Induction of Apoptosis by double-stranded-RNA-dependent Protein Kinase (PKR) Involves the Alpha Subunit of Eukaryotic Translation Initiation Factor 2 and NF-kappaB

Mol Cell Biol. 1999 Jul;19(7):4653-63. doi: 10.1128/mcb.19.7.4653.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Apoptosis*
  • Binding Sites
  • Chlorocebus aethiops
  • Cysteine Endopeptidases
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Eukaryotic Cells / cytology
  • Eukaryotic Initiation Factor-2 / genetics
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Gene Expression
  • Genetic Vectors
  • HeLa Cells
  • Humans
  • I-kappa B Proteins*
  • Mice
  • Multienzyme Complexes
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism*
  • Phosphorylation
  • Proteasome Endopeptidase Complex
  • Protein Biosynthesis
  • RNA, Double-Stranded
  • Serine / metabolism
  • Transcriptional Activation
  • Vaccinia virus
  • eIF-2 Kinase / genetics
  • eIF-2 Kinase / metabolism*

Substances

  • DNA-Binding Proteins
  • Eukaryotic Initiation Factor-2
  • I-kappa B Proteins
  • Multienzyme Complexes
  • NF-kappa B
  • NFKBIA protein, human
  • Nfkbia protein, mouse
  • RNA, Double-Stranded
  • NF-KappaB Inhibitor alpha
  • Serine
  • eIF-2 Kinase
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex