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

The Wnt/β-catenin (cWnt) signaling pathway is one of the most important intercellular signaling pathways in animals. How cWnt components regulate the pathway in bilaterians is well understood, but these processes are poorly characterized in the earliest branching animal clades. We used the cnidarian Nematostella (cnidarians are the closest bilaterian outgroup) to study the cWnt destruction complex (DC) which is the primary mechanism for regulating cytoplasmic concentrations of β-catenin. In bilaterians the DC scaffolding proteins Axin and APC directly interact and bind β-catenin to target it for proteolysis, but all non-bilaterian Axin and APC homologs lack the conserved domains required for bilaterian DC function. Using functional approaches, we show that in Nematostella Axin and APC-like proteins scaffold an active DC that is regulated differently than the bilaterian DC. Additionally, using AlphaFold software we discovered a sequence embedded in the Axin-RGS domain of non-bilaterian Axin that has homology to the bilaterian Axin β-catenin-binding motif (βcatBM). We propose that the bilaterian βcatBM evolved as a low-affinity β-catenin-interacting sequence in the Axin-RGS domain in pre-bilaterians and moved C-terminally in bilaterians where it acquired higher affinity for β-catenin. Our work provides novel insight into how regulatory modules can evolve in metazoan signaling pathways.