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 1ϵ (CK1ϵ) 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 CK1ϵ. Our analysis revealed >50 phosphorylation sites on Dvl3; only a minority of these sites was found dynamically induced after co-expression of CK1ϵ, 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 CK1ϵ 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 CK1ϵ acts via phosphorylation of distinct residues as the activator as well as the shut-off signal of Wnt/β-catenin signaling and (ii) suggest that CK1ϵ acts on Dvl via different mechanism than Fzd5.

Biology Department Developmental Biology Friedrich Alexander University Erlangen Nuremberg Schloßplatz 4 91054 Erlangen Germany

From the Institute of Experimental Biology Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic

From the Institute of Experimental Biology Faculty of Science Masaryk University Kotlarska 2 61137 Brno Czech Republic Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic Kralovopolska 135 61265 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic Research Group Proteomics Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Structural Biology Program Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic and Cellular Protein Chemistry Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands

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