Article Figures & Data
Figures
- FIG 1
DVL-deficient cells are unable to respond to Wnt3a. Both wild-type (WT) and DVL1/DVL2/DVL3 triple knockout (DVL KO) HEK293 cells were treated with Porcupine inhibitor (LGK974) for 24 h to reduce autocrine signaling, and then the cells were treated for 2 h (for Western blotting [WB]) or for 14 h (for the TopFlash reporter assay) by control (ø) or Wnt3a (3a) conditioned medium (CM). (A) In DVL KO HEK293 cells, S1490-LRP6 phosphorylation and active β-catenin were not induced by Wnt3a; total LRP6 (tLRP6 ) levels were not changed. A lack of DVL2 and DVL3 in DVL KO HEK293 cells served as a control for cell identity. (i) Western blots; (ii) quantification of density of pS1490-LRP6 compared to that of actin (n = 3). (B) DVL1 antibody testing. DVL KO HEK293 cells were transfected according to the loading scheme and analyzed by WB. None of the anti-DVL1 antibodies was able to detect DVL1 specifically. All DVL constructs were expressed to a comparable level, as demonstrated by antitag (GFP, FLAG, and HA) staining. (C) TCF/LEF-dependent transcription is not induced in DVL KO HEK293 cells, as analyzed by the TopFlash reporter assay (n = 4). (D) DVL KO HEK293 cells are not able to induce the expression of AXIN2 after Wnt3a treatment, as quantified by RT-PCR (n = 4). Cells were treated with mouse Wnt3a recombinant protein (rmWnt; 100 ng/ml). (E) (Top) Analysis of cells of the DVL KO HEK293 T-REx cell line. DVL KO T-REx cells are not able to phosphorylate S1490-LRP6 after Wnt3a induction. A lack of DVL2 and DVL3 in DVL KO HEK293 cells served as a control for cell identity. (Bottom) The guide RNA (gRNA) design and the sequences of verified deletions in the DVL KO T-REx cell line edited by the CRISPR/Cas9 system are shown. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01).
- FIG 2
DVL overexpression rescues the Wnt3a response in DVL KO HEK293 cells. (A to F) Cells were transfected with the indicated combinations of plasmids (the concentration of each plasmid carrying DVL was 10 ng/sample, if not stated otherwise) and on the next day were treated with control (ø) or Wnt3a (3a) conditioned medium (CM) for 2 h (for Western blotting [WB]) or 14 h (for the TopFlash reporter assay). The response to Wnt3a was monitored by WB (S1490-LRP6) (Ai to Di; n = 3) and the TopFlash reporter system (n = 5 for panel E; n = 4 for panel F). pS1490-LRP6 signals were quantified by densitometry and normalized to those for actin (Aii to Dii; n = 3). (Aiii) Transfection efficiency was monitored by cotransfection of GFP. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01).
- FIG 3
The DVL1 DEP domain is required for the Wnt3a response. (A) Schematic representation of DVL1 deletion mutants used for the rescue experiments; the DIX, PDZ, and DEP domains are indicated. (B to E) HEK293 cells were transfected with the indicated plasmids and treated according to the scheme; in the assay whose results are shown in panel E, cells were sensitized with R-spondin 1 (R-SPO1) to increase the response to Wnt3a. (B) Full-length (FL) DVL1 and the DVL1(1–502) mutant are able to rescue the transcriptional response in DVL KO HEK293 cells induced by Wnt3a conditioned medium (CM), as analyzed by the TopFlash reporter assay (n = 4). (C) Control of expression for the DVL1 mutants used in the assay whose results are shown in panel B was performed by Western blotting for the FLAG tag. (D, E) The DVL1(1–502) deletion mutant can restore the capacity of Wnt3a CM to induce the phosphorylation of pS1490-LRP6; tLRP6 (total LRP6) levels were not changed (Di, E). (Dii) Quantification by densitometry (n = 4). Actin was used as a loading control. (F, G) Rescue experiments in DVL KO T-REx cells. TCF/LEF-dependent transcription (F) and S1490-LRP6 phosphorylation (G) after transfection of the DVL1(1–502) mutant and the DVL1(1–394) mutant lacking the DEP domain were determined. The DVL1(1–394) mutant was not able to rescue either TCF/LEF-dependent transcription or S1490-LRP6 phosphorylation after Wnt3a treatment. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01).
