Structural basis for Ccd1 auto-inhibition in the Wnt pathway through homomerization of the DIX domain

Shin-Ichi Terawaki,Takuya Katsutani,Kensuke Shiomi,Kazuko Keino-Masu,Yoshiki Higuchi,Masayuki Masu,Shohei Fujita,Naoki Shibata,Kaori Wakamatsu

doi:10.1038/s41598-017-08019-5

Shin-Ichi Terawaki, Takuya Katsutani + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-08019-5

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Abstract

Wnt signaling plays an important role in governing cell fate decisions. Coiled-coil-DIX1 (Ccd1), Dishevelled (Dvl), and Axin are signaling proteins that regulate the canonical pathway by controlling the stability of a key signal transducer β-catenin. These proteins contain the DIX domain with a ubiquitin-like fold, which mediates their interaction in the β-catenin destruction complex through dynamic head-to-tail polymerization. Despite high sequence similarities, mammalian Ccd1 shows weaker stimulation of β-catenin transcriptional activity compared with zebrafish (z) Ccd1 in cultured cells. Here, we show that the mouse (m) Ccd1 DIX domain displays weaker ability for homopolymerization than that of zCcd1. Furthermore, X-ray crystallographic analysis of mCcd1 and zCcd1 DIX domains revealed that mCcd1 was assembled into a double-helical filament by the insertion of the β1-β2 loop into the head-to-tail interface, whereas zCcd1 formed a typical single-helical polymer similar to Dvl1 and Axin. The mutation in the contact interface of mCcd1 double-helical polymer changed the hydrodynamic properties of mCcd1 so that it acquired the ability to induce Wnt-specific transcriptional activity similar to zCcd1. These findings suggest a novel regulatory mechanism by which mCcd1 modulates Wnt signaling through auto-inhibition of dynamic head-to-tail homopolymerization.

Highlights

The Wnt signaling pathway plays key roles in cell fate determination during embryonic development, neurogenesis, homeostasis, and oncogenesis[1,2,3,4]
We reported that mouse Ccd[1] showed low level of transcriptional activation compared to zebrafish Ccd[1], suggesting that mCcd[1] signaling activity is controlled by homopolymerization mode distinct from those of other DIX-containing proteins[36]
The zCcd[1] DIX domain contains unique amino acids different from characteristic residues conserved in DIX domains of human and mouse Ccd[1]. These findings suggest a possibility that differences in DIX domain sequences confer distinct activation properties

Summary

DIX domain

Shin-ichi Terawaki[1,2], Shohei Fujita[1], Takuya Katsutani[3], Kensuke Shiomi[4], Kazuko KeinoMasu[4], Masayuki Masu[4], Kaori Wakamatsu 1, Naoki Shibata2,3 &Yoshiki Higuchi 2,3. To test whether the ability for homopolymerization in mCcd[1] DIX was weakened by the insertion of loop β1-β2 into the head-to-tail interface, we generated five mutant proteins in which amino acids in the contact interface of the double-helical polymer were replaced with Ala. Interestingly, two mutations, L402A and W466A, in mCcd[1] DIX increased the molecular diameter of the homopolymer compared with that of the wild type (Fig. 4b). Substitutions of Phe[397] and Trp[466] located at the β1-β5 sheet involved in the heteromeric interaction with Axin and Dvl[1] and mutations of the residues required for head-to-tail homomerization (F442A, D439A, F405D, and V445A/K446A) decreased the transcriptional activity of mCcd[1] both alone and in synergy with Dvl (Fig. 6) These results suggest that the molecular surface created by hydrophobic residues of the β1-β5 sheet plays an important role in the transcriptional activation of the Wnt signaling pathway via heteropolymerization with DIX domains of Axin and Dvl

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