Wang Shuang, Zheng Ying, Yu Yun, Xia Li, Chen Guo-qiang, Yang Yong-zong, Wang Li-shun
Department of Pathophysiology, Xiangya Medical College, Central South University, Changsha, China.
Acta Pharmacol Sin. 2008 Jan;29(1):135-42. doi: 10.1111/j.1745-7254.2008.00753.x.
This study was conducted to reveal new proteins involved in acute myeloid leukemia (AML) cell apoptosis.
Using camptothecin analog NSC606985- induced leukemic U937 cell apoptosis as a model, this study performed a differential proteomic analysis during apoptosis induction. The significantly modulated protein was underwent further investigation in the apoptotic process.
We found that beta-actin protein presented two different spots on the two-dimensional electrophoresis (2-DE) map, which shared similar molecular weight and different pI. Those two spots demonstrated contrary changes (disappeared on the basic-end and increased on the acid-end spot) during apoptosis induction, although the total level of beta-actin kept constant. This observation was further confirmed by immunoblot analysis on 2-DE gel. When NSC606985-treated cell lysate was incubated with alkaline phosphotase, beta-actin on the basic-end spot was restored, indicating increased phosphorylation of beta-actin during NSC606985- induced apoptosis. Moreover, the polymerization of actin also decreased after NSC606985 treatment. The increased beta-actin phosphorylation and decreased actin polymerization was antagonized by pre-treatment of rottlerin, a specific protein kinase C-delta (PKC delta) inhibitor.
All these results indicate that beta-actin was phosphorylated during apoptosis induction, which was mediated by activated PKC delta.
本研究旨在揭示参与急性髓系白血病（AML）细胞凋亡的新蛋白质。
以喜树碱类似物NSC606985诱导白血病U937细胞凋亡为模型，本研究在凋亡诱导过程中进行了差异蛋白质组学分析。对显著调节的蛋白质在凋亡过程中进行进一步研究。
我们发现β-肌动蛋白在二维电泳（2-DE）图谱上呈现出两个不同的斑点，它们具有相似的分子量和不同的等电点。在凋亡诱导过程中，这两个斑点呈现相反的变化（碱性端斑点消失而酸性端斑点增加），尽管β-肌动蛋白的总水平保持恒定。二维凝胶免疫印迹分析进一步证实了这一观察结果。当用碱性磷酸酶孵育NSC606985处理的细胞裂解物时，碱性端斑点上的β-肌动蛋白得以恢复，表明在NSC606985诱导凋亡过程中β-肌动蛋白的磷酸化增加。此外，NSC606985处理后肌动蛋白的聚合也减少。特异性蛋白激酶C-δ（PKCδ）抑制剂rottlerin预处理可拮抗β-肌动蛋白磷酸化增加和肌动蛋白聚合减少。
所有这些结果表明，β-肌动蛋白在凋亡诱导过程中被磷酸化，这是由活化的PKCδ介导的。
