Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo.

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, ß-catenin (ßcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to ßcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds ßcat and the affinity is significantly increased. These distinct APC-ßcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for ßcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.

Cortical localization of APC2 plays a role in actin organization but not in Wnt signaling in Drosophila.

The tumor suppressor Adenomatous polyposis coli (APC) has roles in both Wnt signaling and in actin and microtubule organization. Within the cell, APC proteins have been reported to localize in the cytoplasm, at the cell cortex and in the nucleus. How these localizations relate to the functions of the protein is an aspect of APC biology that is poorly understood. Using Drosophila S2 cells, we have dissected the structural and functional requirements for the cortical localization of Drosophila APC2. Here, we show that both the Armadillo repeats and a novel C-terminal domain are necessary for the cortical localization of APC2 in S2 cells and in the embryo, and that neither domain alone is sufficient for this localization. Furthermore, we show that the Armadillo repeats mediate self-association of APC2 molecules. To test the function of the cortical localization of APC2, we asked whether an APC2 protein deleted for the C-terminal localization domain could rescue APC mutant defects in Wnt signaling and actin organization in the Drosophila embryo. We show that although cortical localization is required for the APC2 function in organizing actin, cortical localization is dispensable for its role in regulating Wnt signaling.