Ribosome targeting of PKR is mediated by two double-stranded RNA-binding domains and facilitates in vivo phosphorylation of eukaryotic initiation factor-2.
Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2alpha. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2alpha. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2alpha, by providing PKR access to its substrate.
Zhu
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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
Romano
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PR
Wek
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RC
D001692:Biological Transport; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase