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      Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled

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          Summary

          Extracellular signals are often transduced by dynamic signaling complexes (“signalosomes”) assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of β-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to β-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

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          Highlights

          • Wnt signalosome assembly by Dishevelled depends on DEP-dependent dimerization

          • DEP dimerization via domain swapping favors unidirectional signaling

          • DEP-dependent cross-linking of Dishevelled polymers triggers phase transition

          • DEP dimerization is mutually exclusive with DEP-dependent binding to Frizzled

          Abstract

          Gammons et al. discover that Wnt signalosome formation by Dishevelled depends on dimerization by its DEP domain via domain swapping. This cross-links DIX-dependent Dishevelled polymers and, thus, promotes phase transition. They propose that DEP domain swapping triggered by high local concentration of Dishevelled in clathrin-coated pits initiates Wnt signal transduction.

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          Most cited references35

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          Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.

          Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.
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            Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques.

            Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.
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              The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.

              The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of beta-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.
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                Author and article information

                Contributors
                Journal
                Mol Cell
                Mol. Cell
                Molecular Cell
                Cell Press
                1097-2765
                1097-4164
                06 October 2016
                06 October 2016
                : 64
                : 1
                : 92-104
                Affiliations
                [1 ]MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK
                Author notes
                []Corresponding author melissag@ 123456mrc-lmb.cam.ac.uk
                [∗∗ ]Corresponding author mb2@ 123456mrc-lmb.cam.ac.uk
                [2]

                Co-first author

                [3]

                Lead Contact

                Article
                S1097-2765(16)30473-7
                10.1016/j.molcel.2016.08.026
                5065529
                27692984
                1bca8072-b184-4e71-aa97-595ef719f6c6
                © 2016 MRC Laboratory of Molecular Biology

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 12 May 2016
                : 15 July 2016
                : 23 August 2016
                Categories
                Article

                Molecular biology
                Molecular biology

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