Characterization and functional analysis of two PKR genes in fugu (Takifugu rubripes)

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.