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

Wnt家族的分泌蛋白在许多基本的发育过程中起着关键作用。Wnt/Wingless(Wg)信号通路的组成包括Frizzled型、蓬头型、糖原合成酶激酶-38、Axin、APC肿瘤抑制因子、β-catenin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。利用我们建立的Wnt/Wg细胞培养方法，我们正在分析Wnt/Wg途径的这些组成部分之间的生化相互作用。酪蛋白激酶I(Casein Kinase I，CKI)是脊椎动物和线虫Wnt信号的正向调节因子(Peters，J.M.et al：Natural 1999 401：345-350)为了阐明果蝇CKI在无翅(WG)途径中的功能，我们用双链RNA介导的干扰(RNAi)干扰了它的功能。虽然以前的研究主要基于CKI的过度表达，但这是第一个令人信服的CKI功能丧失的方法。令人惊讶的是，CKI_α-或CKI_ε-RNAi显著提高了果蝇施耐德S2R+细胞Armadillo(ARM)蛋白水平，而不影响其基因水平。脉冲追逐分析表明，CKI-RNAi确实稳定了ARM蛋白。此外，注射CKI_α双链核糖核酸的果蝇胚胎出现裸露的角质层表型，这与Wg信号的激活有关。这些结果表明，CKI是Wg/ARM信号的负调节因子。在S2R+细胞中，过表达CKI_α可诱导S2R+细胞手臂和头发蓬乱的过度磷酸化，反之，CKI_α-RNAi可减少高修饰形式的数量。体外，CKI_α在一组丝氨酸和苏氨酸残基上被ZEST-White 3磷酸化。因此，我们认为CKI可以磷酸化ARM并刺激其降解。

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.