Distinct functionality of dishevelled isoforms on Ca2+/calmodulin-dependent protein kinase 2 (CamKII) in Xenopus gastrulation

Abstract

Wnt ligands trigger the activation of a variety of β-catenin-dependent and β-catenin-independent intracellular signaling cascades. Despite the variations in intracellular signaling, Wnt pathways share the effector proteins frizzled, dishevelled, and β-arrestin. It is unclear how the specific activation of individual branches and the integration of multiple signals are achieved. We hypothesized that the composition of dishevelled-β-arrestin protein complexes contributes to signal specificity and identified CamKII as an interaction partner of the dishevelled-β-arrestin protein complex by quantitative functional proteomics. Specifically, we found that CamKII isoforms interact differentially with the three vertebrate dishevelled proteins. Dvl1 is required for the activation of CamKII and PKC in the Wnt/Ca(2+) pathway. However, CamKII interacts with Dvl2 but not with Dvl1, and Dvl2 is necessary to mediate CamKII function downstream of Dvl1 in convergent extension movements in Xenopus gastrulation. Our findings indicate that the different Dvl proteins and the composition of dishevelled-β-arrestin protein complexes contribute to the specific activation of individual branches of Wnt signaling.

FIGURE 1:

CamKIIδ interacts with the Arrb2-Dvl2-Gβγ complex. (A) The interaction was confirmed by coimmunoprecipitation of the respective proteins overexpressed in HEK 293T cells. CamKIIδ coimmunoprecipitated with Flag-Arrb2 only in the presence of Dvl2; in cells coexpressing Gβγ, less Myc-Dvl2 and correspondingly less CamKIIδ was detected in the immunoprecipitate. (B) CamKIIδ was coimmunoprecipitated with Flag-Dvl2 from HEK 293T cells; coexpression of Myc-Arrb2 and Gβγ reduced the overall expression levels of CamKIIδ and the amount coprecipitated with Flag-Dvl2; coexpression of Myc-Arrb2 slightly enhanced the interaction of Flag-Dvl2 with CamKIIδ.

FIGURE 2:

Differential interaction between CamKII and Dvl isoforms. (A) Overexpressed Myc-Dvl1, Myc-Dvl2, and Myc-Dvl3 were immunoprecipitated from HEK293T cells and analyzed for coprecipitation of endogenous CamKII. No binding of CamKII to Dvl1 was detectable. CamKII interacted with Dvl2 and very weakly with Dvl3 in unstimulated cells; both interactions were increased upon stimulation with Wnt-5a–conditioned medium for 30 min. To define further the specificity of interaction between CamKII and Dvl isoforms, CamKIIα, CamKIIβ, CamKIIδ, and CamKIIγ were coexpressed with Flag-Dvl2 (B), Flag-Dvl1 (C), or Flag-Dvl3 (D) in HEK 293T cells. The Flag-tagged Dvl isoforms were immunoprecipitated using immobilized anti-Flag antibodies. (B) Flag-Dvl2 bound and coprecipitated CamKIIα, CamKIIβ, and CamKIIδ but not CamKIIγ. (C) Neither CamKIIα nor β, δ, or γ coprecipitated with Flag-Dvl1. (D) Flag- Dvl3 interacted with CamKIIα and CamKIIδ, and weak bands of CamKIIβ and γ were also observed in Dvl3 immunoprecipitates.

FIGURE 3:

Endogenous CamKII interacts with Dvl2 in Xenopus embryos. (A) Xenopus embryos were injected as illustrated in the two dorsal blastomeres at the four-cell stage with RNA encoding Myc-Dvl1, Myc-Dvl2, or Myc-Dvl3. At early-gastrula stage (NF stage 10.5), endogenous CamKII was captured by immunoaffinity and the immunoprecipitates probed for the presence of the Myc-tagged Dvl isoforms (A). Endogenous CamKII coprecipitated with endogenous Dvl2 from Xenopus embryos (B) and from HEK 293T cells (C), confirming the physical interaction.

