Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2α and its response to endoplasmic reticulum stress and IPNV infection

Abstract

The cDNAs of rainbow trout and zebrafish eIF2α have been isolated and found to encode proteins of similar molecular weight and isoelectric point to the α-subunit of the human translational initiation factor, eIF2. The rainbow trout (36.0 kDa) and zebrafish (36.2 kDa) eIF2αs share 93 and 91% identity to the human protein, respectively, and are recognized by antibodies raised to the human form. In mammals, the phosphorylation of the α-subunit of eIF2 plays a key role in the regulation of protein synthesis in response to a range of cellular stresses. Regions corresponding to the human phosphorylation and kinase-docking sites are identical in the proteins of both fish species, as are residues that interact with the eIF2 recycling factor, eIF2B. Moreover, both recombinant rainbow trout and zebrafish eIF2αs can be phosphorylated in vitro by the mammalian heme-sensitive eIF2α-kinase, HRI/HCR, as well as the interferon-inducible, dsRNA sensitive kinase, PKR. Phosphorylation of rainbow trout and zebrafish eIF2α can also occur in vivo. RTG-2 and ZFL cells subjected to endoplasmic reticulum (ER) stress by treatment with the Ca 2+-ionophore A23187 showed increased levels of eIF2α phosphorylation, suggesting similarity between the ER stress response in fish and other higher eukaryotes. Furthermore, RTG-2 cells responded to treatment with poly(I)·poly(C) or to infection by infectious pancreatic necrosis virus, IPNV, by increasing eIF2α phosphorylation. These data imply that RTG-2 cells express the interferon-induced eIF2α-kinase, PKR and suggests that the interferon/eIF2α/PKR response to virus infection may be a conserved vertebrate characteristic. Overall these data are consistent with the premise that fish are able to regulate protein synthesis in response to cellular stresses through phosphorylation of eIF2α.