Gale M, Blakely C M, Hopkins D A, Melville M W, Wambach M, Romano P R, Katze M G
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195-7242, USA.
Mol Cell Biol. 1998 Feb;18(2):859-71. doi: 10.1128/MCB.18.2.859.
The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor. P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2alpha subunit (eIF-2alpha) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein. This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58IPK repressed PKR-mediated eIF-2alpha phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2alpha phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function. Furthermore, P52rIPK inhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58IPK is inhibited through a direct interaction with P52rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.
细胞对环境信号的反应很大程度上依赖于诱导反应性蛋白激酶信号通路。在这些通路中，不同的蛋白质 - 蛋白质相互作用通过调节激酶功能在决定反应的特异性方面发挥作用。干扰素诱导的丝氨酸/苏氨酸蛋白激酶PKR在响应各种环境刺激时被激活。与许多蛋白激酶一样，PKR通过与激活剂和抑制分子的直接相互作用来调节，包括细胞内PKR抑制剂P58IPK。P58IPK通过直接相互作用抑制PKR介导的真核起始因子2α亚基（eIF - 2α）的磷酸化，从而解除PKR对mRNA翻译和细胞生长的抑制。为了进一步确定PKR调节的分子机制，我们利用相互作用克隆策略鉴定了一个编码与P58IPK相互作用蛋白的新cDNA。这种蛋白命名为P52rIPK，与Hsp90的带电结构域具有有限的同源性，并在多种细胞系中表达。P52rIPK和P58IPK在酵母双杂交试验中相互作用，并从哺乳动物细胞提取物中作为复合物回收。当与PKR在酵母中共表达时，P58IPK抑制PKR介导的eIF - 2α磷酸化，抑制与PKR功能相关的正常毒性和生长抑制作用。相反，将P52rIPK引入这些菌株导致PKR活性和eIF - 2α磷酸化恢复，同时由于抑制P58IPK功能而导致生长抑制。此外，在重组的体外PKR调节试验中，P52rIPK抑制P58IPK功能。我们的结果表明，P58IPK通过与P52rIPK的直接相互作用而被抑制，这反过来又导致PKR活性上调。综上所述，我们的数据描述了一种新型的蛋白激酶调节系统，该系统包括干扰素、应激和生长调节通路的交叉点。