FIGURE 4:

Dvl1 activates CamKII in Xenopus embryos. (A) Immunoblotting for active CamKII-pT286 showed that Myc-Dvl1, but not Myc-Dvl2, enhanced CamKII phosphorylation, whereas Myc-Dvl3 reduced pCamKII levels in the embryos. (B) Knockdown of individual Dvl isoforms using specific antisense morpholino oligonucleotides yielded the inverse result. Dvl1 morphants showed reduced and Dvl3 morphants showed increased pCamKII levels, whereas Dvl2 knockdown did not affect pCamKII. In triple Dvl-morphant embryos, increased pCamKII levels were again observed. Ratios of pCamKII/CamKII in A and B were determined densitometrically relative to uninjected controls. Values noted above the respective lanes represent measurements of the blots shown; bar graphs below show the average ± SD from three independent experiments. (C) Xenopus embryos were injected as indicated, and CamKII kinase activity was measured. The graph represents the average activity from at least three independent experiments (average ± SEM); injections and number of experiments (n) are given below the graph. Asterisks indicate statistically significant deviation of means from corresponding wild-type levels (*p > 0.1, **p > 0.05, Wilcoxon rank-sum test).

FIGURE 5:

Dvl1 is required for activation and membrane translocation of PKC in Xenopus embryos. Xenopus embryos were injected with 500 pg of pkcα-gfp RNA and coinjected as indicated above the images. Animal caps were prepared at stage 10 and immunostained as indicated. Nuclei were stained with Hoechst 33258 (blue). Images show representative results of at least three independent experiments with a minimum of six Animal Caps per experiment. Scale bars, 50 μm. (A) PKCα-GFP predominantly localized to the cytoplasm in Xenopus animal caps. (B) Coinjection of 1 ng of fz7 RNA induced translocation of PKCα-GFP to the plasma membrane, indicating activation of PKC. (C) Overexpression of Fz7 was less effective in inducing PKCα-GFP translocation in Dvl1-morphant embryos. Knockdown of Dvl2 (D) or Dvl3 (E) did not impair Fz7-induced PKC-GFP translocation. Injection of subeffective amounts of fz7 RNA (300 ng) did not alter PKCα-GFP localization (F, F′) compared with animal cap cells expressing only PKCα-GFP (A). Coinjection of 200 pg of myc-dvl1 RNA induced robust membrane translocation of PKCα-GFP (G, G′). The tissue was coimmunostained against the Myc epitope, showing that Myc-Dvl1 predominantly localized to the plasma membrane and colocalized with PKCα-GFP (G′′). Coinjection of 200 pg of myc-dvl2 RNA was not sufficient to induce translocation of PKCα-GFP to the plasma membrane (H, H′). Myc-Dvl2 was detected in the cytoplasm and at the plasma membrane (H′′). Coinjection of 200 pg of myc-dvl3 RNA also failed to induce membrane translocation of PKCα-GFP (I, I′). Myc-Dvl3 predominantly localized in the cytoplasm and partially formed large aggregates, which were also positive for PKCα-GFP (I′′ and I).

FIGURE 6:

CamKIIδ acts downstream of Dvl1 in Xenopus convergent extension movements. Xenopus embryos were injected at the four-cell stage in the marginal zone of both dorsal blastomeres as indicated. CE movements in the dorsal mesoderm were monitored by elongation and constriction of Keller open-face explants. The average percentage of explants showing full elongation and constriction (light gray), elongation but impaired constriction (medium gray), or incomplete elongation (dark gray) from at least three independent experiments is shown. Asterisks indicate statistically significant deviations (**p > 0.99, *p > 0.95, χ² test). (A) Dvl1 knockdown predominantly inhibited explant elongation. The Dvl1-knockdown phenotype was rescued by overexpression of Dvl1, PKCα, or CamKIIδ; overexpression of CamKIIδ induced moderate elongation and constriction defects. (B) Dvl2 knockdown resulted in a weaker inhibition of elongation but a significant increase in constriction defects. The Dvl2-knockdown phenotype was not rescued by PKCα or CamKIIδ but was rescued by coinjection of a morpholino-insensitive myc-dvl2 RNA. (C) The Dvl3 knockdown phenotype was similar to the phenotype in Dvl2-depleted explants. Dvl3 MO knockdown was rescued by Dvl3 and partially but statistically not significantly by PKC. CamKIIδ was not able to rescue Dvl3 depletion, but coinjection of an RNA encoding dnCamKIIδ rescued CE movements. (D) Dvl2 overexpression rescued the Dvl1-morphant phenotype, but not vice versa. Explants from Dvl1/Dvl2 double-morphant embryos showed a more severe phenotype than the single knockdowns. Overexpression of CamKIIδ significantly rescued explant elongation but not constriction, thus yielding an explant phenotype highly similar to explants from Dvl2 morphants.