Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1ε and Frizzled5

Background: Phosphorylation of Dishevelled (Dvl) by casein kinase 1E (CK1E) is a key event in Wnt signal transduction. Results: Dvl3 residues phosphorylated by CK1E were identified by proteomics and analyzed functionally. Conclusion: Individual phosphorylation events control different aspects of Dvl biology. Significance: CK1E and Fzd5, a Wnt receptor, act on Dvl via distinct mechanism, suggesting that CK1E is not directly downstream of Frizzled5. Dishevelled-3 (Dvl3), a key component of the Wnt signaling pathways, acts downstream of Frizzled (Fzd) receptors and gets heavily phosphorylated in response to pathway activation by Wnt ligands. Casein kinase 1E (CK1E) was identified as the major kinase responsible for Wnt-induced Dvl3 phosphorylation. Currently it is not clear which Dvl residues are phosphorylated and what is the consequence of individual phosphorylation events. In the present study we employed mass spectrometry to analyze in a comprehensive way the phosphorylation of human Dvl3 induced by CK1E. Our analysis revealed >50 phosphorylation sites on Dvl3; only a minority of these sites was found dynamically induced after co-expression of CK1E, and surprisingly, phosphorylation of one cluster of modified residues was down-regulated. Dynamically phosphorylated sites were analyzed functionally. Mutations within PDZ domain (S280A and S311A) reduced the ability of Dvl3 to activate TCF/LEF (T-cell factor/lymphoid enhancer factor)-driven transcription and induce secondary axis in Xenopus embryos. In contrast, mutations of clustered Ser/Thr in the Dvl3 C terminus prevented ability of CK1E to induce electrophoretic mobility shift of Dvl3 and its even subcellular localization. Surprisingly, mobility shift and subcellular localization changes induced by Fzd5, a Wnt receptor, were in all these mutants indistinguishable from wild type Dvl3. In summary, our data on the molecular level (i) support previous the assumption that CK1E acts via phosphorylation of distinct residues as the activator as well as the shut-off signal of Wnt/-catenin signaling and (ii) suggest that CK1E acts on Dvl via different mechanism than Fzd5.