Fig. 3
The molecular and functional analysis of DVL3 regions interacting with CK1ɛ. a The general workflow of a peptide array analysis: immobilized peptides were incubated with CK1ε, then with anti-CK1ɛ antibody followed by fluorescent secondary antibodies, and detected by reading the fluorescence intensity. Peptide array contained the non-modified or phosphorylated peptide variants from the intrinsically disordered regions (IDRs) according to phosphorylation pattern by CK1ε, as mapped earlier29 (see Supplementary Fig. 3). b Identification of three regions (named RGCF, RGPR, and FRMA regions by their central 4 aa sequences), which bind CK1ε with high affinity. Multiple sequence alignment for the RGCF, RGPR, and FRMA regions of various Dvl/DVL isoforms is shown above. Residues with >80% similarity are highlighted and human DVL3 sequence is denoted by red box; only non-modified (i.e. non-phosphorylated) peptides are shown in this graph. c Generation of the N-terminal FLAG-tagged DVL3 ∆ALL variant lacking the interaction interfaces (RGCF, RGPR, FRMA regions) and its subsequent analysis by d coimmunoprecipitation and e its quantification, f by western blot detection of the pS643 phosphorylation level and f its quantification, g and by Topflash Reporter Assay for the downstream Wnt/β-catenin signaling. h The multiple sequence alignment of Xenopus Dvl3 and human DVL3 sequences in the RGCF, RGPR, and FRMA regions is shown. i Analysis of the activity of the ∆ALL variant derived from Xenopus xDvl3 in the Wnt/β-catenin canonical signaling (in the Xenopus laevis embryos). j Left: Representative image of control (low or no activity of the Wnt/β-catenin pathway; in a gray box) or duplicated (high activity; in a black box) axis in the Xenopus laevis embryos. Right: Quantification of the Xenopus laevis embryos with wild-type xDvl3 and the ∆ALL variant. Experiments in d–f were performed in HEK DVL1-2-3−/− cell line. Data in e, g, h, j represent mean ± S.D. Data in h and j were analyzed by one-way ANOVA test with Gaussian distribution; Tukey's post-test was used for statistical analysis (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, ****, p ≤ 0.0001; ns, not significant, p > 0.05); data in e and g were analyzed by Student's t-test
