Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the a subunit of eukaryotic translation initiation factor 2 andNF-kappa B

The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediat or of antiviral effects of interferon (IFN) and an active player in apoptos is induced by different stimuli. The translation initiation factor eIF-2 al pha (alpha subunit of eukaryotic translation initiation factor 2) and I kap pa B alpha, the inhibitor of the transcription factor NF-kappa B, have been proposed as downstream mediators of PKR effects. To evaluate the involveme nt of NF-kappa B and eIF-2 alpha in the induction of apoptosis by PKR, we h ave used vaccinia virus (VV) recombinants that inducibly express PKR concom itantly with a dominant negative mutant of eIF-2a or a repressor form of I kappa B alpha. We found that while expression of PKR by a VV vector resulte d in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2 alpha (Ser-51- ->Ala) reversed both the PKR-mediated translational block and PKR-induced a poptosis. Coexpression of PKR with a repressor form of I kappa B alpha (Ser -32,36 Ala) also leads to the inhibition of apoptosis by abolishing NF-kapp a B induction, while translation remains blocked. Treating cells with two d ifferent proteasome inhibitors which block I kappa B alpha degradation, pre vented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-kappa B binding and transactivation. In addition, up regulation of Fas mRNA transcription occurred during PKR activation. Our fi ndings provide direct evidence for the involvement of eIF-2 alpha and NF-ka ppa B in the induction of apoptosis by PKR.