Artemis蛋白磷酸化对紫外线C引发的复制检测点的调控  

Regulation of artemis phosphorylation on replication checkpoint induced by UVC irradiation

作　　者：王海勇[1] 张小山[2] 滕理送[1] 

机构地区：[1]浙江大学医学院附属第一医院肿瘤中心,杭州310003 [2]美国德克萨斯大学MD Anderson癌症中心

出　　处：《中华放射医学与防护杂志》2009年第5期543-547,共5页Chinese Journal of Radiological Medicine and Protection

摘　　要：目的研究复制叉前进受阻后Artemis蛋白S516和S645丝氨酸位点的磷酸化和对细胞周期复制检测点的影响。方法Western—blotting检测细胞受紫外线C照射后Artemis S516和S645两个位点的磷酸化变化；突变Artemis蛋白的第516和645位氨基酸，构建非磷酸化和模拟磷酸化的突变体S516—645A和S516—645D表达质粒，转染HEK293细胞建立稳定表达细胞系；流式细胞仪检测受UVC照射后的细胞周期，Western—blotting检测UVC照射后2、6、12h的Chk1、γ-H2AX、Cdk2蛋白的表达变化，免疫共沉淀激酶实验检测Cdk2IP激酶的活性。结果Artemis蛋白S516和S645位点在UVC照射后发生迅速和持久的磷酸化，ATR是Artemis S516和S645磷酸化的主要激酶；S516—645A非磷酸化双突变导致细胞周期在S期复制检测点停留时间延长。Artemis S516—645A双突变不影响Chkl、Cdk2、γ-H2AX蛋白表达水平，却抑制了Cdk2 IP激酶的活性。结论ATR激酶对Artemis蛋白S516和S645位点的磷酸化促进了细胞周期从UVC引发的复制检测点中恢复。Objective To investigate Artemis phosphorylation on S516 and S645 in response to stalled replication forks and its role in regulation of cell cycle replication checkpoint. Methods Western-blotting was used to measure the expression of phosphorylation of Artemis on S516 and S645 after UVC irradiation. The nonphosphorylatable double mutant （S516-645A） and the mimicking phosphorylation mutant （S516-645D） plasmids were constructed. HEK 293 cells with stable expression of wild type Artemis and the corresponding mutants were established by transfection. Cell cycles of the cells treated with UVC irradiation were analyzed by flow cytometry, Western-blotting was used to measure the expression of Chk1, γ-H2AX and Cdk2. IP-kinase assay was used to measure the kinase activity of Cdk2 2, 6 and 12 h after UVC irradiation. Results Artemis got rapid and prolonged phosphorylation on S516 and S645 after treatment with UVC irradiation and the major responsible kinase was ATR. The S516-645A mutant caused prolonged arrest in replication checkpoint in S phase. The Cdk2 IP kinase activity was inhibited in S516-645A mutant cells, but the expression levels of Chkl, Cdk2 and γ-H2AX were not affected. Conclusion The ATR phosphorylation on S516 and S645 of Artemis promotes cell cycle recovery from UVC induced replication checkpoint.

关 键 词：Artemis蛋白 紫外线 磷酸化 复制检测点 细胞周期 

分 类 号：Q343[生物学—遗传学] TN23[电子电信—物理电子学]