ASSEMBLY AND DISASSEMLY OF THE WNT BETA-CATENIN DESTRUCTION COMPLEX

Wnt signaling provides a key example for cell-cell signaling pathways that regulate embryonic development and stem cell homeostasis and then are frequently inappropriately activated in cancers. Wnt signaling acts by regulating levels of β-catenin (βcat), an essential transcriptional coactivator of Wnt target genes. The tumor suppressors APC and Axin, along with the kinases GSK3 and CK1, form the core of the multiprotein destruction complex (DC), which targets βcat for phosphorylation, ubiquitination and destruction. In the presence of Wnt ligands, DC function is down-regulated, allowing levels of βcat levels to rise, eventually entering the nucleus to activate transcription of Wnt target genes. Based on earlier work, we hypothesize that the DC is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulation of assembly and stability of the DC regulates its function. We tested this hypothesis in Drosophila embryos by combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging, and molecule counting. 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 puncta to the membrane, a decrease in the number of Axin molecules, while cytoplasmic Axin levels increase, suggesting altered assembly. GSK3 regulates DC recruitment to the membrane and the release of Armadillo/βcat from the DC. Manipulating Axin or APC2 levels had no effect on DC activity when Wnt signals were absent, but, surprisingly, had opposite effects on the DC when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. We also found that endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels. Hetero-polymerization between Dishevelled and Axin via their DIX domains is essential for Wnt down regulation of the DC, yet the mechanism of regulation is unknown. Our data suggest both absolute levels and the ratio of these three core components affect destruction complex function, supporting models in which competition among Axin partners determines complex activity.Doctor of Philosoph