Abstract
Most colorectal cancers have mutations of the adenomatous polyposis coli (APC) gene or the beta-catenin gene that stabilize beta-catenin and activate beta-catenin target genes, leading ultimately to cancer. The molecular mechanisms of APC function in beta-catenin degradation are not completely known. APC binds beta-catenin and is involved in the Axin complex, suggesting that APC regulates beta-catenin phosphorylation. Some evidence also suggests that APC regulates beta-catenin nuclear export. Here, we examine the effects of APC mutations on beta-catenin phosphorylation, ubiquitination, and degradation in the colon cancer cell lines SW480, DLD-1, and HT29, each of which contains a different APC truncation. Although the current models suggest that beta-catenin phosphorylation should be inhibited by APC mutations, we detected significant beta-catenin phosphorylation in these cells. However, beta-catenin ubiquitination and degradation were inhibited in SW480 but not in DLD-1 and HT29 cells. The ubiquitination ofbeta-catenin in SW480 cells can be rescued by exogenous expression of APC. The APC domains required for beta-catenin ubiquitination were analyzed. Our results suggest that APC regulates beta-catenin phosphorylation and ubiquitination by distinct domains and by separate molecular mechanisms.
Highlights
Colorectal cancer is the second leading cause of cancer-related death in the United States, and the incidence of colorectal cancer is rising in developing countries [1]
We demonstrated that the defect in -catenin ubiquitination and degradation in SW480 cells is due to an adenomatous polyposis coli (APC) truncation
We found that Ad-CBR can rescue -catenin ubiquitination and degradation in SW480 cells, indicating that APC is required for the ubiquitination and degradation of phosphorylated -catenin (Fig. 2C). -Catenin phosphorylation was stronger in SW480 cells, most likely because phosphorylated -catenin is degraded more slowly and accumulates in SW480 cells
Summary
Cell Lines—The colon cancer cell lines, HEK293T, HCT116, SW480, DLD-1, and HT29, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum, at 37 °C, 5% CO2. A, linear representation of different APC truncations in the colon cancer cell lines, SW480, DLD-1, and HT29. The colon cancer cell line, HCT116, contains wild type APC. B, the cytoplasmic lysates from SW480, DLD-1, HT29, and HCT116 cells were analyzed by Western blot using antibodies against -catenin and phosphorylated -catenin. C, the -catenin immunoprecipitation products from HT29 and HCT116 cell lysates were treated with or without CIP and analyzed with antibodies against -catenin and phosphorylated -catenin. Cytoplasmic fractions were analyzed with antibodies against -catenin and phosphorylated -catenin
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