- FIG 4
PCP pathway-specific DEP domain functional units are dispensable for the rescue of the cellular response to Wnt3a. (A) Schematic representation of the DVL1 DEP domain mutants used for the rescue experiments. The alignment of the sequences of individual human DVL isoforms with the Drosophila melanogaster (DROME) Dsh sequence and a summary of previously published Wnt/β-catenin or PCP pathways phenotypes of individual mutants (9, 10, 12, 19–21) are provided. All mutants were generated in the DVL1(1–502) mutant background. (B) (Left) Relocalization of individual DVL mutants to the membrane after cotransfection with Frizzled 5 (FZD5). (Right) Three categories of DVL1 localization after FZD5 coexpression were established: strong, weak, and no membrane localization of DVL1. One hundred cells were counted per condition (n = 3). All mutants showed reduced membrane recruitment compared to that for the control, the DVL1(1–502) mutant. Bar, 7.5 μm. (C to E) HEK293 cells were transfected as indicated and analyzed by the TopFlash reporter assay (C, D) (n = 4 for panel C, n = 3 for panel D) or WB (Ei, blots; Eii, quantification; n = 3). In the assay whose results are presented in panels D and E, cells were pretreated with R-spondin 1 (R-SPO1) and the DIDI mutant was transfected in a 100-ng dose so that its level equaled the protein levels of the other DVL1 mutants. All DVL1 DEP domain mutants except for the ΔLPDSG and DIDI mutants rescued the response to Wnt3a to an extent comparable to that for the DVL1(1–502) mutant. (F and G) Analysis of DEP mutants in DVL KO T-REx cells. All mutants except the ΔLPDSG and DIDI mutants were able to restore Wnt/β-catenin-dependent transcription, as analyzed by the TopFlash reporter assay (n = 4) (F) and S1490-LRP6 phosphorylation (G). Cells for which the results are shown in panels F and G were pretreated with R-spondin1 (R-SPO1). (H) The pattern of localization of full-length (FL) DVL1 and the individual DVL mutants resembles that of the DVL1(1–502) mutant. Colocalization with axin1 was determined by immunocytofluorescence. All mutants except DIDI colocalized with axin1. (Top) DVL1 only (green); (bottom) DVL1 (green) and axin1 (red). ctrl, control. Bars, 7.5 μM. (I) Coimmunoprecipitation of DVL1 mutants in the pulldown of endogenous axin1 and CK1ε. Cells were treated as indicated; unspecific IgG was used as a negative control. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- FIG 5
(A) Schematic representation of the DVL1 DEP domain truncation mutants used for the rescue experiments. (B) DVL KO HEK293 cells were transfected with the indicated DVL1 mutants and V5-FZD5 or HA-axin1 and stained by immunocytochemistry. (Top) DVL1 only; (middle) DVL1 (green) and FZD5 (red); (bottom) DVL1 (green) and axin1 (red). Bar, 7.5 μm. (C) Quantification of DVL1 localization after FZD5 cotransfection. The evaluation criteria used were the same as those described in the legend to Fig. 4B. None of the truncation mutants was recruited to the membrane by FZD5. (D) TopFlash reporter assay analysis of DVL1 mutants with the DEP domain truncation (n = 4). Only the DVL1(1–438) mutant showed a weak ability to rescue TCF/LEF-dependent transcription. (E) WB analysis of expression levels of individual mutants and their ability to rescue phosphorylation of S1490-LRP6 (n = 3). None of the mutants could rescue S1490-LRP6 phosphorylation. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01).
- FIG 6
Model of the role of the Dishevelled DEP domain in Wnt/β-catenin signal transduction. Signal transduction is initiated by the interaction of Wnt3a with the extracellular part of LRP6 and Frizzled. DVL is recruited to the membrane and interacts with FZD. The N-terminal part of the DVL DEP domain (dark blue) is necessary for dynamic DVL membrane recruitment to Frizzled after Wnt3a treatment. Other regions of the DEP domain (light blue) that are important for the stable interaction of DVL with FZD and/or the plasma membrane and that are required for the function of DVL in the Wnt/PCP pathway are indispensable in this process. Via its DIX domain, DVL interacts with axin, which brings axin to the membrane complex and leads to LRP6 phosphorylation and downstream Wnt/β-catenin signaling. This Wnt3a-induced β-catenin signal transduction is indicated by blue arrows. Overexpression of DVL, commonly used in the experiments in the Wnt field, can bypass the requirement for the DEP domain in the Wnt/β-catenin pathway by nucleating endogenous DVL and axin (via the DIX domain) and triggering Wnt ligand-independent β-catenin activation. This pathway is indicated by an orange arrow.
