Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation.
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.
Saelens
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X
Department of Molecular Biology, Unit of Molecular Signaling and Cell Death, Flanders Interuniversity Institute for Biotechnology and Ghent University, 9000 Ghent, Belgium.
Kalai
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Vandenabeele
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P
D017209:Apoptosis; D020169:Caspases / Q000502:physiology*; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D014176:Protein Biosynthesis; D019892:eIF-2 Kinase / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D019892:eIF-2 Kinase; D020169:Caspases