A protein knockdown strategy to study the function of β-catenin in tumorigenesis Journal Article
| Authors: | Cong, F.; Zhang, J.; Pao, W.; Zhou, P.; Varmus, H. |
| Article Title: | A protein knockdown strategy to study the function of β-catenin in tumorigenesis |
| Abstract: | Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic β-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either β-catenin or adenomatous polyposis coli (APC), renders β -catenin resistant to degradation, and has been associated with multiple types of human cancers. Results: A protein knockdown strategy was designed to reduce the cytosolic β-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the β-catenin binding domain of E-cadherin fused to βTrCP ubiquitin-protein ligase, the stable β-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type β-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of βTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of β-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice. Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated β-catenin. Our results suggest that a high concentration of cytoplasmic β-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment. ©2003 Cong et al; licensee BioMed Central Ltd. |
| Keywords: | signal transduction; controlled study; protein expression; gene mutation; human cell; proto-oncogene proteins; nonhuman; protein function; protein localization; cell proliferation; mouse; animals; mice; gene targeting; cell division; cell growth; ubiquitin protein ligase; protein degradation; protein metabolism; transcription initiation; animal experiment; animal model; protein binding; cell fate; in vitro study; cell line, tumor; uvomorulin; carcinogenesis; mus musculus; colorectal neoplasms; ubiquitination; recombinant fusion proteins; mice, nude; colon cancer; myc protein; tumor cell; cellular distribution; trans-activators; oncogene c myc; wnt proteins; beta catenin; wnt protein; ubiquitins; cyclin d; cytoskeletal proteins; chimeric protein; colon polyposis; zebrafish proteins; protein engineering; human; article |
| Journal Title: | BMC Molecular Biology |
| Volume: | 4 |
| ISSN: | 1471-2199 |
| Publisher: | Biomed Central Ltd |
| Date Published: | 2003-09-29 |
| Start Page: | Article 10 |
| Language: | English |
| DOI: | 10.1186/1471-2199-4-10 |
| PUBMED: | 14516475 |
| PROVIDER: | scopus |
| PMCID: | PMC222962 |
| DOI/URL: | |
| Notes: | Export Date: 12 September 2014 -- Source: Scopus |
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