Fig. 5
Phosphorylation of PDZ domain controls the conformational dynamics of DVL3. a Schematic depiction of the closed conformation of DVL proposed here16, where seven last C-terminal aa (sequence: EFFVDIM) interact with the peptide-binding pocket of the PDZ domain. b Measurements of the intramolecular FRET efficiency of the wt DVL3 FlAsH sensors III (aa 1–716 in human DVL3) and the ∆C variant (aa 1–697) in HEK293 wild-type cells. c The western blot-based (above) and MS/MS-based (below) analyses of the CK1ε-induced phosphorylation of serine residues present in the PDZ domain of human DVL3. FLAG-DVL3 wt was overexpressed with/without CK1ε wt in HEK293 wt cells, immunoprecipitated, and the level of phosphorylation was analyzed by MS/MS. CK1ε-induced phosphorylation of S268 and S311 in DVL3 PDZ domain was detected. d In vitro kinase assay with recombinant FLAG-DVL3 and CK1ε analyzed by western blot (above) and MS/MS (below) confirms that S268 and S311 are direct phosphorylation sites of CK1ε. Values in c and d show absolute intensity of phosphorylated peptides plotted on a log10 scale. The detection limit is approximately 1.106, i.e. 6.0. Individual datapoints represent biological replicates. e Comparison of the intramolecular FRET efficiency of wt DVL3 FlAsH III sensor, non-phosphorylatable variant (S268A/S311A), and phosphorylation-mimicking variant (S268E/S311E) in wt HEK293 cells is shown. One data point corresponds to one analyzed cell; datapoints from 3 (b) and 7 (e) independent transfections were merged. Data in b and e represent median ± interquartile range and data in e were analyzed by one-way ANOVA test with Gaussian distribution and Tukey's post-test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, ****, p ≤ 0.0001; ns, not significant, p > 0.05). Data in b were analyzed by Student's t-test. Data in c and d represent mean ± S.D.
