BAD与JNK协同调节UV诱导的细胞凋亡

JNK和BAD(bcl-2相关死亡启动子)都是参与细胞凋亡的重要调控蛋白.然而,二者在功能上的联系及其在细胞凋亡中的相互作用尚未见报导.本研究证明,BAD可作为JNK的磷酸化底物,与JNK相互作用,协同调节紫外线(UV)诱导的细胞凋亡.蛋白质印迹检测PARP(聚ADP核糖聚合酶)裂解,以及流式细胞术检测细胞凋亡结果揭示,UV诱导的MEF细胞凋亡依赖JNK的激酶活性.siRNA敲降BAD的蛋白表达,可增加MEF细胞对UV诱导的细胞凋亡的敏感性.UV处理的野生型MEF细胞抽提液(含JNK激酶活性)可催化GST-BAD底物发生磷酸化修饰,而UV未处理的细胞抽提液却不能.结果提示,UV激活的JNK活性可催化BAD磷酸化;体外合成的持续活化的JNK与GST-BAD体外共孵育结合质谱分析证明,JNK可催化BAD蛋白的Thr-201磷酸化.提示BAD是JNK的底物.此外,野生型和T201A突变的BAD质粒转染BAD-/-细胞结果显示,BAD的T201磷酸化可抑制JNK激酶活性及其底物c-Jun的磷酸化,提示BAD磷酸化对JNK具有负反馈调节作用.上述结果证明,BAD作为底物可被UV激活的JNK激酶磷酸化;磷酸化BAD反过来又可抑制JNK的激酶活性,负性调节细胞凋亡.综上所述,BAD与JNK能够相互影响,协同调控UV诱导的细胞凋亡.

BAD and JNK Regulates UV-induced Apoptosis

JNK and BAD (bcl-2 associated death promoter) are known to regulate apoptosis. We demonstrated that BAD could be a substrate of JNK and involved in the regulation of ultraviolet (UV)-induced apoptosis. Western blotting for PARP (poly ADP-ribose polymerase) cleavage and flow cytometry revealed that JNK was activated in UV-induced apoptosis in MEF cells. RNAi knock-down of BAD increased the sensitivity to apoptosis. The JNK in the lysates from apoptotic cells phosphorylated GST-BAD. By in vitro incubation of synthetic JNK with GST-BAD, the results showed that BAD phosphorylation occurred at Thr-201. Transfection of wild type or T201A mutant of BAD showed that the BAD phosphorylation inhibited the JNK kinase activity and the phophosrylation of c-Jun, suggesting a negative regulation to JNK activity. The data suggested that BAD might be a substrate of JNK to be phosphorylated upon UV exposure. Phosphrylated BAD suppresses the activity of JNK kinase to attenuate apoptotic cell death.