The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.

The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.

An examination of the role of increased cytosolic free Ca2+ concentrations in the inhibition of mRNA translation.

Mobilization of Ca2+ sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca2+ ([Ca2+]i) due to plasmalemmal Ca2+ influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca2+]i per se causes slowing of peptide chain elongation is lacking. L-type Ca2+ channel activity of GH3 pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+]i on eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca2+, resting [Ca2+]i values were high (154-255 nM) and eEF-2 was phosphorylated. The Ca2+ channel antagonist, nisoldipine, lowered [Ca2+]i and reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca2+ channel agonist, Bay K 8644, produced sustained elevations of [Ca2+]i that were associated with 25-50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca2+ influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50-100% above control) and inhibited leucine incorporation by 20-70% but ATP content was reduced by 25-50% and total cell-associated Ca2+ contents rose by 3- to 13-fold. eIF-2alpha was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca2+]i that resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2alpha, not eEF-2. It is concluded that changes in [Ca2+]i occurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.