Functional characterization of pkr gene products expressed in cells from mice with a targeted deletion of the N terminus or C terminus domain of PKR.
The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays an important role in messenger (m) RNA translation by phosphorylating the alpha subunit of eukaryotic initiation factor 2. Through this capacity PKR is thought to be a mediator of the antiviral and antiproliferative actions of interferon. In addition to translational function, PKR has been implicated in many signaling pathways to gene transcription by modulating the activities of a number of transcription factors, including NF-kappa B and STATs. However, experiments with two different PKR knockout (PKR(-/-)) mouse models have failed to verify many of the biological functions attributed to PKR. In addition, results with cells from the two PKR(-/-) mice have been contradictory and confusing. Here, we show that the first PKR(-/-) mouse with deletion of exons 2 and 3, corresponding to the N terminus domain of PKR (N-PKR(-/-)), expresses a truncated protein, resulting from the translation of the exon-skipped mouse PKR (ES-mPKR) mRNA. The ES-mPKR protein is defective in dsRNA binding but remains catalytically active both in vitro and in vivo. Furthermore, we show that the second PKR(-/-) mouse with a targeted deletion of exon 12, which corresponds to the C terminus of the molecule (C-PKR(-/-)), expresses a truncated mPKR produced by alternative splicing of exon 12. Although the spliced form of mPKR (SF-mPKR) is catalytically inactive, it retains the dsRNA-binding properties of the wild type mPKR. Reverse transcription-PCRs demonstrate that SF-mPKR mRNA is expressed in several normal mouse tissues, and appears to be under developmental control during embryogenesis. Our data demonstrate that both PKR(-/-) models are incomplete knockouts, and expression of the PKR variants may account, at least in part, for the significant signaling differences between cells from the two PKR(-/-) mice.
Baltzis
Dionissios
D
Lady Davis Institute for Medical Research, McGill University, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte-Ste-Catherine Street, Montréal, Québec H3T 1E2, Canada.
Li
Suiyang
S
Koromilas
Antonis E
AE
D017398:Alternative Splicing; D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014018:Tissue Distribution; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007527:Isoenzymes; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; D019892:eIF-2 Kinase