Characterization of the Initiation Factor eIF2B and Its Regulation in Drosophila melanogaster

Daniel D Williams,Graham D Pavitt,Christopher G Proud

doi:10.1074/jbc.m008041200

Daniel D Williams, Graham D Pavitt + Show 1 more

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https://doi.org/10.1074/jbc.m008041200

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Abstract

Eukaryotic initiation factor (eIF) 2B catalyzes a key regulatory step in the initiation of mRNA translation. eIF2B is well characterized in mammals and in yeast, although little is known about it in other eukaryotes. eIF2B is a hetropentamer which mediates the exchange of GDP for GTP on eIF2. In mammals and yeast, its activity is regulated by phosphorylation of eIF2alpha. Here we have cloned Drosophila melanogaster cDNAs encoding polypeptides showing substantial similarity to eIF2B subunits from yeast and mammals. They also exhibit the other conserved features of these proteins. D. melanogaster eIF2Balpha confers regulation of eIF2B function in yeast, while eIF2Bepsilon shows guanine nucleotide exchange activity. In common with mammalian eIF2Bepsilon, D. melanogaster eIF2Bepsilon is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purified D. melanogaster eIF2B by glycogen synthase kinase-3 inhibits its activity. Extracts of D. melanogaster S2 Schneider cells display eIF2B activity, which is inhibited by phosphorylation of eIF2alpha, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2alpha phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2alpha phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2alpha phosphorylation is also increased by endoplasmic reticulum stress in S2 cells. These are the first data concerning the structure, function or control of eIF2B from D. melanogaster.

Highlights

The binding of the initiator Met-tRNAi to the 40 S ribosomal subunit is a key control point in the initiation of mRNA translation in both Saccharomyces cerevisiae and mammals [1,2,3]
We report that eIF2␣ phosphorylation occurs in a regulated manner in vivo, that glycogen synthase kinase-3 (GSK-3)␤ phosphorylates eIF2B⑀ in vitro, and that eIF2B activity can be inhibited in vitro by GSK-3␤. eIF2B and its regulation in this species appear to be similar to other eukaryotic organisms that have so far been studied
To assess whether D. melanogaster cells contain a protein with eIF2B activity, we assayed extracts of S2 Schneider cells for their ability to catalyze guanine nucleotide exchange on eIF2 using complexes containing mammalian eIF2 and

Summary

Introduction

The binding of the initiator Met-tRNAi to the 40 S ribosomal subunit is a key control point in the initiation of mRNA translation in both Saccharomyces cerevisiae and mammals [1,2,3] This step is mediated by eukaryotic initiation factor (eIF) , a. A new eIF2␣ kinase, pancreatic eukaryotic initiation factor-2␣ kinase (PEK), was identified in mammalian pancreatic cells and characterized as a membranebound protein localized in the lumen of the endoplasmic reticulum (ER). This kinase has been implicated in the control of translation in response to ER stresses such as improper protein folding. Mammalian cells possess at least four eIF2␣ kinases (heme-regulated inhibitor (HRI; Ref. 9), interferoninduced double-stranded RNA-activated protein kinase (PKR; Ref. 10), general control nonderepressible 2 (GCN2; Ref. 11), chain reaction; 3-AT, 3-amino-1,2,4-triazole; PKR, RNA-activated protein kinase; HRI, heme-regulated inhibitor; PEK, pancreatic eukaryotic initiation factor 2␣ kinase; CK-II, casein kinase II; GCN, general control nonderepressible

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