Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 alpha activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 alpha kinases. Further, only wheat wild-type eIF2 alpha is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 alpha 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 alpha kinases. A truncated version of wild-type wheat eIF2 alpha containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 alpha kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 alpha species and eIF2 alpha kinases and support the presence of a plant eIF2 alpha phosphorylation pathway.