title: Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain.
abstract: The interferon-induced, dsRNA-activated human protein kinase (PKR) exerts antiviral and antiproliferative effects through inhibition of protein synthesis. Studies of structure-function relationships in PKR have shown that two dsRNA binding motifs are important for its autophosphorylation and activation by dsRNA in vitro. To correlate these findings with the activity of PKR in vivo, we examined the function of various PKR deletion mutants in cultured cells by using an inducible expression system. In a reporter gene assay, mutant forms of the kinase lacking amino acids 1-97 (delta 1-97) and 104-157 (delta 104-157), which are required for dsRNA binding in vitro, retained full activity in vivo. Deletion of amino acids 233-271 (delta 233-271), however, abolished the translational inhibitory activity of the kinase and prevented its phosphorylation. Moreover, cells infected with vaccinia virus recombinants expressing wild-type PKR, the mutant delta 104-157, delta 186-222), developed almost complete inhibition of both viral and cellular protein synthesis was upon induction of PKR. This inhibition of viral protein synthesis was not observed in cells infected with a recombinant expressing delta 233-271 mutant PKR. Our findings establish that the region encompassing amino acids 233-271 of PKR is critical for kinase activity in vivo, whereas its dsRNA binding domain is dispensable.
authors.affiliation: Department of Microbiology and Immunology, State University of New York Health Science Center, Brooklyn 11203.
authors.forename: S B
authors.initials: SB
authors.lastname: Lee
authors.forename: S R
authors.initials: SR
authors.lastname: Green
authors.forename: M B
authors.initials: MB
authors.lastname: Mathews
authors.forename: M
authors.initials: M
authors.lastname: Esteban
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D019892:eIF-2 Kinase
chemical_list: D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase