Translation inhibition in apoptosis - Caspase-dependent PKR activation andeIF2-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 synth esis. PKR activation requires binding of double-stranded RNA or PACT/RAX pr oteins to its regulatory domain. Since several reports have demonstrated th at translation is inhibited in apoptosis, we investigated whether PKR and e IF2-alpha phosphorylation contribute to this process. We show that PKR is p roteolysed and that eIF2-alpha is phosphorylated at the early stages of apo ptosis induced by various stimuli. Both events coincide with the onset of c aspase activity and are prevented by caspase inhibitors. Using site-directe d mutagenesis we show that PKR is specifically proteolysed at Asp(251) duri ng 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 c aspase-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.