Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways
- St. Jude Children's Research Hospital, United States
- Sorbonne Universités, France
- Shandong University, China
- David Geffen School of Medicine at University of California, Los Angeles, United States
Figures
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.
Figure 2
Competitive binding experiments.
The KD 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 KI values of the Dvl-C and Dsh-C peptides were obtained by using the equation KDapp = (KD/(1 + [I]/KI)).
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 KD value was averaged from two independent experiments at 25°C.
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.
Figure 5 with 1 supplement
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.
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Figure 5—source data 1
Intermolecular NOEs between the Dvl-C peptide and the PDZ domain obtained from 13C-half-filtered NOESY-HSQC spectraa.
- https://doi.org/10.7554/eLife.08142.008
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Figure 5—source data 2
Structure statistics for the 15 lowest-energy peptide-PDZ complexes.
- https://doi.org/10.7554/eLife.08142.009
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).
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 = ΔmPmax × [P]/([P] + KD), where ΔmP is the polarization change of Rox-DprC, [P] is the concentration of protein, and both KD and ΔmPmax are the fitting variables. For the binding of Rox-DprC to mC1-CΔ7, KD was estimated as 68 ± 5 μM. Because of the limitation in the titration study, to estimate the KD 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, ΔmPmax, was fixed to the value that was obtained in the titration study of Rox-DprC binds to mC1-CΔ7.
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.
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.
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.
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).
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Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways
eLife 4:e08142.
https://doi.org/10.7554/eLife.08142
