Romano P R, Garcia-Barrio M T, Zhang X, Wang Q, Taylor D R, Zhang F, Herring C, Mathews M B, Qin J, Hinnebusch A G
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1998 Apr;18(4):2282-97. doi: 10.1128/MCB.18.4.2282.
The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.
人双链RNA依赖性蛋白激酶（PKR）是干扰素对病毒感染反应的重要组成部分。PKR的激活伴随着多个位点的自身磷酸化，包括N端调节区域中的一个位点（苏氨酸-258），该位点是完全激酶活性所必需的。几种蛋白激酶通过激酶亚结构域VII和VIII之间的区域（称为激活环）中的磷酸化而被激活。我们表明，PKR激活环中的苏氨酸-446和苏氨酸-451在体内和体外都是高水平激酶活性所必需的。将任一残基突变为丙氨酸会损害酵母细胞和COS1细胞中PKR的翻译控制，并导致小鼠肿瘤形成。这些突变还会损害PKR在体外的自身磷酸化和真核起始因子2亚基α（eIF2α）的磷酸化。虽然丙氨酸-446替代显著降低了PKR功能，但含有丙氨酸-451的突变激酶完全无活性。PKR在体外特异性地磷酸化合成肽中的苏氨酸-446和苏氨酸-451，对PKR磷酸肽的质谱分析证实苏氨酸-446是体内的一个自身磷酸化位点。当与丙氨酸-451突变结合时，PKR亚结构域X中的谷氨酸-490替代部分恢复了激酶活性。这一发现表明，先前针对丝裂原活化蛋白激酶描述的亚结构域X与激活环之间的相互作用是PKR中保守的调节特征。我们发现酵母eIF2α激酶GCN2在苏氨酸-882和苏氨酸-887处自身磷酸化，这两个位点位于激活环中，与PKR中的苏氨酸-446和苏氨酸-451处于完全相同的位置。对于GCN2激酶活性，苏氨酸-887比苏氨酸-882更关键，这与PKR中苏氨酸-446和苏氨酸-451的相对重要性相似。这些结果表明，GCN2和PKR在自身磷酸化的重要性以及激活环中保守的苏氨酸残基方面存在显著相似之处。
