Reversible phosphorylation of eukaryotic initiation factor 2? in response to endoplasmic reticular signaling

Abstract

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2 alpha (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostko et al. J. Biol. Chem. 267:16751-16754, 1992). Alterations in eIF-2 alpha phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2 alpha in a manner blocked by actinomycin D. Phosphorylation of eIF-2 alpha in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2 alpha phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2 alpha occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2 alpha and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2 alpha in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2 alpha phosphorylation and translational repression.

Nck-1 Antagonizes the Endoplasmic Reticulum Stress-induced Inhibition of Translation

“…It is described that agents that cause ER stress result in dissociation of polysomes into monosomes and ribosomal subunits and that this is consistent with attenuation of an early step in mRNA translation (23). Therefore, we used the ratio of monosomes to polysomes (M:P) to estimate the translational activity of the various previously described MEFs cell lines treated or not with thapsigargin ( Fig.…”

Using natural variation in Drosophila to discover previously unknown endoplasmic reticulum stress genes

“…In particular, an associated SNP 65 bp downstream of eIF-2β, the beta subunit of the eIF2 translation initiation factor (43), is particularly interesting. In ER stress conditions, the alpha subunit of eIF2 is phosphorylated by PERK, a classic UPR gene, attenuating global translation (44). Although the beta subunit of eIF2 has not been implicated in the UPR, this noncoding SNP downstream of eIF-2β may affect expression and the available amount of eIF2 and the phosphorylation of the alpha subunit.…”

The Role of GADD34 (Growth Arrest and DNA Damage-Inducible Protein) in Regulating Apoptosis, Proliferation, and Protein Synthesis in Human Breast Cancer Cells

“…The phosphorylation of eIF-2a at serine-51 is increased by a variety of cellular stresses, including ER stress induced by defects in protein folding (44). ER stress can be experimentally induced by treatment of cells with tunicamycin, which inhibits protein glycosylation, or thapsigargin, which depletes intracellular calcium stores.…”

“…Aberrations in ER function result in the accumulation of unfolded proteins and activate signal transduction pathways that trigger a complex transcriptional and translational response known as the unfolded protein response (UPR) (30). A hallmark of the UPR is increased phosphorylation of the a. subunit of eukaryotic translation initiation factor 2 (eIF-2a) on serine-51 (44). Phosphorylated eIF-2a inhibits the guanine nucleotide exchange activity of eIF-2B and attenuates global protein synthesis, allowing cells the opportunity to clear misfolded proteins from the ER (40).…”