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

  • ️Sat Jan 01 2005

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

Stefan Rothenburg et al. Proc Natl Acad Sci U S A. 2005.

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 2alpha (eIF2alpha). 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 eIF2alpha kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Zalpha domains. Zalpha 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 Zalpha 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 Zalpha 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.

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Figures

Fig. 1.
Fig. 1.

Proteins containing Zα domain. (A) Domain organization is shown schematically. Numbers to the right indicate the number of residues in human ADAR1, vaccinia virus E3L, human ZBP-1, zebrafish PKZ, and human PKR. Domains containing a Zα motif (Zα and Zβ) are shown in red. dsRNA binding domains are in green. The deaminase domain of ADAR1 (yellow) and the kinase domains of PKR and PKZ (light blue) are labeled. The function of the C-terminal region of ZBP-1 (gray) is unknown. (B) Comparison of Zα domains from PKZ of three fish species with the Zα domains of mouse ZBP-1, human ADAR1, vaccinia virus E3L, and yaba-like disease virus E3L. The crystal structures of the three domains, hZαADAR1, mZαZBP-1, and yabZαE3L, have been determined complexed with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in PKZ, are boxed in light blue. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA-contacting nor structural, but that match the consensus sequence, are highlighted in yellow. Sequences are bracketed by residue numbers relative to the translational start. Full genus, species, and accession numbers are listed in Table 1. For a more extensive comparison, see Fig. 7. (C) Alignment of the kinase domains from fish PKZ and selected mammalian PKRs. The kinase subdomains as defined by Hanks and Hunter (40) are marked with roman numerals. The position of the kinase inserts, specific for eIF2α kinases, and their sizes, are indicated between subdomains IV and V. A conserved lysine, which is essential for kinase activity, is boxed in red. The residues that are autophosphorylated in PKR and the analogous residues in PKZ are boxed in light blue. The highly conserved hydrophobic region is boxed in green. Other residues that match consensus are highlighted in yellow.

Fig. 2.
Fig. 2.

Expression pattern of PKZ 12 h after induction by poly(I:C). RT-PCR was performed on the indicated tissues, by using primers covering the complete ORF. The constitutively expressed receptor for activated C kinase 1 (RACK1) is shown as a loading control at the bottom. Expression patterns in tissues from untreated animals are in Fig. 9.

Fig. 3.
Fig. 3.

Identification of splice variants and genomic organization of PKZ. (A) The exon/intron structure is shown, superimposed on the known domains. Zα and Zβ are encoded by a single exon (E1). The largest part of kinase insert (KI) is encoded by exon 3. Intron sizes were determined by PCR and partial sequencing. The size of intron 3 could not be determined, and genomic organization should be seen as provisional. (B) The structures of splice variants A–D are depicted. In variants C and D, retained intron 2 provides a premature stop codon, which terminates the reading frame as indicated by the hatched bars. (C) The nucleotide and deduced amino acid sequences of variants A–D in the region of intron 2 are shown. The stop codon (TAA) at the beginning of retained intron 2 is marked by an asterisk in the amino acid sequence. The complete sequences of the splice variants, as well as several alleles, are shown in Fig. 10.

Fig. 4.
Fig. 4.

Binding of drZαPKZ to Z-DNA as shown by CD. The spectra of B-DNA (dotted line) and Z-DNA stabilized with 4 M NaCl (dashed line) are shown as controls. Cobalt hexamine was added to stabilize some of the DNA in the Z-form (dashed-dotted line). Protein was added to a final ratio of 1:2.5 (protein/base pair). The protein-bound DNA assumes the Z conformation (solid line). The kinetics of this reaction are shown in Fig. 11, which is published as

supporting information

on the PNAS web site.

Fig. 5.
Fig. 5.

Inhibition of protein synthesis by PKZ. (A) HEK293T and CHO cells were cotransfected with 2.5 μg of the luciferase plasmid pGL3promoter and 0.5 μg of the expression vector pcDNA3.1 containing the indicated inserts. Luciferase activity was normalized for protein content. A control experiment by using the empty pcDNA3.1 vector was used as 100%. An asterisk denotes the position in the constructs of the K199R mutation that abolishes kinase activity. Each bar is the average of three independent experiments. (B) Western blot analysis of HEK293T cells transfected with the expression constructs shown in A. His-tagged proteins were detected by an anti-His-tag antibody. The asterisk marks a nonspecific band detected in all lanes. Silver staining demonstrated comparable loading of proteins (data not shown).

Fig. 6.
Fig. 6.

Evolutionary relationship between PKZ and other protein kinases, shown as a maximum a posteriori phylogram of Bayesian analysis. Posterior probabilities were converted to percentages and are shown above the branches.

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