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+](zeta)) due to plasmalemmal Ca2+ influx increases the phosphorylation of elongation factor (eEF) 2. Im nucleated mammalian cells, depletion of ER Ca2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca2+] per se causes slowing of peptide chain ellongation is lacking. L-type Ca2+ channel activity Of GH(3) pituitary cells, which ape enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+] on eEF-2 phosphorylation and translational rate could be examined for up to 18 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 KCI or KCI pins 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-2 alpha 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-2 alpha, 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.. (C) 1998 Academic Press.