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Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1
J. Harnoš, MCA. Cañizal, M. Jurásek, J. Kumar, C. Holler, A. Schambony, K. Hanáková, O. Bernatík, Z. Zdráhal, K. Gömöryová, T. Gybeľ, TW. Radaszkiewicz, M. Kravec, L. Trantírek, J. Ryneš, Z. Dave, AI. Fernández-Llamazares, R. Vácha, K....
Language English Country England, Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
NV15-34405A
MZ0
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- MeSH
- Single-Cell Analysis methods MeSH
- Biosensing Techniques MeSH
- Enzyme Assays methods MeSH
- Microscopy, Fluorescence methods MeSH
- Phosphorylation physiology MeSH
- Frizzled Receptors metabolism MeSH
- Gene Knockout Techniques MeSH
- HEK293 Cells MeSH
- Casein Kinase 1 epsilon genetics metabolism MeSH
- Humans MeSH
- Mutagenesis, Site-Directed MeSH
- Oocytes MeSH
- PDZ Domains physiology MeSH
- Dishevelled Proteins genetics metabolism MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Wnt Signaling Pathway physiology MeSH
- Molecular Dynamics Simulation MeSH
- Xenopus laevis MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.
CEITEC Central European Institute of Technology Masaryk University Brno 62500 Czech Republic
Department of Experimental Biology Faculty of Science Masaryk University Brno 62500 Czech Republic
References provided by Crossref.org
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- $a Harnoš, Jakub $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, 62500, Czech Republic. Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
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