ACTIVATION OF THE DOUBLE-STRANDED RNA-REGULATED PROTEIN-KINASE BY DEPLETION OF ENDOPLASMIC RETICULAR CALCIUM STORES

Perturbants of the endoplasmic reticulum (ER), including Ca2+-mobilizi ng agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 alpha kinase, Analy sis of extracts derived from cultured cells that had been pretreated w ith Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 al pha phosphatase activity. A peptide of 65-68 kDa, which was phosphoryl ated concurrently with eIF-2 alpha in extracts of pretreated cells, wa s identified as the interferon-inducible, double-stranded RNA (dsRNA)- regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not al ter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed gre ater degrees of phosphorylation of PKR and of eIF-2 alpha than did con trol extracts, The enhanced dsRNA dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with interferon. Lower concentrations of dsRNA were required for maximal phosphorylati on of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.