An Unconventional Nuclear Localization Motif Is Crucial for Function of the Drosophila Wnt/Wingless Antagonist Naked Cuticle

Abstract

Wnt/b-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/b-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the b-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-offunction assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation

“…Thus, the ability of Tum and Pav to translocate into the nucleus is crucial for their downregulation of the Wg/Wnt pathway. Co-expression of Nkd, which is thought to inhibit the Wnt pathway upstream of Arm stabilization (Waldrop et al, 2006), was also unable to block the arm S10 -induced cell-fate transformation, as expected (Fig. 7E,F).…”

Viable Mice with Compound Mutations in the Wnt/Dvl Pathway Antagonists nkd1 and nkd2

“…Mice and humans have two nkd genes, nkd1 and nkd2, that share sequence similarity with fly nkd in two regions: an EF hand-containing domain-termed the EFX domain-that binds Dsh and a C-terminal histidine-rich region (35,(70)(71)(72). Alignments of insect and mammalian Nkd proteins reveal four conserved sequence motifs interspersed by mostly unrelated sequence, suggesting a common arrangement of functional motifs in the ancestral Nkd protein (67,70). Our studies of Drosophila Nkd showed that protein truncations N terminal of Dsh-binding regions produced embryonic lethality with the strongest phenotypic consequences (67, 75).…”

“…Shortly after the onset of Wg action, nkd mutants develop markedly elevated levels of ␤-catenin and expanded domains of Wg target gene expression despite an apparently normal quantity and distribution of Wg, suggesting that nkd mutant cells are hypersensitive to Wg (5,52,67,75). Molecular characterization of nkd revealed a novel gene whose transcript is Wg inducible, thereby forming a negative feedback loop (75).…”

New Advances in Signaling and Pattern Formation

“…At this meeting and in a recent publication in Genetics they provide evidence that Nkd functions, at least in part, within the nucleus to restrain Wg signaling. 10 They find that Nkd contains two nuclear localization sequences that are required for activity. These findings, in conjunction with previous results showing that Nkd must bind to Dsh in order to down regulate the Wg signal, taken together with the finding from vertebrate studies that Dsh nuclear localization is required for its ability to fully activate signaling 11 suggest that Nkd may function as an antagonist of nuclear signaling steps.…”

“…The site for the latest installment of the Drosophila Research Conference (March [7][8][9][10][11]2007) was downtown Philadelphia. The conference offered an illustration of all that is great about the Drosophila field bringing together evolutionary biologists, immunologists and everyone in between.…”