目的 探讨蛋白激酶p38, 蛋白激酶C对几种哺乳动物细胞中水通道蛋白9磷酸化的调控作用,以及这些蛋白因子对细胞中砷摄入含量的影响。方法 采用免疫印迹法和免疫共沉淀技术分别检测细胞株中p38,蛋白激酶C和水通道蛋白9表达水平及其磷酸化水平。电感藕合等离子体质谱法(ICP-MS)测定细胞内砷含量。结果 在ECV-304,AsRE,HepG2和L-02细胞株中,p38蛋白及其磷酸化表达水平在砷的刺激下随时间增加而有显著上升,相同条件下蛋白激酶C末见明显变化。当抑制细胞中p38活性,除了L-02细胞中水通道蛋白9的磷酸化水平受到抑制外,其余细胞中水通道蛋白9的蛋白含量及磷酸化水平都未见明显变化。砷含量也只在L-02细胞中看到有较显著的下降。结论 水通道蛋白9磷酸化水平影响砷进入细胞的速度与含量,但不同的细胞中水通道蛋白9的磷酸化调控机制有所差异。p38蛋白激酶在正常肝细胞L-02中显示对水通道蛋白9磷酸化有一定的调控作用,蛋白激酶C则在本实验条件下未见入调控砷的入胞机制。
Abstract
OBJECTIVE To elucidate the regulation of p38 and PKC for water channel protein AQP9 and the effect on As intake in four kinds of mammal cells, and to investigate the regulation mechanism of AQP9 phosphorylation. METHODS p38, PKC protein and phosphorylation levels were analyzed by Western blotting, and phosphorylation level of AQP9 was detected by immuno-precipitation. Intracellular arsenic content was determined by inductively coupled plasma mass spectrometry (ICP-MS). Experimental data were analysed by SPSS statistical software. RESULTS p38 protein and phosphorylation levels increased significantly with time in four kinds of cells treated with NaAsO2, while no significant change had been observed in PKC protein and phosphorylation levels. AQP9 phosphorylation level was inhibited in L-02 cells when p38 activity was inhibited,and As accumulation also decreased significantly in L-02 cells. CONCLUSION AQP9 phosphorylation has effect on As intake and rate. But the regulated mechanism may be different in different cells. p38 kinase attends partly AQP9 phosphorylation in liver normal cells L-02, PKC has no effect on AQP9 phosphorylation in all kinds cells used in this paper.
关键词
AQP9 /
磷酸化 /
p38 /
PKC /
砷
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Key words
AQP9 /
phosphorylation /
p38 /
PKC /
As
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中图分类号：
R965
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脚注
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基金
国家自然科学基金资助项目(81001227,30860241);浙江省重点创新团队项目(2011R50021);浙江省大学生科技创新活动计划(新苗人才计划)(2013R421040)
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