ATP缺失对大鼠近端肾小管上皮细胞中肌动蛋白磷酸化水平的影响

ATP depletion induced actin phosphorylation in rat renal proximal tubule cells

目的:研究ATP缺失时大鼠近端肾小管上皮细胞(NRK52E)肌动蛋白磷酸化水平的改变。方法:建立细胞的体外ATP缺失模型,用免疫印迹技术检测细胞内骨架组分及胞浆组分中肌动蛋白的分布变化;用二维电泳法分离并比较细胞内磷酸化和非磷酸化的肌动蛋白含量的改变;用免疫共沉淀法及免疫印迹技术检测ATP缺失后细胞内肌动蛋白酪氨酸、苏氨酸及丝氨酸磷酸化水平变化。结果:ATP缺失处理后细胞内F-肌动蛋白含量逐渐增加,G-肌动蛋白含量逐渐减少;ATP缺失细胞内磷酸化肌动蛋白量明显增加,肌动蛋白的磷酸化程度随ATP缺失时间延长而增加;ATP缺失处理后肌动蛋白酪氨酸磷酸化水平逐渐升高,且与ATP缺失程度呈相一致;肌动蛋白苏氨酸磷酸化水平无明显变化。结论:ATP缺失后近端肾小管上皮细胞肌动蛋白出现聚合过程,可能与细胞中肌动蛋白磷酸化酪氨酸水平增加有关。

Objective：To investigate the effect of cellular ATP depletion on actin phosphorylation in rat renal proximal tubule cells （NRK52E）. Methods：NRK52E cells were treated with 0.1 μmol / L Antimycin A to established a cellular ATP depletion model. The contents of G-actin （in cytosolic fraction）and F-actin （in cytoskeletal fraction）in cells were evaluated by Western blot. Two-dimensional electrophoresis was used to separate phosphorylated actin from unphosphorylated actin of cells. With immunoprecipitation （IP）and Western blot analysis,residues of actin phosphorylated was explored by using monoclonal anti-phosphoserine,anti-phosphotyrosine and anti-phosphothreonine antibody. Results：The cellular F-actin was increased and G-actin was decreased after ATP depletion. The proportion of phosphorylated actin after ATP depletion was higher than that of control cells. As compared to the control cells,the level of actin phosphorylation on thyrosine residue （s） was significantly increased during cellular ATP depletion,and these changes were paralleled with the severity of cellular ATP depletion. Conclusion：The thyrosine phosphorylation of actin of NRK52E cells may play an important role in regulation of polymerization of actin during ATP depletion.