Gunnery S, Mathews M B
Department of Biochemistry and Molecular Biology, New Jersey Medical School, UMDNJ, 185 South Orange Avenue, University Heights, Newark, New Jersey, 07103, USA.
Methods. 1998 Jul;15(3):189-98. doi: 10.1006/meth.1998.0623.
PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR.
PKR是一种依赖RNA的蛋白激酶，在哺乳动物细胞中通过干扰素处理诱导产生。它以潜伏或无活性形式存在于哺乳动物细胞中，并被极低浓度的双链（ds）RNA激活。活化的PKR使eIF2（蛋白质合成的必需起始因子）以及其他底物磷酸化，这些底物包括组蛋白IIA、来自兔网织红细胞的一种90 kDa蛋白质、转录因子NF-κB的抑制剂IkappaB以及HIV-1 Tat蛋白。PKR与多种细胞和病毒产物相互作用，这些相互作用调节其被dsRNA激活的过程。在此，我们描述用于研究RNA调节剂对PKR激活或抑制作用的方法。具体而言，我们详细介绍（1）从经干扰素处理的哺乳动物细胞中纯化PKR，（2）使用纯化的酶或粗细胞裂解物在体外进行PKR激活和抑制的功能测定，以及（3）用于评估dsRNA和单链RNA与PKR结合的测定。
