Precise control of Wnt/β-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. Here we identify a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-α3. The Nkd NLS is similar to the Importin-α3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-α3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. Our results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/β-catenin signaling and broaden our understanding of signaling pathways that engage the nuclear import machinery.
