DNA损伤反应中细胞周期检查点蛋白p27~(Kip1)表达及其调控机制

为了研究DNA损伤反应中p2 7Kip1的表达及其调控机制 ,应用免疫印迹的实验结果表明 :10Gy 60 Coγ射线照射后 3h ,HeLa细胞中p2 7Kip1蛋白水平开始下降并持续到 2 4h ,进而失去它对CDKs的抑制功能 .Northern印迹结果显示 ,电离辐射 (IR)对p2 7Kip1mRNA表达水平无明显影响 ,说明电离辐射诱导p2 7Kip1表达水平的降低主要与蛋白质降解相关 ,但其具体的调控机制还不清楚 .已知在G1—S期p2 7Kip1蛋白的降低主要依赖细胞周期蛋白E Cdk2激酶将其磷酸化后的泛素化蛋白酶体途径 (ubiquitin proteasomepathway) .酶动力学研究结果揭示 :电离辐射后细胞周期蛋白E Cdk2激酶活性增高 ,12h细胞周期蛋白E Cdk2激酶活性达到最大 .当在照前用细胞周期蛋白E Cdk2抑制剂olomoucine (10 μmol L)抑制细胞周期蛋白E Cdk2激酶活性时 ,p2 7Kip1蛋白表达水平增加 .此外 ,还观察到电离辐射可诱导p2 7Kip1泛素化水平的增高 ,而在使用蛋白酶体抑制剂MG 132 (5 μmol L)处理HeLa细胞后 ,可抑制辐射诱导p2 7Kip1蛋白水平的下调 .研究结果提示 :泛素化蛋白酶体途径参与了辐射诱导P2 7Kip1蛋白表达下调的降解机制 .

Expression of Cell Cycle Checkpoints Regulator Proteins p27~(Kip1) and Its Mechanism of Regulation in DNA Damage

In the expression of p27 Kip1 and its mechanism of regulation in the DNA damage, the level of p27 Kip1 expression was found to decrease from 3 to 24 hours after 10 Gy γ-irradiation, and lose its inhibition function to cyclin-dependent kinase. Northern blotting results indicated that the mRNA level of p27 Kip1 did not change during ionizing radiation(IR) responses, which showed that the amount of p27 Kip1 was predominantly regulated by posttranslational mechanism, but the precise mechanism of p27 Kip1 regulation was unclear. It was well known that the degradation of p27 Kip1 mainly depends on cyclin E/Cdk2 kinase which could phosphorylate p27 Kip1, and then the phosphorylated p27 Kip1 degraded through ubiquitin-proteasome pathyway. Enzyme kinetics showed that the activity of Cyclin E/Cdk2 kinase complex was upregulated after irradiation, reached to maximum at 12 hours. When Cyclin E/Cdk2 inhibitor Olomoucine inhibited the activity of Cyclin E/Cdk2 kinase before IR, the level of p27 Kip1 increased. The level of ubiquitin-p27 Kip1 conjugates in non-irradiated and irradiated HeLa cells were detected and the level of ubiquitinated p27 Kip1 was found to increase following 10 Gy-irradiation. MG-132 (proteasome inhibitor) inhibited the degradation of p27 Kip1 which was induced by IR in HeLa cells. The data suggested that ubiquitin -proteasome pathway might involve in the regulation of p27 Kip1 during IR responses.