title: Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ϵ and frizzled5.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Bernatík
authors.affiliation: National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Research Group-Proteomics, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
authors.forename: Kateřina
authors.initials: K
authors.lastname: Šedová
authors.affiliation: Biology Department, Developmental Biology, Friedrich-Alexander University Erlangen-Nuremberg, Schloßplatz 4, 91054 Erlangen, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Schille
authors.affiliation: From the Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
authors.forename: Ranjani Sri
authors.initials: RS
authors.lastname: Ganji
authors.affiliation: From the Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
authors.forename: Igor
authors.initials: I
authors.lastname: Červenka
authors.affiliation: Structural Biology Program, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, and Cellular Protein Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Trantírek
authors.affiliation: Biology Department, Developmental Biology, Friedrich-Alexander University Erlangen-Nuremberg, Schloßplatz 4, 91054 Erlangen, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schambony
authors.affiliation: National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Research Group-Proteomics, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.
authors.forename: Zbyněk
authors.initials: Z
authors.lastname: Zdráhal
authors.affiliation: 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, bryja@sci.muni.cz.
authors.forename: Vítězslav
authors.initials: V
authors.lastname: Bryja
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D048149:Casein Kinase 1 epsilon / Q000378:metabolism*; D002853:Chromatography, Liquid; D000072261:Dishevelled Proteins; D024202:Electrophoretic Mobility Shift Assay; D051157:Frizzled Receptors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017510:Protein Folding; D013347:Subcellular Fractions / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014158:Transcription, Genetic; D029867:Xenopus Proteins; D014982:Xenopus laevis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C000606586:DVL1 protein, Xenopus; C000606584:DVL3 protein, human; D000072261:Dishevelled Proteins; C498589:FZD5 protein, human; D051157:Frizzled Receptors; D010750:Phosphoproteins; D029867:Xenopus Proteins; D048149:Casein Kinase 1 epsilon
keywords: Casein Kinase 1ϵ; Cell Signaling; Dishevelled-3; Frizzled5; Mass Spectrometry (MS); Phosphorylation; Post-translational Modification (PTM); Wnt Pathway