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A protein knockdown strategy to study the function of β-catenin in tumorigenesis

Authors
Journal
BMC Molecular Biology
1471-2199
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Volume
4
Issue
1
Identifiers
DOI: 10.1186/1471-2199-4-10
Keywords
  • Research Article
Disciplines
  • Biology
  • Design
  • Engineering

Abstract

1471-2199-4-10.fm ral ss BioMed CentBMC Molecular Biology Open AcceResearch article A protein knockdown strategy to study the function of β-catenin in tumorigenesis Feng Cong*1, Jianxuan Zhang2, William Pao1, Pengbo Zhou*2 and Harold Varmus1 Address: 1Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA and 2Department of Pathology, Weill Medical College and Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA Email: Feng Cong* - [email protected]; Jianxuan Zhang - [email protected]; William Pao - [email protected]; Pengbo Zhou* - [email protected]; Harold Varmus - [email protected] * Corresponding authors 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-

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