Research ArticleBiochemistry

A direct heterotypic interaction between the DIX domains of Dishevelled and Axin mediates signaling to β-catenin

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Science Signaling  10 Dec 2019:
Vol. 12, Issue 611, eaaw5505
DOI: 10.1126/scisignal.aaw5505
  • Fig. 1 Crystal structure of the DAX-DIX dimer.

    (A) Construct in which DVL2 DIX was fused to Axin DIX (DAX) downstream of a flexible linker. The His and GST tags were removed before crystallization by cleaving with the protease HRV3C. (B) Ribbon diagram and space-filling model of the DAX-DIX dimer. Pink, DAX; cyan, DIX; dotted line, flexible linker between DAX and DIX (not structured). The tail of DIX (including the M2 mutation) is not visible in the structure owing to poor electron density.

  • Fig. 2 Comparison of DAX-DIX, DIX-DIX, and DAX-DAX structures.

    (A) Superimposition of DAX-DIX heterodimer onto homodimers of DIX-DIX and DAX-DAX, as indicated in key. Rectangles highlight distinct interaction sites between DIX domains: black, site A (see also inset); red, site B; blue, site C. Amino acid numbering reflects the human proteins, including the DAX-DAX homodimer structure, which was solved using rat Axin (residue numbers for the rat protein are shown in parentheses). (B and C) Close-up views of site B (B) and site C (C). Dashed lines indicate salt bridges and hydrogen bonds.

  • Fig. 3 Fluorescence anisotropy binding assays.

    (A) Effect of dimerization on the fluorescence anisotropy of labeled DIX domains. Fluorescence anisotropy correlates inversely with the rotational mobility of the fluorophores that are excited with polarized light. Assays tested whether an unlabeled DIX or DAX molecule (blue) reduced the mobility of a fluorophore-labeled DIX or DAX molecule (pink), as indicated by an increase in fluorescence anisotropy. The binding interfaces are indicated by colored bars: red bars, disabled head (M4 or M3) or tail (M2) surfaces; purple bars, intact head surfaces; green bars, intact tail surfaces. (B) Fluorescence anisotropy curves of the indicated labeled DIX and DAX proteins upon titration with unlabeled DAX and DIX, respectively. Data represent the mean ± SD. n = 3 independent experiments.

  • Fig. 4 Signaling by DVL2 chimerae.

    (A) Cartoons of GFP-tagged wild-type (WT) DVL2 and corresponding DVL2 chimerae bearing a PB1 or SAM domain in place of the DIX domain. Amino acid numbers in WT DVL2 denote domain boundaries. (B) Representative confocal images of COS-7 cells coexpressing FLAG-tagged Axin and WT GFP-DVL2 or GFP-DVL2 chimerae, fixed and stained as indicated above panels. Images of the other chimerae are presented in the Supplementary Materials (fig. S2). n = 3 independent experiments. Scale bar, 10 μm. (C) SuperTOP reporter assays monitoring signaling activities of WT, M2 or M2M4 mutant GFP-DVL2, and GFP-DVL2 chimerae 1 to 6 in DVL-null HEK293T cells. Fold induction levels relative to empty pEGFP vector control (ev) are shown. Data represent the mean ± SEM. Multiplicity adjusted: *P < 0.043 and **P < 0.0043, one-way ANOVA test. n = 3 independent experiments. Corresponding Western blots are shown to indicate comparable protein levels.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/611/eaaw5505/DC1

    Fig. S1. Models of DAX-DIX polymers based on the dimer structure.

    Fig. S2. Colocalization between coexpressed FLAG-Axin and GFP-DVL2 chimerae bearing PB1 or SAM domains.

    Fig. S3. Complementation of DVL-null cells by GFP-DVL2 chimerae stably expressed at physiological levels.

    Table S1. Data collection and refinement statistics for DAX-DIX dimer.

  • This PDF file includes:

    • Fig. S1. Models of DAX-DIX polymers based on the dimer structure.
    • Fig. S2. Colocalization between coexpressed FLAG-Axin and GFP-DVL2 chimerae bearing PB1 or SAM domains.
    • Fig. S3. Complementation of DVL-null cells by GFP-DVL2 chimerae stably expressed at physiological levels.
    • Table S1. Data collection and refinement statistics for DAX-DIX dimer.

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