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