Functional characterization of pkr gene products expressed in cells from mice with a targeted deletion of the N terminus or C terminus domain of PKR
Functional characterization of pkr gene products expressed in cells from mice with a targeted deletion of the N terminus or C terminus domain of PKR
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DOI：
10.1074/jbc.m203564200
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发表时间：
2002-10-11
影响因子：
4.8
通讯作者：
Koromilas, AE
中科院分区：
文献类型：
--
作者：
Baltzis, D;Lit, SY;Koromilas, AE
干扰素诱导的双链RNA（dsRNA）激活的蛋白激酶PKR通过磷酸化真核起始因子2的α亚基在信使（m）RNA翻译中起重要作用。通过这种能力，PKR被认为是干扰素的抗病毒和抗增殖作用的介质。除了翻译功能之外，PKR还通过调节许多转录因子（包括NF-κ B和STAT）的活性而参与基因转录的许多信号传导途径。然而，用两种不同的PKR敲除（PKR-/-）小鼠模型进行的实验未能验证归因于PKR的许多生物学功能。此外，来自两只PKR-/-小鼠的细胞的结果是矛盾和混乱的。在这里，我们表明，第一个PKR-/-小鼠与外显子2和3的缺失，对应于PKR的N端结构域（N-PYR-/-），表达截短的蛋白质，从外显子跳过小鼠PKR（ES-mPKR）mRNA的翻译。ES-mPKR蛋白在dsRNA结合方面有缺陷，但在体外和体内都保持催化活性。此外，我们表明，第二个PKR-/-小鼠与外显子12的靶向缺失，这对应于分子的C末端（C-PKR-/-），表达截短的mPKR产生的外显子12的选择性剪接。虽然mPKR的剪接形式（SF-mPKR）是无催化活性的，但它保留了野生型mPKR的dsRNA结合特性。反转录-PCR表明，SF-mPKR mRNA表达在几个正常小鼠组织，并出现在胚胎发育过程中的发育控制。我们的数据表明，两种PKR-/-模型都是不完全敲除，并且PKR变体的表达可以至少部分地解释来自两种PKR-/-小鼠的细胞之间的显著信号传导差异。
The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays an important role in messenger (m) RNA translation by phosphorylating the a subunit of eukaryotic initiation factor 2. Through this capacity PKR is thought to be a mediator of the antiviral and antiproliferative actions of interferon. In addition to translational function, PKR has been implicated in many signaling pathways to gene transcription by modulating the activities of a number of transcription factors, including NF-kappaB and STATs. However, experiments with two different PKR knockout (PKR-/-) mouse models have failed to verify many of the biological functions attributed to PKR. In addition, results with cells from the two PKR-/- mice have been contradictory and confusing. Here, we show that the first PKR-/- mouse with deletion of exons 2 and 3, corresponding to the N terminus domain of PKR (N-PYR-/-), expresses a truncated protein, resulting from the translation of the exon-skipped mouse PKR (ES-mPKR) mRNA. The ES-mPKR protein is defective in dsRNA binding but remains catalytically active both in vitro and in vivo. Furthermore, we show that the second PKR-/- mouse with a targeted deletion of exon 12, which corresponds to the C terminus of the molecule (C-PKR-/-), expresses a truncated mPKR produced by alternative splicing of exon 12. Although the spliced form of mPKR (SF-mPKR) is catalytically inactive, it retains the dsRNA-binding properties of the wild type mPKR. Reverse transcription-PCRs demonstrate that SF-mPKR mRNA is expressed in several normal mouse tissues, and appears to be under developmental control during embryogenesis. Our data demonstrate that both PKR-/- models are incomplete knockouts, and expression of the PKR variants may account, at least in part, for the significant signaling differences between cells from the two PKR-/- mice.