Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours

Abstract

The mutation cluster region (MCR) of adenomatous polyposis coli (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the beta-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any beta-catenin binding activity. Similarly, we also show that the tumour-associated truncations abolish the interaction of beta-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to beta-catenin binding.

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

“…Drosophila APC2 has five 20Rs ( Figure 1A); however, prior biochemical studies established that 20R2 of human APC does not bind bcat (Liu et al 2006;Kohler et al 2008). Human 20R2 shares considerable sequence identity with other bcat-binding 20Rs, but lacks an upstream acidic residue (Supporting Information, Figure S1) that contacts one of the "charged buttons" in bcat (Ha et al 2004;Xing et al 2004).…”

“…In designing Drosophila APC2 mutants devoid of particular bcat-binding sites, we were also informed by biochemical work demonstrating that the second 20R of human APC (20R2) lacks any detectable affinity for bcat, even when phosphorylated (Liu et al 2006;Kohler et al 2008). Initially, this finding suggested that 20R2 is a degenerate 20R that lost affinity for bcat through evolution; however, we recently showed that 20R2 is highly conserved in species as diverse as sea snails (Lottia gigantean), fruit flies, and humans (Roberts et al 2011).…”

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

“…A novel truncation mutation G1288* in APC was also detected in 48 (84%) cells in the major clone. The mutation site was in close proximity to the "mutation cluster region" (codon 1300), in which truncation mutations have been shown to abrogate the β-catenin-binding activity of APC [21]. When we assessed the landscape at an individual-cell level within the single tumor tissue, we found that only a few genes (13/186 = ~7.0%) were shared with the cohort results.…”

Amer2 Protein Is a Novel Negative Regulator of Wnt/β-Catenin Signaling Involved in Neuroectodermal Patterning

“…DNA Constructs, siRNAs, and Morpholinos-The following constructs have been described previously: pcDNA-FLAGAmer2, pEGFP-Amer2, pcDNA-FLAG-Amer1, pEGFP-APCArmadillo (Arm), and pEGFP-APC-Arm-N507K (3); pCMV-APC, monomeric YFP (mYFP)-APC, mYFP-␤-catenin, mYFPaxin, mYFP-conductin, and monomeric red fluorescent protein-dominant-active LRP6 (10); pcDNA3.1-FLAG (11); and mYFP-APC1641 (12). To generate mYFP-␤-catenin S33Y, point mutations were introduced into YFP-␤-catenin by PCR.…”