The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle 1 (Nkd), a Dvl-interacting protein.
The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects all the way up to vertebrates.
To test the role of the Nkd EF-hand in Wnt signal integration, we created two different mutations in the zebrafish Nkd: one with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like antagonizes Wnt/β-catenin more robustly than zebrafish Nkd.
The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT, but not the two mutant forms, shows localization changes in the calcium fluxing cells that also host converging Wnt signals versus calcium quiescent cells.
Our functional data suggests that the Nkd EF-hand is important for Wnt signal integration. Interestingly, Nkd only contains one EF-hand, and proteins that bind calcium tend to have multiple. Calcium binding can also be influenced by binding partners. Because of this, we investigate the role of the Nkd binding protein Dvl and their possible calcium affinity. Dvl is a pivotal point in the Wnt signaling network, leading to the output decision of a cell. EF-hand of Nkd binds to the PDZ domain of Dvl. Interestingly, the Dvl PDZ domain contains a region rich in negatively charged amino acids that could aid in binding calcium. In the same manner as Nkd, we generated a Dvl with neutralized putative EF-hand and tested its function and localization relative to wildtype Dvl.
This work elucidates the elegant mechanism by which a cell receiving multiple Wnt signals integrates the information into a specific response. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.