Hanáková K , Bernatík O , Kravec M , Micka M , Kumar J , Harnoš J , Ovesná P , Paclíková P , Rádsetoulal M , Potěšil D , Tripsianes K , Čajánek L , Zdráhal Z , Bryja V .

	Fig. 1. Identification of TTBK2 as a novel DVL kinase. a: HEK293 cells were transfected with FLAG-DVL3 and Myc-DVL2 plasmids with wild type (wt) or kinase dead (KD) TTBK2-GFP. Active TTBK2 promoted phosphorylation-dependent mobility shift of DVL3 on Western blotting. b: Endogenous TTBK2 (green) localized into distal appendages of the mother centriole in hTERT-RPE1 cells (left). Overexpression of FLAG-DVL3 (stained in red) was not able to displace TTBK2 from the centriole (right). Centrioles were stained with CAP350 (blue). c: HEK293 cells were transfected with indicated plasmids, treated with CK1ε inhibitor PF-670462 (10 μM) and subsequently analyzed by Western blotting. TTBK2-induced electrophoretic mobility shift of DVL3 was not diminished upon CK1ε inhibition unlike the mobility shift induced by CK1ε. d: HEK293 cells were transfected with indicated plasmids and by the TopFLASH reporter system. Luminescence in the cell lysates was measured 24 h after transfection. Mean, SD and individual data points are indicated. Statistical differences were tested by One-way ANOVA and Tukey’s post test (* p < 0.05, ** p < 0.01, *** p < 0.001)
	Fig. 2. Validation of the panel of DVL3 kinases. a: Visualization of the kinases used in this study in the phylogenetic kinome tree (http://www.kinhub.org/kinmap/). The individual kinases are representatives of distant kinase groups except for CK1ε and TTBK2 that are members of CK1 superfamily. b. HEK293 cells were transfected by plasmids encoding FLAG-DVL3 and the indicated kinase. Ability of individual kinases to promote DVL3 phosphorylation detected as the electrophoretic mobility shift on WB was assayed. Alpha-tubulin was used as a loading control
	Fig. 3. Experimental design. a: FLAG-DVL3 was overexpressed (with or without kinase) in HEK293 cells. After cell lysis DVL3 was immunoprecipitated using anti-FLAG antibody. Immunoprecipitates were separated on SDS-PAGE gel electrophoresis, stained with Coomassie brilliant blue and the 1D bands corresponding to DVL3 were excised, digested with trypsin and subsequently cleaved by chymotrypsin. In the pipeline #1, the aliquot (1/10) of concentrated sample was directly analyzed by LC-MS/MS in order to analyze site occupancy of the abundant phosphorylated sites. The rest of the sample was enriched for phosphorylated peptides using TiO2 and analyzed by LC-MS/MS to obtain detailed information of DVL3 phosphorylation status. Data from LC-MS/MS were searched, manually validated in Skyline software and further processed by two approaches. In the first approach (pipeline #2) only phosphorylated peptides with the clearly localized phosphorylated site (based on manual inspection of spectra) were considered. In the second approach (pipeline #3) we have considered all phosphorylated peptides that in some cases resulted in the formation of “clusters” of phosphorylated sites. b: The overall sequence coverage of DVL3 across all kinases and replicates. Regions of DVL3 covered by the peptides detected (Mascot score > 20) in any of the MS/MS analyses are highlighted in grey. For sequence coverage in individual samples see Additional file 6: Table S2
	Fig. 4. Site occupancy of the abundant phosphorylated sites. Site occupancy analysis was performed according to the pipeline #1 in Fig. 3a. Fifteen phosphorylated peptides or clusters that were phosphorylated in more than 5% at least in one replicate are plotted. Graphs present individual data points from three biological replicates (two controls/biological sample) and the mean values (horizontal line)
	Fig. 5. Phosphorylation map of DVL3. All identified phosphorylation sites obtained from the pipeline #2 are visualized as a heatmap. Color intensities reflect relative change in the site phosphorylation (red – decrease, green – increase). Following additional information is also provided: a) Corresponding sites in human DVL1 and DVL2. Positions conserved in DVL1 and/or DVL2 either as Ser or Thr are highlighted in yellow. Position of the structured domains (DIX, PDZ and DEP) is indicated. b) The sequence of the phosphorylated epitope. Five amino acids before and after the identified phosphorylated site are shown. c) Mean absolute intensities of the phosphorylation sites in the control (DVL3 without exogenous kinase; N = 6) are expressed in the shades of blue. Numbers indicate decadic logarithm of the mean. ND indicated in white corresponds means “not detected”. All signals lower than 1 × 106, corresponding to log value 6.0 were considered as not detected. d) Nine columns represent heat map of relative change of phosphorylated peptide intensities (in log10 scale) obtained for individual kinases (relative to control). Numbers in the heatmap fields (0, 1, 2, 3) indicate the number of experimental replicates with the positive identification of the given phosphorylated site. e) Grey boxes indicate the position of clusters of sites analyzed also according to the pipeline #3 in the Additional file 2: Figure S2
	Fig. 6. Clusters phosphorylated in more than three sites. The clusters of Ser/Thr where 3 or more phosphorylated sites in one peptide were analyzed (i.e. multiphosphorylated peptides). Graphs indicate total intensities of the multiphosphorylated peptides from all three replicates; signal intensities from pipeline #1 and #3 are merged. Multiple sequence alignment shows the evolutionary conservation of the motif among individual DVL isoforms. All combinations of individual multiphosphorylated peptides detected in the cluster are indicated
