Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways

Abstract

The intracellular signaling molecule Dishevelled (Dvl) mediates canonical and non-canonical Wnt signaling via its PDZ domain. Different pathways diverge at this point by a mechanism that remains unclear. Here we show that the peptide-binding pocket of the Dvl PDZ domain can be occupied by Dvl's own highly conserved C-terminus, inducing a closed conformation. In Xenopus, Wnt-regulated convergent extension (CE) is readily affected by Dvl mutants unable to form the closed conformation than by wild-type Dvl. We also demonstrate that while Dvl cooperates with other Wnt pathway elements to activate canonical Wnt signaling, the open conformation of Dvl more effectively activates Jun N-terminal kinase (JNK). These results suggest that together with other players in the Wnt signaling pathway, the conformational change of Dvl regulates Wnt stimulated JNK activity in the non-canonical Wnt signaling.

Keywords: Dishevelled; NMR; PDZ; Wnt; auto-inhibition; biophysics; cell biology; structural biology; xenopus.

Figure 1.. The C-terminal tail of Dishevelled (Dvl) is a PDZ domain binding motif.

Sequence alignment of the C-terminus of Dvl/Dsh from selected species (Wallingford and Habas, 2005), showing residue numbers. DOI: http://dx.doi.org/10.7554/eLife.08142.003

Figure 2.. Competitive binding experiments.

The K_D value of the fluorescence-labeled Dapper (Dpr)-derived peptide Rox-DprC was obtained by plotting 1/ΔmP vs 1/[PDZ], where ΔmP is the fluorescence polarization change (×1000) of Rox-DprC and [PDZ] is the concentration of the PDZ domain of Dvl. The K_I values of the Dvl-C and Dsh-C peptides were obtained by using the equation K_D^app = (K_D/(1 + [I]/K_I)). DOI: http://dx.doi.org/10.7554/eLife.08142.004

Figure 3.. Isothermal titration calorimetry experiment.

The MicroCal Auto-iTC-200 was used to obtain the binding affinity of Dvl-C peptide and Dvl PDZ protein in 50 mM phosphate buffer. The concentration of Dvl-C peptide in the syringe was 1.05 mM and the concentration of Dvl PDZ domain in the cell was 0.114 mM. The K_D value was averaged from two independent experiments at 25°C. DOI: http://dx.doi.org/10.7554/eLife.08142.005

Figure 4.. Direct interaction of the Dvl C-terminus and PDZ domain.

(A) Overlap of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled PDZ domain without (blue) and with the unlabeled peptide (SEFFVDVM) derived from the extreme C-terminus of Dvl. Free: blue; final: red; ratio of peptide:protein = 20:1. (B) Overlap of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled PDZ domain without (blue) and with unlabeled peptide (QDVSVSNYVL) derived from the C-terminus of Drosophila Dsh (Dsh-C). Blue: free; red: final; ratio of peptide:protein = 20:1. DOI: http://dx.doi.org/10.7554/eLife.08142.006

Figure 5.. Solution NMR structure of Dvl PDZ domain in complex with Dvl-C peptide.

(A) 2D plane of 3D ¹³C-F1-half-filtered F2-edited NOESY-HSQC spectrum (mixing time, 300 ms) at 15°C. The ratio of peptide:protein was 10:1. [¹³C,¹⁵N-PDZ] = ∼1 mM. (B) A stereo view of the backbone of 15 superimposed structures of the Dvl PDZ–Dvl-C peptide complex. (C) Ribbon diagram of the lowest-energy structure of the Dvl PDZ/Dvl-C peptide complex. (D) Surface of Dvl-1 PDZ bound to Dvl-C peptide (carbon, green; nitrogen, blue; sulfur, yellow; oxygen, red; hydrogen atoms are omitted for clarity). (E) Structural details of the Dvl-C peptide–PDZ domain complex. The side chain of Asp(-2) in the Dvl-C peptide forms a hydrogen bond with the side chain of Arg322 on the αB-structure. DOI: http://dx.doi.org/10.7554/eLife.08142.007

Figure 5—figure supplement 1.. The mutant Dvl-1 PDZ (R322A) domain binds more weakly than wild-type Dvl-1 PDZ domain to the Dvl-C peptide.

Overlap of the ¹H-¹⁵N HSQC spectra of the labeled mutant Dvl PDZ (R322A) domain and the Dvl-C peptide. The mutation dramatically weakens interaction with the Dvl-C peptide (free: blue; final titration: red; final peptide:protein ratio = 4:1). DOI: http://dx.doi.org/10.7554/eLife.08142.010

Figure 6.. The binding pocket of the Dvl PDZ domain can be occupied by its intrinsic C-terminus.

