Abstract
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.
Highlights
One of the best characterized interferon (IFN)1 inducible proteins is the double-stranded RNA-activated protein ki
Detection of the ES-mPKR Product in N-PKRϪ/Ϫ mouse embryonic fibroblasts (MEFs)—To test whether N-PKRϪ/Ϫ cells express the PKR fragment produced from the translation of exon-skipped mouse PKR mRNA (Fig. 1A) (6), we performed Western blot analysis with protein extracts from strain-matched PKRϩ/ϩ and N-PKRϪ/Ϫ MEFs (Fig. 1B)
Immunoblot analysis with an anti-PKR monoclonal antibody specific for an epitope within the C terminus half of the kinase2 detected the expression of full-length PKR in PKRϩ/ϩ cells and the presence of a 45-kDa protein in isogenic N-PKRϪ/Ϫ MEFs, which was highly induced by IFN treatment
Summary
One of the best characterized interferon (IFN) inducible proteins is the double-stranded (ds) RNA-activated protein ki-. The susceptibility of C-PKRϪ/Ϫ MEFs to apoptotic death in response to dsRNA, virus infection, lipopolysaccharide, or TNF-␣ treatment is normal compared with genetically matched PKRϩ/ϩ MEFs (7), as is normal the activation of NF-B by dsRNA (19) These striking signaling differences between the two types of PKRϪ/Ϫ cells prompted us to investigate the possible expression of truncated PKR proteins resulting from incomplete disruption of the pkr gene. Using antibodies specific to the C terminus of mPKR we demonstrate that ES-mPKR is expressed in N-PKRϪ/Ϫ MEFs. We show that the ES-mPKR is inducible by IFN-␣/ and contains eIF-2␣ kinase activity both in vitro and in vivo. Our data demonstrate the expression of partially functional products in both types of PKRϪ/Ϫ cells, providing an explanation for the striking signaling differences between cells from the two PKRϪ/Ϫ models
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