The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-kappaB activation.
The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-kappaB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-kappaB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-kappaB induction. In PKR(0/0) cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-kappaB. An inactive PKR mutant (K296R), was unable to induce NF-kappaB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-kappaB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2alpha phosphorylation and NF-kappaB activation. However, PKR mutants unable to activate NF-kappaB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-kappaB.
Gil
J
J
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, (CSIC), Campus Universidad Autónoma, 28049 Madrid, Spain.
Rullas
J
J
García
M A
MA
Alcamí
J
J
Esteban
M
M
D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D051550:I-kappa B Kinase; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D016328:NF-kappa B; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse