-catenin-binding motifs required for these interactions raising questions about the origin of BDC function. Using functional assays in Nematostella vectensis, a representative of the Cnidaria, the outgroup to bilaterians, we demonstrate that both NvAxin and NvAPC regulate Wnt/β-catenin signaling, indicating the presence of a BDC despite the lack of canonical bilaterian binding motifs. In vitro experiments revealed that NvAxin binds Nvβ-catenin through previously unrecognized low-affinity interactions. Guided by AlphaFold3 predictions, we identified two β-catenin-binding motif-like sequences in cnidarian Axin including one within the Axin-RGS domain, and a single motif in the Axin-RGS domain of placozoans, sponges, and ctenophores. Functional analyses showed that a conserved residue within these motifs is required for Axin-β-catenin interaction. Our results support a model where an ancestral β-catenin-binding motif-like sequence in Axin-RGS acquired weak β-catenin-binding capacity in early-emerging metazoans followed by motif duplication in the cnidarian-bilaterian last common ancestor. In bilaterians, the duplicated β-catenin-binding motif evolved higher affinity for β-catenin, while the ancestral sequence was lost. By integrating phylogenetics, AI-based structural prediction, and experimental validation, we have revealed for the first time the molecular steps underlying functional evolution of a complex signaling pathway.