Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

Abstract

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

“…In our recent work, we explored the role of Dsh. We found that overall protein levels of Axin, APC2 and Dsh in Drosophila embryos experiencing active Wnt signaling are within a few-fold of one another, suggesting that competition is a plausible mechanism for destruction complex down-regulation (Schaefer et al, 2018). The competition model is also consistent with the effects of elevating Axin levels, which makes the destruction complex more resistant to turn-down (Cliffe et al,…”

“…Wnt signaling triggers down-regulation of the destruction complex, and the ability to do so depends on relative Axin levels. Thus, while a 3-4 fold increase in Axin levels is tolerated by the developing embryo (Wang et al, 2016;Schaefer et al, 2018), more substantial increases in Axin levels prevent inactivation of the destruction complex even in cells exposed to the Wnt ligand (Willert et al, 1999;Cliffe et al, 2003;Schaefer et al, 2018). Dsh is a key positive effector of Wnt signaling and its ability to homopolymerize and to heteropolymerize with Axin are critical for downregulating the destruction complex (Schwarz-Romond et al, 2007a;Schwarz-Romond et al, 2007b;Fiedler et al, 2011;Mendoza-Topaz et al, 2011).…”

“…Work in cultured cells suggests Dsh can compete with APC for association with Axin, providing a potential mechanism for Dsh's role in destruction complex downregulation (Fiedler et al, 2011;Mendoza-Topaz et al, 2011). However, surprisingly, in Drosophila embryos elevating Dsh levels 7-fold has only modest effects on viability and cell fate choices (Cliffe et al, 2003;Schaefer et al, 2018), suggesting that Dsh may need to be "activated" by Wnt signaling in order to compete for Axin binding.…”

“…Our recent work provided mis-expression tools allowing us to control for both these variables, and provide quantitative assessments of relative levels of the two proteins under different conditions (Schaefer et al, 2018).…”

“…Constitutive activation of Wnt signaling leads to expansion of naked cuticle (Fig 7B, left) while inactivation of Wnt signaling expands denticle cell fates ( Fig 7B, right). We used cell fate scoring criteria assessing this which we previously developed ( Fig 7C; (Schaefer et al, 2018). As we previously found, mild zygotic Axin overexpression (Mat>RFP x Axin; 2-fold increase in Axin levels (Schaefer et al, 2018); crosses used and cross nomenclature are in the Methods) had little or no effect on embryonic viability ( Fig 7A) or cell fate choices, as assessed by cuticle pattern (Fig.…”