Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.
Bottom Line: The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3β, and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation.The destruction complex forms macromolecular particles we termed the destructosome.These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.Show MeSH
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Mentions: To test this hypothesis, we generated a mutant version of Drosophila APC2 lacking this region (APC2-∆ASAD; Figure 2A). In addition, we disrupted potential coiled-coil formation by changing four key hydrophobic leucine residues to proline (APC2-ASADPro; Figures 1C and 2A). To determine whether this domain is necessary to mediate APC2 self-association, we performed immunoprecipitation assays from transiently transfected Drosophila S2 cells. Previously we showed that mCherry-tagged (mCh) APC2-FL (full length) and APC2-N (aa 1–490) could coprecipitate untagged APC2-FL, unlike mCh-APC2-C (aa 491–1067; Zhou et al., 2011; Figure 2B). Neither mCh-APC2-∆ASAD nor mCh-APC2-ASADPro was able to coprecipitate untagged APC2-FL, demonstrating that the N-terminal coil is necessary for self-association (Figure 2B). Because human OD-1 mediates dimer formation through a direct protein–protein interaction (Joslyn et al., 1993), we asked whether ASAD mediates direct APC2–APC2 binding. Consistent with that model, APC2-N (Figure 2A) self-associated in a yeast two-hybrid (Y2H) assay, and this interaction was disrupted in both ASAD mutants (APC2-N-ΔASAD and APC2-N-ASADPro; Figure 2C).
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.