Feedback Regulation of Wnt Signaling By Naked Cuticle (Nkd) During Drosophila Embryogenesis

Wnt/beta -catenin signals are essential for many developmental and physiological processes in animals. Deregulation of the Wnt signaling pathway in mammals can cause diseases such as birth defects, cancer, osteoporosis, and diabetes. In Drosophila, the naked cuticle (nkd) gene antagonizes the Wnt/beta -catenin signaling in every segment of the embryo. Nkd is a modular, evolutionarily conserved protein that uses an EF-hand motif and adjacent sequences to target the cytoplasmic Wnt signal transducer Disheveled (Dsh). The mechanism by which Nkd antagonizes Wnt signaling in Drosophila embryos is not well understood. The abundance and bulk distribution of Dsh is not altered in nkd mutants as compared to wild type embryos, and overexpression of Nkd transgenes in nkd mutants did not alter Dsh distribution or abundance by confocal microscopy. Nkd transgenes lacking Dsh-binding regions were mostly able to rescue nkd mutants, suggesting that the Dsh-binding regions of Nkd contribute little to Nkd activity, at least when the transgenes were overexpressed. In this thesis, I have investigated non-Dsh binding regions that are critical for Nkd function. Our lab's findings indicate that a conserved 30 amino acid motif is essential for Nkd nuclear localization and function. Substitution of the 30aa motif with a heterologous nuclear localization sequence (NLS) rescued some nkd mutants to adulthood. In support of Nkd's role in the nucleus, Nkd binds to Importin-alpha 3, an adaptor for the canonical nuclear import apparatus. I identified that Nkd associates with Importin-alpha 3 via a motif ("D6") that is conserved between D. melanogaster and D. pseudoobscura. NkdΔD6, lacking the Importin-alpha 3-binding motif, was defective in nuclear localization and in rescuing nkd mutants. RNAi knockdown of importin-alpha 3 prevented the nuclear localization of Nkd. The findings that Nkd possesses two NLSs, each of which is required for function, and that Nkd associates with a component of the nuclear import apparatus, suggest that Nkd antagonizes the Wnt/beta -catenin signaling in the nucleus. Furthermore, I also addressed the function of the N-terminus of Drosophila Nkd. Unlike mammalian Nkd homologs that have N-terminal myristoylation consensus sequences responsible for membrane association, the N-terminus of Drosophila Nkd, also conserved in mosquito Nkd, lack such a sequence. Nonetheless, Nkd's N-terminus was required for function and membrane association. Substitution of the N-terminus with heterologous myristoylation sequences did not restore nkd function, indicating that the mechanism by which Drosophila Nkd associates with the membrane is different than mammalian Nkds. Therefore, Nkd appears to function in the membrane, in the cytoplasm to target Dsh, and in the nucleus to antagonize Wg signaling.