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.
Highlights
Dishevelled (DVL) is the key component of the Wnt signaling pathway
Using fluorescein arsenical hairpin binder (FlAsH) Förster resonance energy transfer (FRET) DVL3 sensors we describe the conformational dynamics of DVL3 in living cells upon Wnt pathway stimulation and the key function of Casein kinase 1 ɛ (CK1ɛ) in this process
Biological and signaling properties of all four DVL3 FlAsH sensors were indistinguishable from wild-type ECFP-DVL3 in the following assays: activation of the Wnt/β-catenin pathway monitored by Dual Luciferase TopFlash/Renilla reporter gene assay (Supplementary Fig. 1c); CK1ɛ-dependent DVL electrophoretic mobility shift assay (Supplementary Fig. 1d); and changes of DVL subcellular localization induced by CK1ε (Supplementary Fig. 1e)
Summary
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. It has been shown in vitro that at least two regions located in the C-terminus of DVL— (i) a nuclear export signal and (ii) the terminal 7 aa in the C-terminus of DVL—can interact with the PDZ domain[15,16,17]. These reports suggested that DVL can exist in a closed and open conformation (s) undergoing multiple structural transitions that control DVL function. There is neither an experimental approach to study this phenomenon directly nor clear evidence that DVL exists in multiple conformations in vivo
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