%0 Journal Article
%T A protein knockdown strategy to study the function of ¦Â-catenin in tumorigenesis
%A Feng Cong
%A Jianxuan Zhang
%A William Pao
%A Pengbo Zhou
%A Harold Varmus
%J BMC Molecular Biology
%D 2003
%I BioMed Central
%R 10.1186/1471-2199-4-10
%X A protein knockdown strategy was designed to reduce the cytosolic ¦Â-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the ¦Â-catenin binding domain of E-cadherin fused to ¦ÂTrCP ubiquitin-protein ligase, the stable ¦Â-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 ¦Â-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of ¦ÂTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of ¦Â-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.We have designed a novel approach to induce degradation of stabilized/mutated ¦Â-catenin. Our results suggest that a high concentration of cytoplasmic ¦Â-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.Wnt signaling plays diverse roles at many stages of development by regulating the stability of ¦Â-catenin [1]. In cells that do not receive a Wnt signal, cytoplasmic ¦Â-catenin is bound to a multi-protein ¦Â-catenin destruction complex that contains several proteins including Axin, APC, and glycogen synthase kinase-3 (GSK3), and it is constitutively phosphorylated at a cluster of Ser and Thr residues at its N-terminus by GSK3. Phosphorylated ¦Â-catenin is recognized by ¦ÂTrCP, a component of the SCF¦ÂTrCP ubiquitin-protein ligase complex, and degraded by the ubiquitin-proteasome pathway. Wnt signaling disassembles the ¦Â-catenin destructio
%U http://www.biomedcentral.com/1471-2199/4/10