An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Abstract

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+]iper se causes slowing of peptide chain elongation is lacking. L-type Ca2+channel activity of GH3pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+]ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca2+, resting [Ca2+]ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca2+channel antagonist, nisoldipine, lowered [Ca2+]iand 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+]ithat 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-2α 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+]ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca2+]ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.