Dishevelled (DVL) is the central signal transducer in both Wnt/β-catenin-dependent and independent signalling pathways. DVL is required to connect receptor complexes and downstream effectors. Since proximal Wnt pathway components and DVL itself are upregulated in many types of cancer, DVL represents an attractive therapeutic target in the Wnt-addicted cancers and other disorders caused by aberrant Wnt signalling. Here, we discuss progress in several approaches for the modulation of DVL function and hence inhibition of the Wnt signalling. Namely, we sum up the potential of modulation of enzymes that control post-translational modification of DVL - such as inhibition of DVL kinases or promotion of DVL ubiquitination and degradation. In addition, we discuss research directions that can take advantage of direct interaction with the protein domains essential for DVL function: the inhibition of DIX- and DEP-domain mediated polymerization and interaction of DVL PDZ domain with its ligands.

• Klíčová slova
• Casein kinase 1, DIX oligomerization, Dishevelled, PDZ inhibitors, Wnt signalling-related diseases,
• MeSH
• adaptorové proteiny signální transdukční MeSH
• fosfoproteiny * metabolismus   MeSH
• lidé MeSH
• protein dishevelled * metabolismus   MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• fosfoproteiny * MeSH
• protein dishevelled * MeSH

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

• Klíčová slova
• CK1, DVL3, Dishevelled, Kinase, Mass spectrometry, NEK2, Phosphorylation, TTBK2, Wnt,
• MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• hmotnostní spektrometrie MeSH
• kinasy NEK metabolismus   MeSH
• konformace proteinů MeSH
• kultivované buňky MeSH
• lidé MeSH
• protein dishevelled chemie   metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• kinasy NEK MeSH
• NEK2 protein, human MeSH Prohlížeč
• protein dishevelled MeSH

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.

• Klíčová slova
• Casein Kinase 1ϵ, Cell Signaling, Dishevelled-3, Frizzled5, Mass Spectrometry (MS), Phosphorylation, Post-translational Modification (PTM), Wnt Pathway,
• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• chromatografie kapalinová MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• frizzled receptory metabolismus   MeSH
• genetická transkripce MeSH
• HEK293 buňky MeSH
• kaseinkinasa Iepsilon metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• protein dishevelled MeSH
• proteiny Xenopus MeSH
• retardační test MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• subcelulární frakce metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• DVL1 protein, Xenopus MeSH Prohlížeč
• DVL3 protein, human MeSH Prohlížeč
• fosfoproteiny MeSH
• frizzled receptory MeSH
• FZD5 protein, human MeSH Prohlížeč
• kaseinkinasa Iepsilon MeSH
• protein dishevelled MeSH
• proteiny Xenopus MeSH