Naked and Dishevelled with Zinc but Not Calcium

Science's STKE  17 Dec 2002:
Vol. 2002, Issue 163, pp. tw473-TW473
DOI: 10.1126/stke.2002.163.tw473

Rousset et al. investigated interactions between the Drosophila proteins naked cuticle (Nkd) and dishevelled (Dsh) and discovered that, unexpectedly, binding was insensitive to Ca2+ but sensitive to Zn2+. The Wnt signaling pathway plays a critical role in regulating gene expression during development and inappropriate activation of genes under Wnt control has been implicated in cancer pathogenesis. Nkd, a protein that contains a single EF-hand, a motif associated with Ca2+ binding, participates in a negative feedback loop to attenuate Wnt signaling by binding to the Wnt effector Dsh in a region that contains a sequence high in basic residues as well as a PDZ domain. The authors used Nkd deletion mutants in both yeast two-hybrid and glutathione S-transferase (GST) pull-down assays to determine that both the EF-hand domain and an adjacent region were involved in binding to Dsh. Using transgenic flies that misexpressed mutant Nkd, the authors determined that both of these regions contributed to Nkd activity in vivo. The divalent cation chelator EDTA inhibited Nkd association with Dsh in the GST pull-down assay, whereas EGTA, which binds Ca2+ with an affinity similar to EDTA's but has a much lower affinity for Zn2+, did not. Zn2+, but not Ca2+ or Mg2+, restored the association between Nkd and Dsh. Autoradiographic analysis on purified Nkd deletion mutants indicated that Nkd bound 65Zn2+ but not 45Ca2+, and that Zn2+ binding was mediated by a domain in the region adjacent to the EF-hand domain that contributed to Nkd interaction with Dsh.

R. Rousset, K. A. Wharton, G. Zimmermann, M. P. Scott, Zinc-dependent interaction between dishevelled and the Drosophila Wnt antagonist naked cuticle. J. Biol. Chem. 277, 49019-49026 (2002). [Abstract] [Full Text]