Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling

Abstract

APC is a key tumor suppressor and Wnt signaling regulator, but its mechanism of action remains mysterious. We combined parallel assays in Drosophila and cultured human colon cancer cell lines to test hypotheses regarding APC function and to develop novel hypotheses, using mutants altering its structure in specific ways.

The -Catenin Destruction Complex

“…Peifer and colleagues generated a Drosophila APC2 variant in which the SAMP repeats were deleted cleanly rather than by truncation, so that all b-cateninbinding sites were retained. This mutant APC was nonfunctional in animals, although b-catenin destruction and TCF signaling down-regulation were retained in SW480 cells (Roberts et al 2011). Curiously, Roberts et al 2011 found that APC2 lacking SAMP repeats colocalized with Axin, but the mutant APC2 did not coimmunoprecipitate with Axin, suggesting a weak and indirect interaction.…”

“…This mutant APC was nonfunctional in animals, although b-catenin destruction and TCF signaling down-regulation were retained in SW480 cells (Roberts et al 2011). Curiously, Roberts et al 2011 found that APC2 lacking SAMP repeats colocalized with Axin, but the mutant APC2 did not coimmunoprecipitate with Axin, suggesting a weak and indirect interaction. It is possible that bcatenin itself bridged Axin and the mutant APC2, because it can interact with Axin and the APC 15-mer or nonphosphorylated 20-mer repeats simultaneously (Ha et al 2004).…”

“…In addition, deletion of the Axin RGS domain in both vertebrates and Drosophila compromises b-catenin destruction in vivo (Fagotto et al 1999;Peterson-Nedry et al 2008), demonstrating the importance of the Axin-APC interaction. However, the APC interaction mediated by the RGS domain is dispensable when Axin is overexpressed in SW480 colon cancer cells (see below) (Kohler et al 2009;Roberts et al 2011). Measurements of destruction complex components in mammalian kidney epithelial cells and Xenopus oocytes gave different absolute and relative concentrations of APC and Axin (Salic et al 2000;Lee et al 2003;Tan et al 2012), suggesting that Wnt signaling operates over a range of component concentrations that vary with cell type.…”

“…Equivalent experiments have not been performed with heterotrimeric PP2A, however, and the effect of PP2A interactions with Axin and/or APC have not been assessed in this context. Finally, Peifer and colleagues showed that an APC2 construct in which only the highest affinity 20-mer repeat is retained is unable to promote b-catenin destruction (Roberts et al 2011), indicating that high-affinity b-catenin binding by phosphorylated APC is not sufficient for destruction complex function.…”

“…Ubiquitination requires the presence of APC 20-mer repeat 2 (20R2), which, importantly, does not bind to b-catenin Kohler et al 2008). Furthermore, transfection of SW480 cells with a construct of APC truncated after 20R3 can still down-regulate b-catenin in a cell culture system (Yang et al 2006;Kohler et al 2008), whereas deletion of 20R2 and the strongly conserved sequence that follows it, designated the "catenin inhibitory domain" (CID; also called "sequence B" by Peifer and coworkers), prevents b-catenin destruction (Kohler et al 2009;Roberts et al 2011). (Ha et al 2004), Axin ( purple) (Xing et al 2003), and phosphorylated APC R3-P (red) (Ha et al 2004;Xing et al 2004) bound to the armadillo repeat domain of b-catenin (blue and gray) were superimposed to show the relationship between the bound ligands.…”