A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.
A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.
复制标题
研究β-连环蛋白在肿瘤发生中的功能的蛋白质敲除策略。
DOI：
10.1186/1471-2199-4-10
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发表时间：
2003-09-29
影响因子：
--
通讯作者：
Varmus, H
中科院分区：
文献类型：
--
作者：
Cong, F;Zhang, JX;Pao, W;Zhou, PB;Varmus, H
Wnt信号通路在许多发育阶段的细胞增殖和细胞命运决定中起关键作用。Wnt信号传导的关键下游靶标是胞质β-连环蛋白，其在Wnt活化后稳定并促进包括c-myc和细胞周期蛋白D在内的多种靶基因的转录。由β-连环蛋白或腺瘤性结肠息肉病（APC）突变引起的异常Wnt信号传导使β-连环蛋白对降解具有抗性，并且与多种类型的人类癌症相关。设计了一种蛋白质敲低策略，通过加速β-连环蛋白的周转率来降低胞浆β-连环蛋白水平。通过将E-cadherin的β-catenin结合结构域与βTrCP泛素-蛋白连接酶融合，构建嵌合蛋白，将稳定的β-catenin突变体募集到细胞SCF（Skp 1、Cullin 1和含F-box底物受体）泛素化机制中，进行泛素化和降解。DLD 1结肠癌细胞由于APC的丧失而以异常高的水平表达野生型β-连环蛋白。值得注意的是，在DLD 1细胞中，在四环素抑制性启动子的控制下，βTrCP-E-钙粘蛋白的条件表达选择性地敲低了β-连环蛋白的胞质亚群，而不是膜相关亚群。结果，DLD 1细胞在体外的生长和克隆形成能力受损，并且在裸鼠体内失去了致瘤潜力。我们设计了一种新的方法来诱导稳定/突变的β-连环蛋白的降解。我们的研究结果表明，高浓度的细胞质β-catenin对结直肠肿瘤细胞的生长至关重要。蛋白质敲除策略不仅可以作为一种新的方法来剖析癌蛋白在肿瘤发生中的作用，而且还可以作为一种独特的工具来描绘定位于特定亚细胞区室的蛋白质亚群的功能。
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. 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. 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.
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影响因子：
10.5
作者：
Winston, JT;Strack, P;Harper, JW
通讯作者：
Harper, JW
影响因子：
56.9
作者：
He, TC;Sparks, AB;Kinzler, KW
通讯作者：
Kinzler, KW
DOI：
10.1073/pnas.93.15.7950
发表时间：
1996-07-23
影响因子：
11.1
作者：
Morin, PJ;Vogelstein, B;Kinzler, KW
通讯作者：
Kinzler, KW
影响因子：
64.5
作者：
NUSSE, R;VARMUS, HE
通讯作者：
VARMUS, HE
影响因子：
64.5
作者：
van de Wetering, M;Sancho, E;Clevers, H
通讯作者：
Clevers, H