Harper Nicholas, Hughes Michelle A, Farrow Stuart N, Cohen Gerald M, MacFarlane Marion
Medical Research Council Toxicology Unit, University of Leicester, Hodgkin Bldg., P. O. Box 138, Lancaster Rd., Leicester LE1 9HN, United Kingdom.
J Biol Chem. 2003 Nov 7;278(45):44338-47. doi: 10.1074/jbc.M307376200. Epub 2003 Aug 14.
We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.
我们进一步研究了佛波酯介导的蛋白激酶C（PKC）激活对肿瘤坏死因子（TNF）相关凋亡诱导配体（TRAIL）诱导的细胞毒性的保护机制。我们现在报告，PKC的激活靶向死亡受体信号复合物的形成。用12-O-十四酰佛波醇-13-乙酸酯（PMA）预处理可抑制TRAIL诱导的HeLa细胞凋亡，其特征是磷脂酰丝氨酸（PS）外化减少、半胱天冬酶-8加工减少以及半胱天冬酶-3不完全成熟和激活。PKC抑制剂双吲哚马来酰亚胺I（Bis I）完全消除了PMA的这些作用，明确表明PKC参与了PMA的保护作用。PMA可阻断TRAIL诱导的凋亡介质细胞色素c和Smac从线粒体释放。这与观察到的Bid裂解减少一起表明，PKC激活可调节线粒体上游TRAIL信号传导中的顶端事件。对TRAIL死亡诱导信号复合物形成的分析证实了这一点，在PMA处理的细胞中该复合物被破坏，这表现为Fas相关死亡结构域蛋白（FADD）募集显著减少，这种效应无法用FADD磷酸化状态的任何变化来解释。在体外结合试验中，TRAIL-R1和TRAIL-R2的细胞内结构域均与FADD结合：PKC的激活显著抑制了这种相互作用，表明PKC可能靶向死亡受体信号传导的关键顶端成分。值得注意的是，这种效应并不局限于TRAIL，因为天然TNF受体信号复合物的分离显示，PKC激活也抑制了TNF受体相关死亡结构域蛋白向TNF-R1的募集以及TNF诱导的IkappaB-α磷酸化。综上所述，这些结果表明，PKC激活特异性抑制关键的含死亡结构域衔接蛋白向其各自的膜相关信号复合物的募集，从而分别调节TRAIL诱导的凋亡和TNF诱导的NF-κB激活。
