title: 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+]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.
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, New Jersey, 08854, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Laitusis
authors.forename: C O
authors.initials: CO
authors.lastname: Brostrom
authors.forename: A G
authors.initials: AG
authors.lastname: Ryazanov
authors.forename: M A
authors.initials: MA
authors.lastname: Brostrom
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism; D020746:Calcium Channels, L-Type; D003600:Cytosol / Q000502:physiology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007930:Leucine / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational / Q000502:physiology; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D015852:Eukaryotic Initiation Factor-2; D007476:Ionophores; D012333:RNA, Messenger; D015759:Ionomycin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D007930:Leucine; D002118:Calcium