摘要
目的:研究端粒结合因子-1(TRF1)丝氨酸219位磷酸化对细胞周期的影响。方法:突变TRF1的219位氨基酸,构建模拟磷酸化和非磷酸化状态的突变体TRF1S219D-GFP和TRF1S219A-GFP,核苷酸测序和免疫印迹验证目的基因序列及蛋白表达。将野生型和突变体质粒用脂质体转染法转入H eLa细胞,观察它们在细胞中的定位、流式细胞仪检测各细胞周期的细胞数量、免疫印迹检测转染后ATM蛋白水平的改变。结果:测序证实突变成功,GFP抗体免疫印迹证实了突变体的蛋白表达。荧光显微镜下观察到TRF1S219A-GFP和TRF1S219D-GFP均呈点状定位于细胞端粒。流式细胞仪结果显示,过表达野生型和非磷酸化突变体的H eLa细胞产生G 2/M期的阻滞(P<0.05)。转染野生型和突变体后H eLa细胞中ATM蛋白含量均比对照组增高。结论:ATM激酶对TRF1丝氨酸219位的磷酸化可以抑制由于TRF1过量表达而引起的细胞周期G 2/M期的阻滞。
Objective： To investigate the role of Ser 219 phosphorylation of TRF1 （telomere repeat binding factor 1 ） in regulation of cell cycle. Methods： The mimicking phosphorylation mutant （TRF1^S219D-GFP） and the non-phosphorylatable mutant （TRF1^S219A-GFP） were constructed; the mutant genes and corresponding proteins were checked by sequencing and Western blot,respectively. Immunofluorescence staining was performed to detect the localization of mutants in HeLa cells. Cell cycle was analyzed by flow cytometry and ATM level was evaluated by immunoblotting. Results： The mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting. TRF1^S219A-GFP and TRF1^S219D-GFP were both localized in telomere of HeLa cells. Moreover,overexpression of TRF1- GFP or TRF1^S219A-GFP resulted in an accumulation of HeLa cells in G2/M （P（0. 05）. The protein level of ATM was increased when overexpression the wide type or mutants. Conclusion： The Ser 219 phosphorylation of TRF1 by ATM could result in cell cycle arrest in G2/M,which is related to overexpression of TRF1.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2007年第4期325-330,共6页
Journal of Zhejiang University(Medical Sciences)
基金
国家重点基础研究发展计划(973计划)资助项目(2002CB713700)
国家自然科学基金资助项目(39870339)
关键词
端粒
端粒结合蛋白类
端粒结合因子-1
ATM
磷酸化
细胞周期
Telomere
Telomere-binding proteins
Telomere repeat binding faetor 1
ATM
Phosphorylation
Cell eyele
