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.
- MeSH
- analýza jednotlivých buněk metody MeSH
- biosenzitivní techniky MeSH
- enzymatické testy metody MeSH
- fluorescenční mikroskopie metody MeSH
- fosforylace fyziologie MeSH
- frizzled receptory metabolismus MeSH
- genový knockout MeSH
- HEK293 buňky MeSH
- kasein kinasa 1 epsilon genetika metabolismus MeSH
- lidé MeSH
- mutageneze cílená MeSH
- oocyty MeSH
- PDZ domény fyziologie MeSH
- protein dishevelled genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- signální dráha Wnt fyziologie MeSH
- simulace molekulární dynamiky MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
