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Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation
X Saelens1, M Kalai, P Vandenabeele
1Department of Molecular Biology, Unit of Molecular Signaling and Cell Death, Flanders Interuniversity Institute for Biotechnology and Ghent University, 9000 Ghent, Belgium.
Abstract:
The protein kinase PKR is a major player in the cellular antiviral response, acting mainly by phosphorylation of the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2-alpha) to block de novo protein synthesis. PKR activation requires binding of double-stranded RNA or PACT/RAX proteins to its regulatory domain. Since several reports have demonstrated that translation is inhibited in apoptosis, we investigated whether PKR and eIF2-alpha phosphorylation contribute to this process. We show that PKR is proteolysed and that eIF2-alpha is phosphorylated at the early stages of apoptosis induced by various stimuli. Both events coincide with the onset of caspase activity and are prevented by caspase inhibitors. Using site-directed mutagenesis we show that PKR is specifically proteolysed at Asp(251) during cellular apoptosis. This site is cleaved in vitro by recombinant caspase-3, caspase-7, and caspase-8 and not by the proinflammatory caspase-1 and caspase-11. The released kinase domain efficiently phosphorylates eIF2-alpha at the cognate Ser(51) residue, and its overexpression in mammalian cells impairs the translation of its own mRNA and of reporter mRNAs. Our results demonstrate a new and caspase-dependent activation mode for PKR, leading to eIF2-alpha phosphorylation and translation inhibition in apoptosis.
Insights
Protein kinase PKR is proteolyzed during apoptosis, leading to eIF2-alpha phosphorylation and translation inhibition. This novel, caspase-dependent activation of PKR contributes to the apoptotic process.
Area of Science:
- Cellular biology
- Molecular mechanisms of apoptosis
- Protein kinase regulation
Background:
- Protein kinase PKR (PKR) is crucial for antiviral responses by phosphorylating eIF2-alpha, inhibiting protein synthesis.
- PKR activation typically involves double-stranded RNA or PACT/RAX proteins.
- Translation inhibition is observed during apoptosis, suggesting a potential role for PKR.
Purpose of the Study:
- To investigate the involvement of PKR and eIF2-alpha phosphorylation in apoptosis.
- To elucidate the mechanism of PKR regulation during programmed cell death.
Main Methods:
- Induction of apoptosis using various stimuli.
- Analysis of PKR proteolysis and eIF2-alpha phosphorylation.
- Site-directed mutagenesis to identify PKR cleavage sites.
- In vitro cleavage assays using recombinant caspases.
- Overexpression of PKR kinase domain in mammalian cells.
Main Results:
- PKR undergoes proteolysis and eIF2-alpha is phosphorylated early in apoptosis.
- These events correlate with caspase activity and are inhibited by caspase inhibitors.
- PKR is specifically cleaved at Asp(251) by caspases-3, -7, and -8.
- The released PKR kinase domain phosphorylates eIF2-alpha and inhibits translation.
Conclusions:
- A novel, caspase-dependent activation pathway for PKR in apoptosis is identified.
- This pathway involves PKR proteolysis, release of its kinase domain, eIF2-alpha phosphorylation, and translation inhibition.
- PKR plays a significant role in regulating protein synthesis during apoptosis.