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

Abstract

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.

Functional Complementation by Wheat eIF2α in the Yeast GCN2-Mediated Pathway

“…This study indicates that the basic processes of translational initiation are highly conserved among all eukaryotes and taken together with previous studies (11,12) supports the concept of a plant eIF2␣ phosphorylation pathway. We are currently evaluating transgenic plants that overexpress wild type eIF2␣ and the 51A mutant as a means to dissect the physiological significance of eIF2␣ phosphorylation in plants.…”

“…Other members of the GCN2 kinase family include Drosophila and mammalian homologs (9,10). The model plant Arabidopsis also encodes a putative GCN2 kinase (Accession number CAB91611) and plants encode a biochemical and immunological homolog of the mammalian dsRNA dependent protein kinase R (PKR) that in vitro phosphorylates plant and yeast eIF2␣ specifically on serine 51 (11)(12)(13)(14)(15).…”

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana

“…On the other hand, studies in mammals have shown that L18 interacts with a double-stranded RNA-activated protein kinase (PKR) and that this interaction inhibits PKR activity. A putative PKR-like protein was identified in plant extracts using antibodies raised against human PKR (Langland et al, 1995(Langland et al, , 1996 but no protein containing double-stranded RNA binding and kinase domains has been identified so far (although the two domains could be present on separate proteins in plants). The possible existence of a PKR-like activity in plants was reinforced by the discovery of a plant PKR inhibitor and the fact that its inactivation favours viral pathogenesis (Bilgin et al, 2003).…”

Mechanism of Cytoplasmic mRNA Translation

“…eIF2B (see below) cannot easily dissociate from phosphorylated eIF2α and therefore guanine nucleotide exchange is inhibited depleting available eIF2 for ternary complex formation (reviewed in Donnelly et al, 2013). Despite early reports of a "PKR-like" activity in virus-infected plants (Hiddinga et al, 1988;Langland et al, 1995;Langland et al, 1996;Chang et al, 1999), no specific kinase could be purified and the sequence of a putative PKR ortholog is absent from plant genomes (Immanuel et al, 2012). GCN2 is therefore the only recognizable plant eIF2α kinase at this time and targets a similar serine residue in plant eIF2α (Halford et al, 2004;Byrne et al, 2012;Li et al, 2013a;Wang et al, 2014).…”