论文部分内容阅读
目的:研究Plk的330/597位丝氨酸的模拟磷酸化突变体Plk1S330/597D和不能磷酸化突变体Plk1S330/597A的绿色荧光融合蛋白在HeLa细胞中的定位以及对有丝分裂的影响。方法:将野生型Plk1-GFP质粒、模拟磷酸化型突变体Plk1S330/597D-GFP质粒和不能磷酸化突变体Plk1S330/597A-GFP质粒分别转染HeLa细胞株,免疫荧光法检测突变体融合蛋白Plk1S330/597D-GFP和Plk1S330/597A-GFP的细胞定位的定位,West-ern blot法检测上述突变体融合蛋白的表达。通过有丝分裂期的细胞计数,流式细胞仪检测细胞周期,观察转染突变体后HeLa细胞的有丝分裂进程。结果:模拟磷酸化突变体Plk1S330/597D和不能磷酸化突变体Plk1S330/597A的融合蛋白能正确表达。突变体在细胞有丝分裂过程中均能正确定位于动点和中体,但不能磷酸化突变体Plk1S330/597A融合蛋白对HeLa细胞的有丝分裂有明显的抑制作用,使胞质分裂期的细胞数量较对照组明显增多,产生G2/M期阻滞(P<0.01),并造成染色体分离滞后的现象。结论:Plk1激酶自身的磷酸化状态对其有丝分裂期功能具有重要作用,其330和597位丝氨酸去磷酸化能抑制细胞的有丝分裂,使细胞周期发生G2/M期阻滞。
Aims: To investigate the localization of Plk1S330 / 597D, a phosphorylated mutant of Plk serine 330/597 serine, and the green fluorescent fusion protein Plk1S330 / 597A, which can not phosphorylate Plk1S330 / 597A, in HeLa cells and its effect on mitosis. Methods: HeLa cell lines were transfected with wild-type Plk1-GFP plasmid, Plk1S330 / 597D-GFP mutant plasmid and Plk1S330 / 597A-GFP without phosphorylation mutant respectively. The immunofluorescence assay was used to detect the expression of the mutant fusion protein Plk1S330 / 597D-GFP and Plk1S330 / 597A-GFP, West blotting was used to detect the expression of the mutant fusion protein. Cell cycle was detected by flow cytometry and the mitotic progression of HeLa cells was observed by transfection of mitotic cells. RESULTS: The fusion protein of Plk1S330 / 597D, a phosphorylation mutant, and Plk1S330 / 597A, a phosphorylation mutant, was correctly expressed. Mutants can be correctly located in both the moving point and the middle body during cell mitosis. However, the phosphorylation mutant Plk1S330 / 597A fusion protein significantly inhibited the mitosis of HeLa cells, resulting in more cytokinesis than the control Group significantly increased, resulting in G2 / M phase arrest (P <0.01), and cause chromosome lagging phenomenon. Conclusion: The phosphorylation state of Plk1 kinase plays an important role in its mitotic function. The dephosphorylation of serine 330 and 597 can inhibit the mitosis of cells and block the cell cycle in G2 / M phase.