JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells
JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells
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DOI：
10.1074/jbc.m510644200
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发表时间：
2006-07-28
影响因子：
4.8
通讯作者：
Song, Byoung-Joon
中科院分区：
文献类型：
--
作者：
Kim, Bong-Jo;Ryu, Seung-Wook;Song, Byoung-Joon
在用许多细胞死亡刺激剂处理后或在凋亡诱导条件下，在凋亡之前促凋亡Bax的线粒体易位被很好地建立。然而，Bax的线粒体易位的机制仍然未知。本研究的目的是探讨Bax激活和线粒体转位启动人肝癌HepG 2和猪肾LLC-PK 1细胞凋亡的机制。Bax的磷酸化JNK和p38激酶激活后处理星形孢菌素，H2 O2，依托泊苷，紫外线被证明了在二维凝胶上的Bax的pI值的移动，并证实了代谢标记与无机[P-32]磷酸在HepG 2细胞。JNK和p38激酶的特异性抑制剂可显著抑制Bax磷酸化和线粒体转位，并抑制HepG 2细胞凋亡。MAPKK 4（JNK和p38激酶的上游蛋白激酶）的特异性小干扰RNA显著降低MAPKK 4和MAPKK 3/6的水平，阻断JNK或p38激酶的激活，并抑制Bax磷酸化。然而，阴性对照小干扰RNA没有引起这些变化。各种Bax突变体的共聚焦显微镜显示星形孢菌素治疗前后Bax的线粒体易位率不同。在Bax突变体中，T167 D在星形孢菌素暴露后没有易位到线粒体，表明Thr（167）是潜在的磷酸化位点。总之，我们的研究结果表明，第一次，Bax是由应激激活的JNK和/或p38激酶磷酸化，Bax的磷酸化导致线粒体易位凋亡前。
Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H2O2, etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [P-32] phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr(167) is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.