Dynamic recruitment of axin by Dishevelled protein assemblies

Abstract

Dishevelled (Dvl) proteins are cytoplasmic components of the Wnt signalling pathway, which controls numerous cell fate decisions during animal development. During Wnt signalling, Dvl binds to the intracellular domain of the frizzled transmembrane receptors, and also to axin to block its activity, which results in the activation of ␤-catenin and, consequently, in a transcriptional switch. We have previously reported that the DIX domain of mammalian Dvl2 allows it to form dynamic protein assemblies. Here, we show that these Dvl2 assemblies recruit axin, and also casein kinase I⑀. Using photobleaching experiments of GFP-tagged Dvl2 and axin to study the dynamics of their interaction, we found that the recruitment of axin-GFP by Dvl2 assemblies is accompanied by a striking acceleration of the dynamic properties of axin-GFP. We also show that the interaction between Dvl2 and axin remains highly dynamic even after Wnt-induced relocation to the plasma membrane. We discuss how the recruitment of casein kinase I⑀ by Dvl2 assemblies might impact on the recruitment of axin to the plasma membrane during Wnt signalling.

Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta

“…As we had observed previously, expression of axin-RFP resulted in destruction of b-catenin (Figure 6a, left) that was prevented by inhibition of the proteasome, resulting in detection of b-catenin in axin puncta (Figure 6a, right). We found that axinDDIX did not form puncta but instead was distributed diffusely in the cytoplasm (Figure 6b), consistent with previous reports that the DIX domain is required for efficient puncta formation (Schwarz-Romond et al, 2007b). Importantly, expression of axinDDIX-RFP did not result in reduced bcatenin, but instead b-catenin was predominantly cytosolic with a distribution that mirrored that of axinDDIX (Figure 6b).…”

“…Using a commercially available polyclonal axin antibody, we observed endogenous axin in distinct puncta in the cytoplasm of Madin-Darby canine kidney (MDCK) cells (Figures 1b and c). Both endogenous and recombinant axin puncta were evenly distributed throughout the cytoplasm (Figures 1b-e), consistent with previous reports for endogenous axin in HEK293 cells (Levina et al, 2004;Wiechens et al, 2004) and recombinant axin (Fagotto et al, 1999;Smalley et al, 1999;Schwarz-Romond et al, 2005, 2007b. Axin-RFP was localized to cytoplasmic puncta when expressed in both MDCK ( Figure 1e) and HEK293 T cells (Supplementary Figures S1, S2).…”

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…This property correlates well with DVL aggregates under the overexpression condition, and it has been argued that the endogenous DVL may form such aggregates, which are highly dynamic (Schwarz-Romond et al 2005). DIX oligomerization/polymerization is proposed to provide a DVL platform for dynamic assembly of protein -protein interactions of low avidity, such as between DVL and Axin (Schwarz-Romond et al 2007b), and is a main underpinning for the receptor "signalosome" hypothesis (Bilic et al 2007) (see below). However, some have suggested that the DVL "aggregates" (or "dots" under microscopes) represent endocytic vesicles (Capelluto et al 2002;Taelman et al 2010).…”

“…2), and additional proteins in the assembly of the "b-catenin destruction complex," in which b-catenin phosphorylation (and degradation) is performed (MacDonald et al 2009;Stamos and Weis 2012). In relevance to Wnt receptor and DVL functions, Axin has a carboxy-terminal DIX domain, which was also called for convenience DAX to distinguish it from the DVL DIX domain (Schwarz-Romond et al 2007b). DAX shows an oligomerization/ polymerization property similar to but less dynamic than DIX (Schwarz-Romond et al 2007a).…”

“…Studies in mammalian and Drosophila models argue that DAX oligomerization is critical for Axin function in b-catenin phosphorylation/degradation (Kishida et al 1999b;Fiedler et al 2011). Axin binds directly with DVL and is recruited into DVL aggregates (under overexpression) via, in part, DAX -DIX interaction (Kishida et al 1999b;Schwarz-Romond et al 2007b), which may represent one mechanism by which DVL inhibits Axin (Kishida et al 1999b;Fiedler et al 2011). But it is unclear whether/how Wnt signaling regulates DVLAxin interaction.…”