Nuclear localization of Duplin, a β-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway

Abstract

Duplin binds to β-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the β-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin α was isolated as a binding protein of Duplin. Importin α bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (DuplinΔ500-584) retained Duplin in the cytoplasm. DuplinΔ500-584) bound to β-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt- dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a β-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed β-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin a, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream β-catenin target genes.