Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB.
The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2alpha (alpha subunit of eukaryotic translation initiation factor 2) and IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-kappaB and eIF-2alpha in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2alpha or a repressor form of IkappaBalpha. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2alpha (Ser-51-->Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IkappaBalpha (Ser-32, 36-Ala) also leads to the inhibition of apoptosis by abolishing NF-kappaB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IkappaBalpha degradation, prevented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-kappaB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2alpha and NF-kappaB in the induction of apoptosis by PKR.
Gil
J
J
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, 28049 Madrid, Spain.
Alcamí
J
J
Esteban
M
M
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000166:cytology; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D051379:Mice; D009097:Multienzyme Complexes; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded; D012694:Serine / Q000378:metabolism; D015533:Transcriptional Activation; D014616:Vaccinia virus; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D012330:RNA, Double-Stranded; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; D019892:eIF-2 Kinase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex