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 (elF2 alpha), and activates NF kappa B by inducing NF kappa B-inducing kinase and I kappa B (inhibitor of NF kappa B) 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 (Polyl:C) or lipopolysaccharides (LPS). The fPKRs were highly expressed in the skin and fPKR2 was significantly induced in PBLs by Polyl: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 NF kappa B 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. (C) 2011 Elsevier Ltd. All rights reserved.