Independent Signaling of grp78 Gene Transcription and Phosphorylation of Eukaryotic Initiation Factor 2α by the Stressed Endoplasmic Reticulum

Abstract

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.