Molecular Mechanism Of Wnt Activation By Ccd1

The Wnt signaling pathway plays central roles in embryonic development, adult tissue homeostasis and many other processes. The canonical Wnt signaling is one of the best characterized pathways, whereβ-catenin is the key effector molecule. In response to Wnt stimulation, Frizzled/LRP6 receptors can associate with Dvl, which then recruits Axin, along with GSK-3β, to the plasma membrane via direct interaction involving their conserved DIX domains, where Dvl and Axin/GSK-3β, among other components, constitute the initiation complex, leading to stabilization of cytoplasmicβ-catenin that subsequently enters the nucleus and ultimately modulates expression of specific target genes.Both Axin and Dvl contain a conserved DIX domain, which mediates their homo- and/or hetero-oligomerization and is indispensable for the canonical Wnt signaling transduction. The cytoplasmic Dvl/Axin has a great tendency to form puncta, dynamic protein assemblies, via its DIX domain. Coiled-coil-DIX1 (Ccd1), the other member of the DIX-domain protein family, can form heteromeric complexes with Dvl and Axin and positively regulate the canonical Wnt signaling during zebrafish neural patterning.However, the structural basis for the three DIX-domain proteins to interplay with one another is unclear. To explore the function of the DIX domains in Wnt signaling in the structural terms, we have determined the structures of Ccd1-DIX and Dvl1-DIX(Y17D) and carried out extensive studies on how the three DIX domain proteins can dynamically modulate each other, with emphasis on the abilities and the structural attributes therein of Ccd1 to form a ternary complex with Axin and Dvl and to control the polymeric nature of Dvl. The crystal packing of Ccd1-DIX suggested that Dvl and Ccd1 directly interact with each other through the'head'of Dvl-DIX and the'tail'of Ccd1-DIX, and revealed critical residues for DIX-DIX interaction from two sites, a primary one (Site I) and an auxiliary site (Site II). Systematic mutagenesis studies suggest that the integrity of the interface is indispensable required for Dvl homo-assembly, puncta formation and Wnt signal transduction. The ability of Ccd1 to interact with Dvl correlates precisely with its function to activate Wnt signaling. Moreover, Ccd1 was found to depolymerize the homo-assembly of Dvl in vitro and induces its diffuse cytoplasmic distribution in vivo. Importantly, evidence exists to suggest the formation of a Dvl-Ccd1-Axin ternary complex, which involves not only the DIX domains but also the CC domain of Ccd1. Our data, together with prior work, lead to a bifunctional model for Ccd1 in Wnt signal activation, in that Ccd1 not only provides a bridge for Axin to interact with Dvl, but also serves as a determinant for the length of Dvl polymer.