TheNematostellaWnt/β-catenin destruction complex provides insight into the evolution of the regulation of a major metazoan signal transduction pathway

Abstract 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. Competing Interest Statement The authors have declared no competing interest. Footnotes In this revised manuscript, we have expanded our investigation into the evolutionary origins of the Wnt/β-catenin signaling pathway by integrating new functional experiments, structural predictions, and updated visualizations. Our primary objective was to resolve how the destruction complex (DC) acquired signaling activity in early-branching metazoans despite lacking previously defined bilaterian binding motifs. Functional Validation: We performed new assays to test the necessity of Axin β-catenin binding motifs. By mutating a conserved leucine residue in NvAxin to match the sequence found in ctenophores (which we demonstrate lack Axin-β-catenin interaction), we successfully abolished the interaction between NvAxin and Nvβ-catenin. This provides direct evidence for the functional importance of these ancestral motifs. Expanded Phylogenetic Scope: Our analysis now includes broader comparisons across Placozoa, Porifera, and Ctenophora, identifying single βcatBM-like sequences within the Axin-RGS domains of these early lineages. Revised Figures: Figures 1, 6, 7, and 8 have been updated to incorporate new experimental data and refined structural models. Supplementary Figure 8 has been updated provide detailed support for the lack of interaction in ctenophore Axin and β-catenin. Supplementary Figure 9 (New) has been added more cnidarians and ctenophore for comparative analysis of identified βcatBM-like sequences.