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J Biol Chem August 22, 2014; 289 (34): 23520-33.

Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ϵ and frizzled5.

Bernatík O , Šedová K , Schille C , Ganji RS , Červenka I , Trantírek L , Schambony A , Zdráhal Z , Bryja V .

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.

PubMed ID: 24993822
PMC ID: PMC4156093
Article link: J Biol Chem

Species referenced: Xenopus
Genes referenced: ctnnb1 cxxc4 dvl1 dvl2 dvl3 fzd5 fzd7 tmem88 tptep2-csnk1e
Antibodies: Csnk1e Ab1 Dvl3 Ab2 FLAG Ab2 FLAG Ab3 V5 Ab1

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References [+] :
Angers, The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. 2006, Pubmed, Xenbase