Identification of zinc-finger BED domain-containing 3 (Zbed3) as a novel axin-interacting protein that activates Wnt/β-catenin signaling

Abstract

Axin, a key modulator of the Wnt/β-catenin pathway, acts as a scaffold protein in phosphorylating and degrading cytoplasmic β-catenin. Canonical Wnt proteins appear to stabilize β-catenin by inducing the interaction of LRP5/6 with Axin. This interaction requires the phosphorylation of the Ser or Thr residues in the PPPP(S/T)PX(T/S) motifs at the intracellular domain of LRP5/6. In this work, we identified a novel Axin-interacting protein, zinc-finger BED domain-containing 3 (Zbed3), by yeast two-hybrid screening. The interaction was confirmed in co-immunoprecipitation experiment in mammalian cells and in vitro pulldown assays. Moreover, we found Zbed3 also contains a PPPPSPT motif, which is crucial to its binding to Axin. The Ser and Thr residues in the motif appear to be also phosphorylated by glycogen synthase kinase 3β (GSK3β) and the CKI family kinases, as GSK3β and CKIε could enhance the interaction of Zbed3 with Axin. Mutation of the Ser (SA) or Thr (TA) residue to Ala in the motif markedly impaired its ability to interact with Axin. Expressing Zbed3, but not these mutants, led to inhibition of GSK3β-mediated β-catenin phosphorylation, cytoplasmic β-catenin accumulation, and activation of lymphoid enhancer binding factor-1-dependent reporter gene transcription. Furthermore, knockdown of Zbed3 with RNA interference attenuated Wnt-induced β-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and the Wnt target gene expression. These results together indicate that Zbed3 is a novel Axin-binding protein that is involved in Wnt/β-catenin signaling modulation. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.