Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2alpha and its response to endoplasmic reticulum stress and IPNV infection.
The cDNAs of rainbow trout and zebrafish eIF2alpha have been isolated and found to encode proteins of similar molecular weight and isoelectric point to the alpha-subunit of the human translational initiation factor, eIF2. The rainbow trout (36.0kDa) and zebrafish (36.2kDa) eIF2alphas 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 alpha-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 eIF2alphas can be phosphorylated in vitro by the mammalian heme-sensitive eIF2alpha-kinase, HRI/HCR, as well as the interferon-inducible, dsRNA sensitive kinase, PKR. Phosphorylation of rainbow trout and zebrafish eIF2alpha 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 eIF2alpha 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 eIF2alpha phosphorylation. These data imply that RTG-2 cells express the interferon-induced eIF2alpha-kinase, PKR and suggests that the interferon/eIF2alpha/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 eIF2alpha.
Garner
Joseph N
JN
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA.
Joshi
Bhavesh
B
Jagus
Rosemary
R
D000595:Amino Acid Sequence; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018061:Infectious pancreatic necrosis virus / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000276:immunology* / Q000821:virology*; D010766:Phosphorylation; D011070:Poly I-C / Q000494:pharmacology; D013312:Stress, Physiological / Q000378:metabolism; D015027:Zebrafish / Q000276:immunology* / Q000821:virology*; D019892:eIF-2 Kinase / Q000378:metabolism
D015852:Eukaryotic Initiation Factor-2; D000001:Calcimycin; D019892:eIF-2 Kinase; D011070:Poly I-C