Plk1330／597位丝氨酸磷酸化突变体对胞质分裂的影响

Effect of Plk1 330/597 serine phosphorylated mutant on cytokinesis during mitosis

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。

AIM： To explore the locations of pseudo- phosphorylated mutant （ Plk1^S330/597D） and non-phosphorylat- able mutant （Plk1^S330/597A） of Plkl （residues 330 and 597 serine） in HeLa cells and the effect on mitosis. METHODS： We transfected wide-type Plkl-GFP, Plkl^S330/597D-GFP and PIk^S330/597A-GFP into HeLa cells, respectively, and detected the locations of wide type and mutants in HeLa cells using immunofluorescence staining and the protein expressions of Plk1^S330/597D-GFP and Plk1^S330/597A-GFP with immunoblotting. HeLa cells transfected with Plki mutants were observed and analyzed in mitotic phase for cell count and cell cycle by flow cytometry. RESULTS： The protein expressions of Plkl mutants were verified. Immunofluorescence microscopy revealed that mutagenesis of these phosphorylation sites did not affect targeting of Plkl to kinetochore and midbody dur- ing mitosis. However, after transfection of Plk1^S330/597A-GFP, mitosis was inhibited and arrested at cytokinesis compared to control cells. Flow cytometry showed that Plk1^S330/597A- GFP expression led to an increased G2/M arrest （P 〈 0.01）. CONCLUSION： Non-phosphorylatable mutant of PIk] （PIk1^S330/597A） induces cytokinesis failure and causes the cell cycle arrest at G2/M phase.