| dc.description.abstract | The canonical Wnt signaling pathway is a master regulator of metazoan development and disease. The tumor suppressor APC negatively regulates Wnt pathway activation, and is conventionally thought to function primarily in the cytosolic β-catenin destruction complex. APC has also been shown to inhibit constitutive, ligand-independent receptor activation, and this is proposed to be via inhibition of clathrin/AP2-mediated signalosome formation. First, we ask if APC directly interacts with the cargo recognition subunit of AP2, and if this interaction is required for APC regulation of Wnt receptors. We find that the central region of APC, which is functional in the β-catenin destruction complex, interacts with AP2μ2. We find that this interaction is mediated by two tyrosine-based sorting motifs present on the central region of APC, and we also investigate the minimal fragments of APC required for this interaction. Additionally, the intracellular domain of LRP6, which is known to interact with AP2 during Wnt receptor activation, competitively blocks the APC-AP2 interaction. Furthermore, overexpression of the central region of APC inhibits Wnt ligand dependent activation, and this inhibition is dependent on APC interactions with AP2. These studies provide biochemical insight into the novel mechanism of APC regulation of Wnt receptor activation. We next ask if proteins controlling Wnt receptor homeostasis regulate APC-deficient Wnt pathway activation, given the LRP6 requirement pathway activation upon APC deletion in cells. The deubiquitylase USP46 promotes Wnt pathway activation by deubiquitylating LRP6 and stabilizing the receptor. We find that USP46 is required for pathway activation in APC knockdown and mutant cells, and this is conserved in APC mutant Drosophila midgut, and in organoids derived from APCmin mice. USP46 knockdown, however, did not reduce β-catenin and LRP6 levels in APC mutant human cell lines. Epistasis studies indicate a potential receptor-independent regulatory function for USP46. These findings provide evidence for an as of yet determined function for USP46 in the Wnt pathway, independent of receptor regulation. Together, these studies unravel a novel role for APC in the regulation of Wnt receptor activation. | |
