Induction of Apoptosis by Double-Stranded-RNA-Dependent Protein Kinase (PKR) Involves the α Subunit of Eukaryotic Translation Initiation Factor 2 and NF-κB

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-2α (α subunit of eukaryotic translation initiation factor 2) and IκBα, the inhibitor of the transcription factor NF-κB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-κB and eIF-2α 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-2α or a repressor form of IκBα. 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-2α (Ser-51→Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IκBα (Ser-32,36-Ala) also leads to the inhibition of apoptosis by abolishing NF-κB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IκBα 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-κB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2α and NF-κB in the induction of apoptosis by PKR.

ACKNOWLEDGMENTS

We thank R. E. Randall (University of Glasgow, Glasgow, Scotland) for the SV5 monoclonal antibody (from the NIBSC-MRC AIDS Reagent Project), J. Hershey (University of California) for the eIF-2α plasmid, M. J. Clemens (St. George’s Hospital, London, United Kingdom) and César de Haro (CBMSO) for the eIF-2α monoclonal antibody, and Fernando Arenzana (Institut Pasteur, Paris, France) for providing the NF-κB reporter plasmids. We also thank C. Weissman (University of Zurich, Zurich, Switzerland) for the generous gift of the 3T3-like PKR^0/0 and PKR^+/+ cells. We especially thank Juan Pablo Albar from the Department of Immunology and Oncology, Centro Nacional de Biotecnologia, for the production of the PKR synthetic peptide. We thank Manuel Collado, Carmen Rivas, Don Roth, and Isabel Vázquez for their critical reading of the manuscript and helpful suggestions and Victoria Jiménez and Laura Giménez for expert technical assistance.

This investigation was supported by grants SAF 95 0022 and PM98-0112 from the Comision Interministerial de Ciencia y Tecnologia of Spain (to M.E.) and grants from Fundación Caja de Madrid and Ministerio de Educación y Ciencia MEC (SAF 96/186) (to J.A.). J.G. was the recipient of an FPI fellowship from the Spanish MEC.