1. Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1.
- Author
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Harnoš J, Cañizal MCA, Jurásek M, Kumar J, Holler C, Schambony A, Hanáková K, Bernatík O, Zdráhal Z, Gömöryová K, Gybeľ T, Radaszkiewicz TW, Kravec M, Trantírek L, Ryneš J, Dave Z, Fernández-Llamazares AI, Vácha R, Tripsianes K, Hoffmann C, and Bryja V
- Subjects
- Animals, Biosensing Techniques, Casein Kinase 1 epsilon genetics, Dishevelled Proteins genetics, Enzyme Assays methods, Fluorescence Resonance Energy Transfer, Frizzled Receptors metabolism, Gene Knockout Techniques, HEK293 Cells, Humans, Microscopy, Fluorescence methods, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Oocytes, Phosphorylation physiology, Single-Cell Analysis methods, Xenopus laevis, Casein Kinase 1 epsilon metabolism, Dishevelled Proteins metabolism, PDZ Domains physiology, Wnt Signaling Pathway physiology
- Abstract
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.
- Published
- 2019
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