Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin-Independent Targets by Proteomics

Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of beta-catenin, but the extent to which APC exerts Wnt/beta-catenin-independent tumor-suppressive activity is unclear. To identify interaction partners and beta-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed tandem mass tag-based mass spectrometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of beta-catenin. Combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an APC-containing destruction complex. We validated the interaction between endogenous MINK1 and APC and further confirmed the negative, and beta-catenin-independent, regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared with wild-type tissue/cells. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease.