Expression of unphosphorylated form of human double-stranded RNA-activated protein kinase in Escherichia coli.
Interferon (IFN)-inducible, double-stranded (dsRNA)-activated protein kinase (PKR) is a key mediator of the antiviral and antiproliferative effects of IFN. PKR is present within cells in a latent state. In response to binding dsRNA, the enzyme becomes activated, causing autophosphorylation and an increase in specific kinase activity. In order to study PKR and its inhibitors, a large amount of the enzyme in its latent, unphosphorylated state is required. When PKR is fused to glutathione S-transferase (GST-PKR) and the fusion protein is expressed in Escherichia coli, the PKR obtained is fully activated by autophosphorylation. Therefore, we have developed an expression plasmid in which both GST-PKR and bacteriophage lambda protein phosphatase (lambda-PPase) genes were placed downstream of a T7 promoter. After induction of expression, unphosphorylated GST-PKR was obtained in good yield, and purified to near homogeneity. The purified enzyme has dsRNA-dependent activation and phosphorylates the translation initiation factor eIF2 alpha. Using the recombinant protein, we analyzed the inhibition mechanisms of two viral inhibitors, vaccinia virus K3L protein and adenovirus virus-associated RNA I (VAI RNA). K3L inhibited both autophosphorylation of PKR and phosphorylation of eIF2 alpha, whereas VAI RNA inhibited only autophosphorylation. The separation of autophosphorylation and catalytic activity shows that the recombinant PKR is useful in analyzing the functions of PKR, its inhibitors, and its regulatory molecules. The coexpression system of protein kinase with lambda-PPase described here will be applicable to obtaining unphosphorylated and unactivated forms of other protein kinases.
Matsui
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Department of Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.
Tanihara
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Date
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D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D012367:RNA, Viral / Q000494:pharmacology; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000494:pharmacology; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C052835:adenovirus associated RNA; C081646:pK3 protein, Vaccinia virus; D005982:Glutathione Transferase; D019892:eIF-2 Kinase; C516868:PTPRU protein, human; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2