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-2α (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostkoet al. J. Biol. Chem. 267: 16751–16754, 1992). Alterations in eIF-2α 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-2α in a manner blocked by antinomycin D. Phosphorylation of eIF-2α 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-2α 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-2α 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-2α 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-2α 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-2α phosphorylation and translational repression.
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Prostko, C.R., Brostrom, M.A. & Brostrom, C.O. Reversible phosphorylation of eukaryotic initiation factor 2α in response to endoplasmic reticular signaling. Mol Cell Biochem 127, 255–265 (1993). https://doi.org/10.1007/BF01076776
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DOI: https://doi.org/10.1007/BF01076776