Phosphorylation of plant eukaryotic initiation factor-2 by the plant- encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro

Regulation of protein synthesis by eukaryotic initiation factor-2α (eIF- 2α) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2α have been characterized from mammals and yeast. However, the phenomenon of eIF2-α-mediated regulation of protein synthesis and the presence of an eIF-2α kinase has not been demonstrated in higher plants. We show that plant eIF-2α (peIF-2α) and mammalian eIF-2α (meIF- 2α) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2α kinase, PKR. By several criteria, phosphorylation of peIF-2α is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser⁵¹ in a synthetic eIF- 2α peptide, a key characteristic of the eIF-2α kinase family. Taken together, these data support the concept that pKR is a member of the eIF-2α kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2α, meIF-2α, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2α.