Barber G N, Tomita J, Garfinkel M S, Meurs E, Hovanessian A, Katze M G
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Virology. 1992 Dec;191(2):670-9. doi: 10.1016/0042-6822(92)90242-h.
The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.
P68蛋白激酶（因其在人细胞中的相对分子质量为68,000而被称为P68）是一种丝氨酸/苏氨酸激酶，由干扰素处理诱导并被双链RNA激活。该激酶在病毒感染的细胞中受到严格调控，因为一旦被激活，它就会磷酸化其天然底物真核生物起始因子2（elF-2），导致功能性elF-2受到潜在限制，并降低蛋白质合成起始。为了进一步阐明激酶调控的分子机制，我们尝试使用杆状病毒载体在昆虫细胞中表达P68蛋白激酶。多次尝试分离含有野生型激酶的重组杆状病毒均失败，而表达具有催化结构域II突变的无功能激酶的重组体则很容易分离出来。当用于感染草地贪夜蛾细胞时，重组病毒表达的外源突变蛋白占合成总蛋白的近5%-10%。然后，我们通过免疫亲和层析纯化该激酶，以制备单特异性抗血清，通过免疫印迹和免疫沉淀分析确定，该抗血清不仅能识别人类天然野生型P68，还能识别小鼠、牛和猴细胞中的激酶同源物。幸运的是，由于免疫沉淀中存在的人和非人类激酶同源物会发生自身磷酸化并磷酸化天然底物elF-2α，因此也可以使用该抗体检测激酶功能。此外，与现有的单克隆抗体不同，该抗血清识别整个分子中的表位，包括氨基和羧基末端。使用多克隆抗体进行的体外试验揭示了氨基末端，尤其是氨基酸1-97在激酶与病毒RNA激活剂和抑制剂结合中的重要性。最后，我们确定P68氨基末端对于结合双链RNA既是必要的也是充分的，因为我们能够将双链RNA结合特性转移到先前无法结合RNA的报告基因产物上。
