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. 2011 Feb 1;108(5):1937-42.
doi: 10.1073/pnas.1017063108. Epub 2011 Jan 18.

Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin

Marc Fiedler  1 , Carolina Mendoza-Topaz, Trevor J Rutherford, Juliusz Mieszczanek, Mariann Bienz
Affiliations

Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin

Marc Fiedler et al. Proc Natl Acad Sci U S A. .

Abstract

Wnt/β-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, β-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic head-to-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/β-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo- and heterotypic interactions. This result implies that Dishevelled has the potential to act as a "natural" dominant-negative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Polymerization-blocking DAX mutations attenuate Axin's response to Dvl and its effector function in down-regulating β-catenin in human cells. (A) Alignment of amino acid sequences of DAX (from human and Drosophila Axin) with DIX (from human Dvl2), with secondary structure elements of head and tail regions underneath (blue, head; turquoise, tail) and polymerization-blocking DAX mutations indicated. Invariant (yellow) and semiconserved (gray) residues are shaded; surface residues engaged in close DAX–DAX interactions (20) are marked by arrows (with the closest interactions in gray). (B) Ribbon representation of DAX filament, with head regions of individual DAX monomers in blue, tail regions in turquoise, and N-termini marked by green spheres, and positions of M2 and M3 indicated (in one DAX–DAX interface). (C) MALLS of purified wt and mutant DAX domains. (D–G) Confocal images of fixed HeLa cells, expressing wt or M3 mutant Axin-GFP (plus recruitment mixture; refs. 6 and 22) upon exposure to control or WCM (for 2 h) (D and E), or coexpressing wt or M3 mutant Flag-Axin with GFP-Dvl2, as indicated (F and G). (H and I) Confocal images of fixed SW480 cells, expressing wt or M3 mutant Axin-GFP (individual transfected cells indicated by arrows), stained for β-catenin (red). (J) Western blot analysis of total lysates from FACS-sorted SW480 cells expressing low levels of wt or M3 mutant HA-Axin-GFP, probed with antibodies as indicated [active β-catenin (ABC)]; numbers indicate levels at right relative to left (set to 1) as measured by densitometry. (K) HeLa cells cotransfected with Flag-β-catenin plus wt or mutant HA-Axin-GFP, as indicated; equal amounts of total protein were loaded in each lane; note that HA-M3-GFP is as inactive in down-regulating Flag-β-catenin as an Axin deletion mutant lacking its entire DAX domain (HA-ΔDAX).
Fig. 2.
Fig. 2.
Attenuated rescue activity of polymerization-defective Axin mutants in axin null mutant Drosophila embryos. (A–C) Confocal sections of the lateral epidermis of ≈6-h-old embryos, after fixation and staining with α-Wg antibody (red), expressing wt (green) (A) or mutant Axin-GFP in the embryonic epidermis, as indicated (B and C) (with arm.GAL4, which mediates somewhat patchy expression, with nonexpressing cells interspersed between GFP-expressing cells; only the green channel is shown for A′, B, and C). Arrowheads point to Dsh-dependent PM association of Axin puncta in the Wg-expressing zones (marked by arrows in A–C); brackets indicate zones with cytoplasmic Axin puncta, likely reflecting the Axin destruction complex (because they also contain APC; ref. 5). (D–G) Dark-field views of the ventral epidermis of fully developed wt or axin null mutant embryos, +/− overexpression of wt or M4 mutant Axin-GFP, as indicated. Arrows point to naked zones (i.e., the output of embryonic Wg signaling in the cuticle), and brackets indicate the denticle belts (i.e., the output of Axin effector function). The majority of Axin-expressing axin mutants show normal denticle belts plus ectopic denticles (F) (5), whereas most M4-expressing mutants show merely rudimentary denticle belts (G, asterisks). (H) Semiquantitative analysis of rescue activities of wt and mutant Axin-GFP. +++, normal denticle belts with ectopic denticles interspersed; ++, normal denticle belts; +, <4 rudimentary denticle belts; −, naked (no rescue activity). (I) Western blots (from two independent experiments) of total embryonic extracts after expression of wt or mutant Axin-GFP.
Fig. 3.
Fig. 3.
Polymerization-blocking DAX mutations attenuate Axin's effector function in reducing Wg target gene expression in axin null mutant wing disk clones. Fixed wing discs from third instar Drosophila larva, stained with α-Sens antibody (A), to visualize the Armadillo-dependent expression of this Wg target gene on either side of Wg along the prospective wing margin, double-stained as indicated (B), to reveal ectopic Sens (blue) in axin null mutant tissue (absence of red, α-lacZ staining), expressing wt or M2 mutant Axin-GFP (green) as indicated (C and D). (C′ and D′) Boxed areas are at high magnification, revealing that M2 fails to reduce Sens in this axin mutant clone (D′, Right).
Fig. 4.
Fig. 4.
NMR spectroscopy reveals direct interaction between the polymerization surfaces of DIX and DAX. (A and B) Overlays of HSQC spectra of 100 μM 15N-DAX_M3 (red) plus 125 μM of unlabeled DAX_M3 (A) or DIX_M2 (blue) (B), with assigned DAX residues annotated. Numerous line broadenings in B signify direct interaction between the head surface of DIX_M2 and the tail surface of 15N-DAX_M3; no perturbations are detectable if the latter is probed with head-surface mutants DAX_M3 (A) or DIX_M4 (Fig. S3). (C) Shift perturbation map, indicating shifting (black) or line broadening (red) of DAX domain residues (Axin DAX sequence underneath, with symbols as in Fig. 1A; turquoise, undetectable prolines).
Fig. 5.
Fig. 5.
Projections of the key head and tail interactions between DIX and DAX onto the DAX crystal structure. (A and D) Ribbon representations of two adjacent DAX monomers (translated apart), with head in blue, and tail in turquoise (Fig. 1 A and B); DIX-interacting residues are colored in red (line broadening) or magenta (highly significant shifts), based on maps in Fig. 4C and Fig. S5. Head (B and E) and tail (C and F) surface representations of individual DAX monomers (rotated by angles as indicated), colored as in A and D (with key interacting residues indicated by arrows).
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References

    1. Clevers H. Wnt/β-catenin signaling in development and disease. Cell. 2006;127:469–480. - PubMed
    1. MacDonald BT, Tamai K, He X. Wnt/β-catenin signaling: Components, mechanisms, and diseases. Dev Cell. 2009;17:9–26. - PMC - PubMed
    1. Ikeda S, et al. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin. EMBO J. 1998;17:1371–1384. - PMC - PubMed
    1. Dajani R, et al. Structural basis for recruitment of glycogen synthase kinase 3β to the axin-APC scaffold complex. EMBO J. 2003;22:494–501. - PMC - PubMed
    1. Cliffe A, Hamada F, Bienz M. A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling. Curr Biol. 2003;13:960–966. - PubMed

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