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Artemis是1个具有多种生物学功能的磷酸化蛋白,它在基因毒性应激引发的细胞周期检测点调控中起重要作用,但其调控机制知之甚少.为了探讨UVC等DNA复制阻滞应激引发的Artemis磷酸化及蛋白表达水平对细胞周期蛋白E的调控作用和调控机制.首先以Western印迹方法检测Artemis S516-645A突变细胞和Artemis表达降低细胞的细胞周期蛋白E的表达水平,发现ArtemisS516-645A突变细胞和多种Artemis siRNA转染细胞的细胞周期蛋白E表达水平均高于对照细胞.在此基础上,为分析细胞周期蛋白E表达受调控的分子机制,在稳定表达各种磷酸化状态Artemis的HEK-293细胞中导入外源性启动子转录驱动的细胞周期蛋白E表达质粒,发现表达Artemis S516-645A突变体的细胞中外源性的细胞周期蛋白E蛋白表达水平也高于野生型细胞.进一步的研究发现在Artemis蛋白表达降低的细胞中与泛素结合的细胞周期蛋白E减少而蛋白稳定性增加.本研究还发现Artemis蛋白对细胞周期蛋白E的调控过程是不依赖于p53和p21表达的.这些结果表明,Artemis S516-645A突变和Artemis表达降低都可以引起细胞周期蛋白E蛋白水平升高,该调控作用是在转录后水平发生的,可能是干扰了细胞周期蛋白E的泛素化介导的蛋白降解过程,并且该调控作用是独立于p53-p21信号通路的.
Artemis is a phosphorylated protein with a variety of biological functions that plays an important role in genotoxic stress-induced cell cycle checkpoint regulation, but its regulatory mechanism is poorly understood.In order to investigate whether DNA replication arrest Stimulated phosphorylation of Artemis and protein expression level on the regulation and action mechanism of cyclin E.Firstly, the expression of Artemis S516-645A mutant cells and Artemis expression was detected by Western blotting to reduce the expression of cell cycle protein E, and found that ArtemisS516 -645A mutant cells and a variety of Artemis siRNA transfected cells were higher than control cells.On this basis, in order to analyze the molecular mechanism of the regulation of cyclin E expression in the stable expression of various phosphorylation Exogenous promoter transcription driven cyclin E expression plasmids were introduced into Artemis HEK-293 cells and the expression of exogenous cyclin E protein was also found to be higher in cells expressing Artemis S516-645A mutant than in wild type Cells.Further studies found that the reduction of ubiquitin-bound cyclin E in cells with decreased Artemis protein expression While the stability of protein was increased.The study also found that the regulation of cyclin E by Artemis protein is independent of the expression of p53 and p21.These results indicate that both Artemis S516-645A mutation and Artemis expression decrease can induce the expression of cyclin E Elevated protein levels, which occur at the post-transcriptional level, may interfere with ubiquitination of cyclin E-mediated protein degradation and are independent of the p53-p21 signaling pathway.