【作者】 高磊；

【导师】 吴畏；

【作者基本信息】 清华大学 ， 生物学， 2009， 硕士

【摘要】 经典Wnt信号通路是一条非常保守的信号转导途径,在许多生理过程中都发挥着重要作用,因此,对经典Wnt信号通路的充分了解认识是非常必需的。在从非洲爪蟾胚胎cDNA文库中筛选能够影响经典Wnt信号的基因时,我们发现Skip(ski interacting protein)蛋白的C端缺失型(SkipΔC)能够很好地与经典Wnt信号特异的转录激活调控因子β-catenin协作,激活Wnt信号;而过量表达的野生型Skip却对β-catenin信号起抑制作用。对于Skip参与经典Wnt信号通路这一现象,本课题对其作用机制进行了初步的研究。我们使用免疫共沉淀的方法检测到SkipΔC能够与β-catenin相互作用并能稳定β-catenin蛋白,而全长的Skip与β-catenin之间没有相互作用存在。同时,我们也检测到Skip及SkipΔC与LEF1(经典Wnt信号特异的转录调控因子)之间的相互作用。我们还发现Skip不影响β-catenin与LEF1的结合,排除了Skip蛋白过量表达抑制β-catenin与LEF1的结合而起作用的可能性。利用RNA干扰技术,我们发现Skip对于经典Wnt信号通路的传递是必需的。在爪蟾胚胎中,我们发现SkipΔC能够一定程度上模拟经典Wnt信号抑制头部发育,而缺失Skip蛋白后的胚胎神经发育有严重缺陷,并且伴随着轻微的脊索发育缺陷。更多还原

【Abstract】 Wnt signaling pathway is a conserved signaling pathway, playing very important roles in diverse physiological processes. Therefore, it is of great interesting for us to understand this pathway completely. In a functional screening of genes affecting Wnt signaling from a Xenopus embryonic cDNA library, we found that C terminal deleted Skip (ski interacting protein) can cooperate withβ-catenin, a specifical transcriptional activator, to active Wnt signaling, while overexpression of full-length Skip inhibitedβ-catenin induced Wnt signaling. In this study, we analysized the molecular mechanism of Skip in regulating Wnt signaling. Using Co-immunoprecipitation assay, we found that SkipΔC can interact withβ-catenin, and stabilizeβ-catenin in HEK293T cell. However, this interaction does not exist between full-length Skip andβ-catenin. Meanwhile, we also detected that both SkipΔC and Skip can interact with LEF1, the transcriptional factor of Wnt signaling pathway. Our results also indicated that Skip does not interfere with the interaction betweenβ-catenin and LEF1. Moreover, using RNAi we found that Skip is required for Wnt signaling pathway in culture cells. In Xenopus embryo, we found that SkipΔC, to some extend, can mimic the active of Wnt signal during anterio-posterior patternin, and the embryos without expressing Skip of which the develop of head is severely abnormal, frequently accompany with some defects in notochord.更多还原