title: Characterization and functional analysis of two PKR genes in fugu (Takifugu rubripes).
abstract: PKR (protein kinase R) is a serine-threonine kinase that inhibits protein synthesis by the phosphorylation of the eukaryotic translation initiation factor 2-alpha (eIF2α), and activates NFκB by inducing NFκB-inducing kinase and IκB (inhibitor of NFκB) kinase. This can lead to antiviral and anti-proliferative effects. In this study, the complete sequence and organization of two fugu PKR genes (fPKRs) were determined by in silico analysis and conventional PCR. The full-length fPKR1 and fPKR2 genes were 3832 bp and 4325 bp, which encoded 523 and 492 amino acids, respectively. Both encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain, and showed very high similarity to green spotted puffer PKRs. Gene expression of the two fPKRs was measured by quantitative real-time PCR on tissue samples from healthy fish and peripheral blood leukocytes stimulated with polyinosinic:polycytidylic acid (PolyI:C) or lipopolysaccharides (LPS). The fPKRs were highly expressed in the skin and fPKR2 was significantly induced in PBLs by PolyI:C but not by LPS. The fPKRs inhibited translation of a luciferase reporter gene in a dose-dependent manner and induced transcriptional activity of a mammalian NFκB luciferase reporter. These results demonstrate that two PKRs in a single species can both be independently, but not equally, functional and support the hypothesis that fish PKRs have roles in the innate immune response similar to those of mammalian PKRs.
authors: Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, South Korea.
authors: Carmelo S
authors: CS
authors: del Castillo
authors: Jun-ichi
authors: J
authors: Hikima
authors: Maki
authors: M
authors: Ohtani
authors: Tae-Sung
authors: TS
authors: Jung
authors: Takashi
authors: T
authors: Aoki
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D023061:Gene Order; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010802:Phylogeny; D011070:Poly I-C / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D023741:Takifugu / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000276:Adjuvants, Immunologic; D008070:Lipopolysaccharides; D016328:NF-kappa B; D008156:Luciferases; D011494:Protein Kinases; D011070:Poly I-C