	Fig. 7. Comparison of individual methods and their validation by phospho-specific antibodies. a-d. Comparison of methods. Existing phosphorylation-specific antibodies were used to visualize the level of phosphorylation of DVL3 with our kinase panel. HEK293 cells were transfected by the indicated combination of plasmids and analyzed by Western Blotting. Reactivity of individual phosphoantibodies against DVL3 phosphorylated by individual kinases is shown. Western blots are quantified using ImageJ software as absolute values for peak areas of corresponding bands and the intensities were normalized to the control. Mean intensities of the phosphorylated peptides obtained by MS/MS via pipelines #1 and #2 are shown in the shades of grey. Numbers indicate decadic logarithm of the mean peptide intensity. “Not detected” (ND) indicated in white corresponds to the signals below 1 × 106, i.e. 6.0. a. anti-pS192 (this study), b. anti-pS280, c. anti-pS643, d. anti-pS697
	Fig. 8. Phosphoplots - phosphorylation barcodes of DVL3 with the individual kinases. Visualization of absolute intensity of phosphorylated peptides corresponding to the individual phosphorylated sites plotted on the primary sequence of DVL3 (only Ser and Thr are shown). Black bars represent a control condition, red bars the intensities in the presence of the kinase. Intensities are plotted on a log10 scale
	Fig. 9. Phosphorylation of S268 and S311 contributes to the activation of the Wnt/b-catenin pathway. a, b: HEK293 cells were transfected by indicated plasmids together with TopFlash and Renilla reporter plasmids. The ability of individual kinases to induce activation of Wnt/β-catenin pathway either alone (a) or in combination with DVL3 (b) was analyzed. Mean, SD and individual data points are indicated. Statistical differences were tested by One-way ANOVA and Tukey’s post test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). c, d: Rescue experiments with DVL3 S268/S311 mutants. WT and D1/2/3 TKO HEK293 T-Rex cells were transfected as indicated and treated according to the scheme with 80 ng/ml recombinant human Wnt3a (rWnt3a). All samples were treated with 0.1 μM LGK974 inhibitor and 250 ng/ml R-SPONDIN1 and analyzed by TopFlash assay (c). Mean, SD and individual data points are indicated. Statistical differences were tested by paired t-test (* p < 0.05, ** p < 0.01). Samples were also used for WB analysis (d)
	Fig. 10. Phosphorylation of S630-S643 promotes even localization of DVL3. a: HEK293 cells were transfected in the indicated combinations and the subcellular localization of DVL3 was assessed by immunocytochemistry. DVL3 was localized in two typical patterns – either in cytoplasmic puncta or evenly dispersed in the cytoplasm (upper panel). Scale bar, 7.5 μm. b: The effects of individual kinases on DVL3 localization is shown in the bottom panel (HA-DVL3 was used for PLK1 and TTBK2, FLAG-DVL3 for the rest of kinases). c: Phosphorylation patterns associated with the even localization of DVL3 were analyzed by mutation of cluster of serine residues to alanine. Statistical data represent mean + SD from three independent experiments (N = 3× 200 cells). Statistical significance was confirmed by the comparison of the corresponding control (DVL3-FLAG or DVL3-HA without kinase) and DVL3 with individual kinases by One-way ANOVA and Tukey’s post test (* p < 0.05, ** p < 0.01, n.s. - not significant)
	Fig. 11. NEK2 phosphorylation in PDZ domain promotes open conformation of DVL3. a: PDZ sequence alignment of the three human DVL isoforms. The conserved serine residues phosphorylated specifically by NEK2 are highlighted in magenta. b: Crystal structure of DVL2 PDZ domain bound to internal ligand (PDB: 3CC0). The two serines prone to NEK2 phosphorylation are shown in magenta, the bound peptide in cyan, and PDZ residues of the binding loop or close to the binding loop in orange. c: NMR titrations of DVL3 C-terminal peptide to DVL2 wildtype PDZ or S281E/S298E phosphomimicking mutant. Magnified insets show the qualitative differences in peptide binding for PDZ residues annotated in (b). d: Schematic depiction of the FRET efficiency experiment of the ECFP DVL3 FlAsH III construct. In the default conformation, the DVL molecule is less phosphorylated and closed/compact, which is reflected in the close proximity of the FRET pair (ECFP and FlAsH (F) tags) – leading to the high intramolecular FRET efficiency (depicted as orange dash lines). In the presence of the active kinase (e.g. NEK2), DVL is phosphorylated in the PDZ domain, which promotes the open/loose conformation by the disruption of the C-terminus/PDZ domain interaction. This is reflected in the high proximity of ECFP and F tags thus leading to the low intramolecular FRET efficiency (no orange dash lines). e: HEK293 cells were transfected in the indicated combinations and the intramolecular FRET efficiency of the ECFP-DVL3 FlAsH III construct (schematically depicted in D) was measured. The data represent median ± interquartile range from three independent experiments (numbers of analyzed cells are indicated below). Statistical significance was confirmed by One-way ANOVA and Tukey’s post test (** p < 0.01, n.s. - not significant). FRET eff. Stands for Förster-Resonance-Energy-Transfer efficiency, ECFP for Enhanced Cyan Fluorescent Protein, F for FlAsH tag, and kd for kinase dead

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