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Biochemical analysis of Wnt/Wingless signal transduction pathway with tissue culture system

利用组织培养系统对Wnt/Wingless信号转导通路进行生化分析

基本信息

批准号：
12680635
负责人：
YANAGAWA Shin-ichi
金额：
$ 2.43万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680635/
关键词：
Wnt Development Casein kinase I Signal transduction β-catenin Drosophila Wingless Casein Kinase I Signal transduction Drosophila RNAi

项目摘要

The Wnt family of secretory protein plays pivotal roles in a number of basic developmental processes. Components of the Wnt/Wingless (Wg) signaling pathway are Frizzled, Dishevelled, Glycogen synthase kinase-38, Axin, APC tumor suppressor, β-catenin, and T-cell transcription factor. These genes conserved in vertebrates and invertebrates. Using cell culture assay for Wnt/Wg that we have established, we are analyzing biochemical interactions among these components of the Wnt/Wg pathway. Casein kinase I (CKI) was recently reported as a positive regulator of Wnt signaling in vertebrates and C. elegans (Peters, J. M. et al : Nature 1999 401 : 345-350) To elucidate the function of Drosophila CKI in the wingless (wg) pathway, we have disrupted its function by double-stranded RNA-mediated interference (RNAi). While previous studies mainly based on CKI overexpression, this is the first convincing loss-of-function approach of CKI. Surprisingly, CKI_α- or CKI_ε-RNAi markedly elevated the Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting its mRNA levels. Pulse-chase analysis showed that CKI-RNAi indeed stabilizes Arm protein. Moreover Drosophila embryos injected with CKI_α-double-stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKI_α induced hyper-phosphorylation of both Arm and Dishevelled in S2R+ cells and conversely, CKI_α-RNAi reduced the amount of hyper-modified forms. His-tagged Arm was phosphorylated by CKI_α in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste-white 3. Thus, we propose that CKI phosphorylates Arm and stimulates its degradation.

分泌蛋白Wnt家族在许多基本发育过程中起着关键作用。Wnt/Wingless（Wg）信号传导途径的组分是卷曲、紊乱、糖原合成酶激酶-38、轴蛋白、APC肿瘤抑制因子、β-连环蛋白和T细胞转录因子。这些基因在脊椎动物和无脊椎动物中是保守的。使用我们已经建立的Wnt/Wg细胞培养试验，我们正在分析Wnt/Wg途径的这些组分之间的生物化学相互作用。酪蛋白激酶I（caseinkinase I，CKI）是近年来发现的脊椎动物Wnt信号的正调控因子，而C. elegans（Peters，J. M.等：Nature 1999 401：345-350）为了阐明果蝇CKI在无翅（wg）途径中的功能，我们通过双链RNA介导的干扰（RNAi）破坏了其功能。虽然以前的研究主要基于CKI过表达，但这是第一个令人信服的CKI功能丧失方法。令人惊讶的是，CKI_α-或CKI_ε-RNAi显著提高果蝇Schneider S2 R+细胞中Armadillo（Arm）蛋白的水平，而不影响其mRNA水平。脉冲追踪分析表明CKI-RNAi确实稳定了Arm蛋白。此外，注射CKI_α-双链RNA的果蝇胚胎表现出裸露角质层的表型，这与Wg信号的激活有关。这些结果表明CKI作为Wg/Arm信号传导的负调节剂起作用。CKI_α过表达可诱导S2 R+细胞中Arm和Dishevelled的过度磷酸化，相反，CKI_α-RNAi可减少Arm和Dishevelled的过度磷酸化。His标记的臂在体外被CKI_α在一组丝氨酸和苏氨酸残基上磷酸化，所述丝氨酸和苏氨酸残基也被Zeste-white 3磷酸化。因此，我们提出CKI磷酸化Arm并刺激其降解。