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

Abstract

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in .80% of colorectal cancers. APC participates in a multiprotein "destruction complex" that targets the proto-oncogene b-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity b-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all b-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that b-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all b-catenin-binding sites (including the 15Rs) and find that a direct b-catenin/APC interaction is also not essential for b-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby b-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock b-catenin in the destruction complex to increase the efficiency of b-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of b-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of b-catenin likely accounts for this difference.

Reconstituting regulation of the canonical Wnt pathway by engineering a minimal β-catenin destruction machine

“…In earlier work, we were surprised to learn that APC’s multiple βcat-binding sites are dispensable for targeting βcat for destruction, although they do modulate signaling by retaining βcat in the cytoplasm (Roberts et al. , 2011; Yamulla et al. , 2014).…”

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2

“…Yeast Two-hybrid Analysis-Yeast Two-Hybrid (Y2H) analysis was performed using the Matchmaker System (Clontech) as previously described (21). Briefly, the pGBKT7 and pGADT7 yeast vectors were engineered to be Gateway compatible, and APC2 entry vectors LR cloned into pGBKT7-W, whereas Armadillo, Axin, and TNKS were LR cloned into pGADT7-W. pGBKT7-W constructs were transformed into the Y2HGold yeast strain and pGADT7-W into Y187 using the SC Easy Transformation kit (Life Technologies).…”

Transcriptomics and machine learning predict diagnosis and severity of growth hormone deficiency

“…Loss-of-function mutations in anaphase-promoting complex 2 (APC2) have been associated with a Sotos syndrome-like phenotype of overgrowth and neurodevelopmental delay (29), and it is a negative regulator of WNT signaling through its targeting of β-catenin for ubiquitin-mediated proteolysis (30). WNT signaling is known to be involved in pituitary development, promoting the expression of PITX2 and proliferation of pituitary precursors (31).…”