Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1

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.

Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs

“…FlAsH labeling of the DVL3 FlAsH III sensor was performed as previously described [24]. Shortly, transfected cells were washed once with Hank’s Balanced Salt Solution (HBSS) containing 1.8 g/l glucose and then incubated at 37 °C for 1 h with HBSS supplemented with 500 nM FlAsH; 12.5 μM 1,2-ethanedithiol (EDT).…”

“…For NMR studies, cells were grown in minimal medium (M9) supplemented by 15 NH 4 Cl (1 g/l) and/or 13 C 6 glucose (2 g/l) and induced with 0.5 mM IPTG for 24 h at 16 °C. Proteins were purified as described before [24] and stored in 20 mM Hepes (pH 6.8) and 50 mM KCl for NMR studies. The DVL3_C peptide 698–716 with phosphorylated S700 (DVL_C) used in NMR studies was purchased from KareBay Biochem, Inc. (New Jersey, USA).…”

“…We have selected DVL3 as a candidate because its absence shows the strong phenotypes in vivo [22]. We have studied the phosphorylation of this DVL isoform earlier [10, 16, 23, 24], which allows for a direct comparison. The studied kinases included six previously reported DVL kinases, Aurora A that was reported with DVL in the same complex [25] and TTBK2, a DVL kinase identified in this study.…”

Decoding Dishevelled-Mediated Wnt Signaling in Vertebrate Early Development

“…These findings suggest an importance of the C-terminus in intra-or inter-molecular interaction, which may be subjected to regulation by other partners to switch pathway specificity. Indeed, recent studies show that casein kinase 1ε (CK1ε) and NIMA-related kinase 2 (NEK2) function as scaffold proteins and regulate the dynamics of Dvl conformational changes by phosphorylation of the PDZ domain and modulation of its interaction with the extreme C-terminal tail (Harnoš et al, 2019;Hanáková et al, 2019).…”

“…Moreover, the last 3 residues represent a type II PDZ-binding (PDZ-B) motif that can occupy the peptide-binding pocket of the PDZ domain, inducing Dvl to adopt a closed conformation and an auto-inhibited state ( Lee et al, 2015 ; Qi et al, 2017 ). Dvl variants with an opened conformation show efficient membrane recruitment and reduced activity in Wnt/ß-catenin signaling but display increased activity in Wnt/PCP signaling ( Qi et al, 2017 ; Harnoš et al, 2019 ). The function of Dvl C-terminal region in Wnt signaling is further demonstrated in autosomal-dominant Robinow syndrome caused by de novo frameshift mutations in human DVL1 and DVL3 genes, which delete and replace the C-terminal region after the DEP domain ( Bunn et al, 2015 ; White et al, 2015 , 2016 ; Danyel et al, 2018 ).…”