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目的观察HCMV感染细胞周期素依赖性蛋白激酶2(Cdk2)的亚细胞定位,研究HCMV感染对Cdk2蛋白水平及对细胞周期蛋白E(CyclinE)/Cdk2激酶活性的影响。方法通过密度抑制使细胞同步化于G0/G1期,用HCMVAD169毒株感染人胚肺成纤维细胞(HEL),用免疫细胞化学技术分别测定HCMV感染前及感染后24hCdk2亚细胞定位;WesternBlot法测定HCMVCdk2蛋白丰度;用免疫沉淀,激酶活性分析法检测HCMV感染细胞内Cdk2的活性。结果接触抑制阻止在G0期细胞Cdk2游离在细胞质,HCMV感染24h内导致Cdk2从细胞质移位到细胞核。同时HCMV感染可引起cyclinE/Cdk2激酶的强烈激活,但HCMV感染并不诱导Cdk2蛋白丰度增加。结论HCMV感染G0/G1细胞,在24h内导致Cdk2从细胞质移位到细胞核,使之与细胞核内的调节亚单位CyclinE结合,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,进展至晚G1期。
Objective To investigate the subcellular localization of cyclin-dependent kinase 2 (HCKV) -infected cells and the effect of HCMV infection on the Cdk2 protein level and the activity of CyclinE / Cdk2 kinase. Methods Cells were synchronized to G0 / G1 phase through density inhibition. Human embryonic lung fibroblasts (HEL) were infected with HCMVAD169 strain. The subcellular localization of HCMV before and 24 hours after infection was detected by immunocytochemistry. HCMVCdk2 protein abundance. The activity of Cdk2 in HCMV infected cells was detected by immunoprecipitation and kinase activity assay. Results Contact inhibition prevented Cdk2 from dissociating in the cytoplasm during G0 phase, leading to Cdk2 translocation from the cytoplasm to the nucleus within 24h of HCMV infection. At the same time HCMV infection can cause strong activation of cyclinE / Cdk2 kinase, but HCMV infection does not induce Cdk2 protein abundance. CONCLUSION: HCMV infection of G0 / G1 cells led to the translocation of Cdk2 from the cytoplasm to the nucleus within 24 hours, binding to the CyclinE regulatory subunit in the nucleus and activating the CyclinE / Cdk2 kinase, thereby allowing the cell cycle to cross the G1 / S limit and progress to Late G1.