红细胞蛋白激酶C活性变化对锚蛋白的影响

Effects of Protein Kinase C on Ankyrin in Erythrocytes

目的本研究探讨红细胞蛋白激酶C(PKC)活性变化对锚蛋白的影响。方法采用γ-P32标记和免疫沉淀法检测锚蛋白磷酸化情况;采用间接免疫荧光标记方法观察PKC和锚蛋白的细胞内分布情况;利用红细胞变形仪测定红细胞的变形能力;测量红细胞衍射斑的长短轴之比表示红细胞变形指数。结果PKC激活剂佛波酯(PMA)处理实验组红细胞1min、5min、10min、30min、1h、2h,PMA处理后即刻发生锚蛋白的磷酸化、PKC和锚蛋白细胞内同步移位及共分布现象,磷酸化高峰值和发生这种分布改变的红细胞百分率于30min达最高。PMA处理的红细胞在不同切应力(50,100,200,300N/m2)下,其变形指数都于30min达最大;不同切应力下的变形指数都与发生PKC和锚蛋白细胞内同步移位的细胞百分率显著负相关。结论红细胞PKC活化后可以对锚蛋白进行磷酸化,并引起锚蛋白和PKC发生细胞内同步移位和共分布,这可能是造成红细胞变形性发生改变的新机制。

Objective To study the effects of protein kinase C （PKC）on ankyrin in erythrocytes. Methods Phosphorylation of ankyrin was detected by γ-P^32 marking and immunoprecipitation, and cellular distribution of PKC and ankyrin was observed by indirect immunofluorescence. Erythrocyte deformability was measured by erythrocyte deformation meter,and erythrocyte deformation index（DI ） was calculated by the ratio between long axis and short axis of erythrocyte diffractive spot. Results Erythrocytes in test group were treated with Phorbol-12-myrisrate- 13-acetate （PMA）, a PKC activator, for 1 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour and 2 hours, respectively. The phosphorylafion of ankyrin, synchronous migration and co-localization of PKC and ankyrin occurred instantly after PMA treatment and reached to the high peak at 30 min. DI increased to the highest value at 30 min under various shear suesses （50,100,200,300 N/m^2）. Moreover,DI was negatively correlative significantly with the pereentage of cellular migration of PKC and ankyrin in erythrocytes under various shear stresses. Conclusion PKC can phosphorylate ankyrin and therefore lead to the cellular migration and co-localization of ankyrin along with PKC after PKA activation, which may be the new mechanism of erythrocyte deformation decrease.