(A) Schematic representation of protein constructs mC1 (residues 251–695) and mC1-CΔ7 (residues 251–688) numbered according to the mouse Dvl-1 protein sequence. (B) Polarization change of the fluorescence-labeled peptide Rox-DprC (Rox-SGSLKLMTTV, derived from the C-terminus of Dpr) after addition of mC1-CΔ7 and mC1 proteins in 50 mM phosphate with 0.3 M NaCl and 6 mM β-mercaptoethanol. For the binding of Rox-DprC to mC1, KD is 3.8 ± 0.5 μM the value was obtained by fitting the titration data with the equation: ΔmP = ΔmP_max × [P]/([P] + K_D), where ΔmP is the polarization change of Rox-DprC, [P] is the concentration of protein, and both K_D and ΔmP_max are the fitting variables. For the binding of Rox-DprC to mC1-CΔ7, K_D was estimated as 68 ± 5 μM. Because of the limitation in the titration study, to estimate the K_D value, although we used the same equation to fit the titration data, in the fitting, Kd was the only variable and the maximum polarization change, ΔmP_max, was fixed to the value that was obtained in the titration study of Rox-DprC binds to mC1-CΔ7. DOI: http://dx.doi.org/10.7554/eLife.08142.011

Figure 7.. Effect of wild-type XDsh and XDsh-CΔ8 activity on canonical Wnt signaling.

Luciferase assay using a Siamois promoter reporter (Sialuc). Sialuc DNA (200 pg) was injected alone or with myc-tagged Xdsh or Xdsh-CΔ8 mRNA (500 pg) into the animal pole region of 2-cell Xenopus embryos. Ectodermal explants were dissected at the early gastrula stage for luciferase assay. Values are the means ±SD from four independent experiments (p < 0.05). Inset shows a representative western blot using anti-myc antibody (9E10) to control for XDsh and XDsh-CΔ8 protein expression in the four experiments. DOI: http://dx.doi.org/10.7554/eLife.08142.012

Figure 8.. The open conformation of Dvl significantly enhances gain-of-function planar cell polarity (PCP) signaling.

(A) Xenopus embryonic abnormal convergent extension (CE) phenotypes induced by injection of wild-type XDsh and XDsh-CΔ8 mRNA at three increasing concentrations (arrows at bottom represent 80 pg, 200 pg and 500 pg of injected mRNA; above are numbers of embryos injected from two independent experiments). Phenotypes are severe (green), mild (red), and normal (blue). (B) Comparison of phenotypes induced by dose-equivalent injections (500 pg mRNA) of XDsh, XDsh-CΔ8, and Xdd1 (a well-established dominant-negative XDsh mutant). XDsh-CΔ8 and Xdd1 induced similar phenotypes. The numbers of embryos injected from three independent experiments are listed on the top of each column. DOI: http://dx.doi.org/10.7554/eLife.08142.013

Figure 9.. The open conformation of Dvl disrupts CE by activating Jun N-terminal kinase (JNK).

(A) Western blot of phosphorylated JNK in ventral mesoderm cells overexpressing wild-type XDsh or its mutants. At equivalent protein level, Xdd1 and XDsh-CΔ8 more potently induce JNK phosphorylation than wild-type XDsh. (B) Xenopus 4-cell stage embryos were dorsally coinjected with equal quantities of wild-type XDsh mRNA or XDsh-CΔ8 mRNA (500 pg) and the AP1-luciferase reporter DNA (200 pg); luciferase activity was assayed at the late gastrula stage. Inset shows a representative Western blot using anti-myc antibody to control XDsh and XDsh-CΔ8 protein levels. Values are the mean and SD from three independent experiments (XDsh vs XDsh-CΔ8, p < 0.05). (C–H) The dominant negative JNK mutant (dnJNK) rescues activin-induced explant elongation blocked by overexpression of XDsh-CΔ8 or Xdd1. (C) Uninjected explants treated with activin show extensive elongation. (D) XDsh-injected explants treated with activin show moderate inhibition of explant elongation. (E) Injection of XDsh-CΔ8 strongly inhibits explant elongation. (F) Injection of Xdd1 similarly inhibits explant elongation as injection of XDsh-CΔ8. (G, H) dnJNK rescues explant elongation inhibited by XDsh-CΔ8 or Xdd1. (I) dnJNK also recues CE defects produced by overexpression of XDsh-CΔ8 or Xdd1 in whole embryos. Phenotypes are severe (green), mild (red), and normal (blue). Numbers on the top indicate total embryos scored from three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.08142.014

Figure 10.. The open conformation of Dvl induced by targeting the Dvl PDZ domain potentiates Wnt/JNK signaling.

(A) Regulation of XDsh-mediated PCP signaling by a PDZ-binding small molecule or peptide is shown by the gain-of-function CE phenotypes of whole embryos that were uninjected (controls) or injected with XDsh mRNA with or without treatment with the Dvl inhibitors 3209–8625 or coinjected with XDsh and TMEM88-C mRNAs. (B) Inhibiting the Dvl PDZ domain blocks canonical Wnt signalling induced by Dvl overexpression. Wild-type XDsh mRNA was injected alone or coinjected with an equal quantity of TMEM88-C mRNA in the animal pole region of two-cell stage Xenopus embryos, and ectodermal explants were dissected at the late blastula stage. TOPFLASH luciferase activity values are the mean and SD from three independent experiments (p < 0.05). (C) Inhibition of the Dvl PDZ domain by TMEM88 opens the conformation of Dvl and potentiates Wnt/JNK signaling induced by Dvl overexpression. Xenopus 4-cell stage embryos were injected dorsally with wild-type XDsh mRNA or coinjected with equal quantities of wild-type XDsh mRNA and TMEM88-C mRNA. AP1 luciferase activity was assayed at the late gastrula stage. Values are the mean and SD from three independent experiments (p < 0.05). DOI: http://dx.doi.org/10.7554/eLife.08142.015

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DOI: http://dx.doi.org/10.7554/eLife.08142.018