Chen Gang, Ma Cuiling, Bower Kimberly A, Ke Zunji, Luo Jia
Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown, West Virginia 26506, USA.
J Biol Chem. 2006 Jun 9;281(23):15909-15. doi: 10.1074/jbc.M600612200. Epub 2006 Mar 29.
Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.
乙醇暴露会抑制发育中的中枢神经系统中的蛋白质合成并导致细胞死亡。双链RNA（dsRNA）激活的蛋白激酶（PKR）是一种丝氨酸/苏氨酸蛋白激酶，在翻译调控和细胞存活中发挥重要作用。PKR因其抗病毒反应而广为人知。在被病毒感染或dsRNA激活后，PKR会使其底物——真核翻译起始因子2（eIF2α）的α亚基磷酸化，从而导致翻译起始受到抑制。最近有研究表明，在没有病毒或dsRNA的情况下，PKR可通过与它的蛋白激活剂PACT或其小鼠同源物RAX直接相互作用而被激活。我们已经证明，暴露于乙醇会增加发育中小脑内PKR和eIF2α的磷酸化。乙醇对PKR/eIF2α磷酸化的影响与培养的神经元细胞中PACT/RAX的表达呈正相关。使用PKR抑制剂和PKR基因敲除小鼠成纤维细胞，我们证实乙醇诱导的eIF2α磷酸化是由PKR介导的。野生型RAX的过表达显著增强了对乙醇诱导的PKR/eIF2α磷酸化、翻译抑制和细胞死亡的敏感性。相比之下，突变型（S18A）RAX的过表达则抑制了乙醇介导的PKR/eIF2α激活。乙醇促进了表达野生型RAX的细胞中PKR与RAX的结合，但在表达S18A RAX的细胞中则没有。S18A RAX起到了显性负性蛋白的作用，并阻断了乙醇诱导的蛋白质合成抑制和细胞死亡。我们的结果表明，PKR与PACT/RAX之间的相互作用调节了乙醇对中枢神经系统中蛋白质合成和细胞存活的影响。
