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: For these FRAP experiments, we chose similarly sized puncta for each condition and kept the bleached area constant. For the âˆ†ASAD mutant, where the puncta can be interconnected at higher expression levels, we chose cells with relatively low expression, for which we could see isolated, individual puncta. To compare the Axin-GFP fluorescence recovery among the three conditions, we first normalized for the starting postbleach fluorescence by calculating âˆ†F/F for each time point in each condition and plotted this over time (Figure 5, Aâ€“C). To compare the rates of recovery, we compared the mean slope of the regression lines for each condition (Figure 5D). Axin-GFP/APC2-âˆ†ASAD puncta displayed a significantly greater slope than either Axin-GFP alone or Axin-GFP/APC2-FL puncta. Conversely, Axin-GFP/APC2-FL puncta exhibited a significantly reduced slope compared with the other conditions (Figure 5D). Axin-alone puncta exhibited the greatest variation in rate of recovery; some puncta displayed Axin-GFP/APC2-FLâ€“like properties, whereas others exhibited Axin-GFP/APC2-âˆ†ASADâ€“like properties (Figure 5A). Cells expressing Axin-GFP alone express significantly more Axin than the low level of endogenous APC2 in these cells (Zhou and McCartney, unpublished data). This suggests that at a high Axin:APC2 ratio, Axin turnover rates are not well controlled and fluctuate as a consequence. When the Axin:APC2 ratio is closer to 1, as in the case of Axin-GFP/APC2-FL puncta, Axin-GFP is stabilized, and the overall rates of recovery decrease significantly (Figure 5D). On the other hand, disrupting APC2 self-association appears to drive Axin-GFP toward the opposite end of its dynamic spectrum (Figure 5D).
Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.