Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells.
Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells.
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DOI：
10.1073/pnas.89.22.10837
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发表时间：
1992-11
影响因子：
11.1
通讯作者：
D. Thomis;C. E. Samuel
中科院分区：
文献类型：
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作者：
D. Thomis;C. E. Samuel
在转染的猴细胞和无细胞蛋白质合成系统中检测了人RNA依赖性P1/eIF-2 α蛋白激酶（PKR）的分子cDNA克隆（P1 KIN）的表达。将编码活性蛋白激酶的野生型（wt）P1 KIN cDNA的表达与不编码活性激酶的磷酸转移催化结构域II Lys-296->Arg（K296 R）突变体cDNA的表达进行比较。野生型和K296 R突变体P1 mRNA通过体外转录与T7 RNA聚合酶编程的P1核糖体相关蛋白的无细胞合成在兔网织红细胞系统中具有可比的效率。K296 R突变体P1蛋白也在转染的COS猴细胞中体内有效合成。然而，与K296 R突变体P1蛋白相比，在转染的COS细胞中，wt P1蛋白的合成减少了约30倍。野生型P1 KIN cDNA与K296 R突变型P1 KIN cDNA或呼肠孤病毒S4 cDNA共转染分别显著降低了K296 R突变型P1蛋白和呼肠孤病毒sigma 3蛋白的合成。虽然野生型和K296 R突变型P1 KIN质粒表达载体在COS细胞中复制效率相当，但与K296 R突变型P1 KIN cDNA相比，野生型转染的COS细胞中P1 mRNA的稳态量约为3倍。这些结果表明，RNA依赖性P1蛋白激酶的表达在体内的转染的哺乳动物细胞中主要在翻译水平上的一种机制，这可能是依赖于催化活性P1激酶的自我调节。
The expression of a molecular cDNA clone (P1 KIN) of the human RNA-dependent P1/eIF-2 alpha protein kinase (PKR) was examined in transfected monkey cells and in cell-free protein-synthesizing systems. Expression of the wild-type (wt) P1 KIN cDNA, which encodes an active protein kinase, was compared with that of the phosphotransfer catalytic domain II Lys-296-->Arg (K296R) mutant cDNA, which does not encode an active kinase. wt and K296R mutant P1 mRNAs prepared by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of P1 ribosome-associated protein with comparable efficiency in the rabbit reticulocyte system. The K296R mutant P1 protein was also efficiently synthesized in vivo in transfected COS monkey cells. However, synthesis of the wt P1 protein was reduced about 30-fold in transfected COS cells as compared with the K296R mutant P1 protein. Cotransfection of wt P1 KIN cDNA with either K296R mutant P1 KIN cDNA or reovirus S4 cDNA greatly reduced the synthesis of K296R mutant P1 protein and reovirus sigma 3 protein, respectively. Although the wt and K296R mutant P1 KIN plasmid expression vectors replicated with comparable efficiencies in COS cells, the steady-state amount of P1 mRNA was about 3-fold less in COS cells transfected with the wt as compared with the K296R mutant P1 KIN cDNA. These results suggest that RNA-dependent P1 protein kinase expression is autoregulated in vivo in transfected mammalian cells primarily at the level of translation by a mechanism that is likely dependent upon catalytically active P1 kinase.