A PKR-like eukaryotic initiation factor 2α kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains

Abstract

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is induced as part of the IFN response in mammals and acts to shut down protein synthesis by the phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣). In fish, a PKR-like kinase activity has been detected, but the enzyme responsible has eluded characterization. Here, we describe a PKR-like kinase from zebrafish. Phylogenetic analysis shows that the C-terminal kinase domain is more closely related to the kinase domain of PKR than to any of the other three known eIF2␣ kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Z␣ domains. Z␣ domains specifically bind dsDNA and RNA in the left-handed Z conformation, often with high affinity. They have been found previously in two other IFN-inducible proteins, the dsRNA editing enzyme, ADAR1, and Z-DNA binding protein 1 (ZBP1), as well as in the poxvirus virulence factor, E3L. This previously undescribed kinase, designated PKZ (protein kinase containing Z-DNA binding domains), is transcribed constitutively at low levels and is highly induced after injection of poly(inosinic)-poly(cytidylic) acid, which simulates viral infection. Binding of Z-DNA by the Z␣ domain of PKZ was demonstrated by circular dichroism. PKZ inhibits translation in transfected cells; site-directed mutagenesis indicates that this inhibition depends on its catalytic activity. Identification of a gene combining Z␣ domains with a PKR-like kinase domain strengthens the hypothesis that the ability to bind left-handed nucleic acid plays a role in the host response to viruses.E3L ͉ interferon response ͉ viral infection ͉ Z-RNA ͉ Z␣ domain

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

“…Recently, cDNAs encoding PKZ have been isolated from crucian carp, zebrafish and Atlantic salmon [17,19,28]. Crucian carp PKZ is detected at a very low level of constitutive expression and can be induced by GCRV, poly(I:C) or interferon in CAB cells [28,29].…”

“…Zebrafish PKZ is transcribed constitutively at low levels and is highly induced after injection of poly(I:C). It also inhibits translation of proteins in transfected cells and plays a role in the host response to viruses [17]. Atlantic salmon PKZ expression is upregulated by interferon in Atlantic salmon TO cells and by poly(I:C) in head kidney.…”

“…Fish appear to have a PKR-like protein that has Z-DNA binding (Zalpha) domain(s) instead of dsRNA binding (DSRM) domain(s) in the regulatory domain, and has thus been designated Z-DNA binding protein kinase (PKZ) [17]. Zalpha domain(s) specifically bind dsDNA and dsRNA in the lefthanded Z conformation, often with high affinity [17,18].…”

“…Zalpha domain(s) specifically bind dsDNA and dsRNA in the lefthanded Z conformation, often with high affinity [17,18]. PKZ is able to phosphorylate eIF2a [19] and may play a role, like PKR, in host defense against virus infection [17,19].…”

Comparative analysis of vertebrateEIF2AK2(PKR) genes and assignment of the equine gene to ECA15q24-q25 and the bovine gene to BTA11q12-q15

“…These include chicken (Gallus gallus; GGA) [14], goldfish (Carassius auratus; CAU) [9] and zebrafish (Danio rerio; DRE) [22]. Amphibian EIF2AK2 gene sequences have not been described previously.…”