Cloning and Characterization of a cDNA Encoding a Protein Synthesis Initiation Factor-2α (eIF-2α) Kinase fromDrosophila melanogaster

Abstract

Phosphorylation of the ␣ subunit of the eukaryotic initiation factor 2 (eIF-2␣) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions. In Drosophila, such a regulatory mechanism has not been elucidated. We report the molecular cloning and characterization of DGCN2, a Drosophila eIF-2␣ kinase related to yeast GCN2 protein kinase. DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2␣ kinases in subdomain V. A large insert of 94 amino acids, which is characteristic of eIF-2␣ kinases, is also present between subdomains IV and V. It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2 expression is developmentally regulated. During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues. Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system. Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2␣ kinase activity and recognized an ϳ175 kDa phosphoprotein in Western blots of Drosophila embryo extracts.Protein synthesis is mainly regulated at the initiation of mRNA translation. The best-characterized mechanism of translational regulation in eukaryotes involves the phosphorylation of the ␣-subunit of eukaryotic initiation factor 2 (eIF-2␣)

Phosphorylation of Eukaryotic Initiation Factor 2 by Heme-Regulated Inhibitor Kinase-Related Protein Kinases in Schizosaccharomyces pombe Is Important for Resistance to Environmental Stresses

“…Another bZIP protein, ATF4, is preferentially translated in response to phosphorylation of mammalian eIF2␣ by a mechanism involving upstream open reading frames, although it is uncertain whether the mechanism of leaky scanning described previously for GCN4 translation also functions in mammals (27). In invertebrate metazoans such as Drosophila melanogaster and Caenorhabditis elegans, the only eIF2␣ kinases present are GCN2 and PEK/Perk, which are proposed to be important for stresses impacting the cytoplasm and endoplasmic reticulum, respectively (52,61,65).…”

Translational control of hippocampal synaptic plasticity and memory by the eIF2α kinase GCN2

“…In view of the need for translation for modulation of synaptic activity and strong evidence that eIF2α phosphorylation controls translation of ATF4 mRNA 6,11,12 , eIF2α kinase(s) may regulate synaptic plasticity. Because GCN2 is the most evolutionarily conserved eIF2α kinase and GCN2 mRNA is enriched in the brain of flies 13 and mammals (as well as in liver) 14,15 (see supplementary Fig. 1), we explored the role of GCN2 in synaptic plasticity and behavioral learning.…”

The Interferon-induced Double-stranded RNA-activated Protein Kinase PKR Will Phosphorylate Serine, Threonine, or Tyrosine at Residue 51 in Eukaryotic Initiation Factor 2α

“…The active, phosphorylated form of PKR can then phosphorylate eIF2␣ on Ser-51 and convert eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, resulting in the inhibition of translation initiation (1,2). The other members of the eIF2␣ kinase family are the mammalian heme-regulated inhibitor of translation (HRI) that is activated by heme deprivation, the apparently ubiquitous kinase GCN2, first identified in yeast but also found in flies and mammals, which is activated under conditions of amino acid or purine nucleotide deprivation (1)(2)(3)(4), and the newly identified mammalian kinase PERK or PEK, a transmembrane kinase located in the endoplasmic reticulum that is activated under conditions of endoplasmic reticulum stress (5,6). In the yeast Saccharomyces cerevisiae, low level phosphorylation of eIF2␣ by GCN2 alters the pattern of translation reinitiation on the GCN4 mRNA and induces GCN4 expression (2).…”