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 a 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-kappaB 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-PYR-/-), 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.