The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR)
The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR)
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DOI：
10.1042/bj20020204
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发表时间：
2002-08-15
影响因子：
4.1
通讯作者：
Patel, RC
中科院分区：
文献类型：
--
作者：
Huang, X;Hutchins, B;Patel, RC
双链RNA依赖性蛋白激酶（PKR）是干扰素抗病毒和抗增殖作用的关键介质之一，PKR活性还参与细胞增殖、凋亡和信号转导的调节。我们最近已经确定PACT，PKR的一种新的蛋白激活剂，作为一个重要的调节剂PKR活性在细胞中的病毒感染的情况下。PACT与PKR异源二聚化并通过直接的蛋白质-蛋白质相互作用激活PKR。内源性PACT作为PKR的激活剂响应于不同的应激信号，如血清饥饿和过氧化物或亚砷酸盐处理，因此是一种新的，应激调节的PKR生理激活剂。在这项研究中，我们的特点是PKR激活所需的PACT的功能域。我们的研究结果表明，与N端保守结构域1和2不同，PACT的第三个保守结构域由于其与双链RNA的结合以及与PKR的相互作用而被破坏。然而，结构域3的缺失导致PKR活化能力的丧失，尽管与PKR的正常相互作用，从而表明结构域3在PKR活化中起重要作用。纯化的重组结构域3在体外也能有效激活PKR。我们的研究结果表明，虽然PACT的高亲和力与PKR的相互作用，第三个基序是必不可少的PKR激活。此外，结构域3和真核起始因子2，X都通过PKR内的相同区域与PKR相互作用，我们已经将其定位于氨基酸残基318和551之间。
One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR), PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein-protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and interaction with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that, although dispensable for PACT's high-affinity interaction with PKR, the third motif is essential for PKR activation. In addition, domain 3 and eukaryotic initiation factor 2,X both interact with PKR through the same region within PKR, which we have mapped to lie between amino acid residues 318 and 551.