Activation of the Double-stranded RNA-regulated Protein Kinase by Depletion of Endoplasmic Reticular Calcium Stores

Abstract

Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing 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. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 alpha than did control 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 phosphorylation 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.

Phosphorylation of Eukaryotic Translation Initiation Factor 2 Mediates Apoptosis in Response to Activation of the Double-stranded RNA-dependent Protein Kinase

“…PKR activation was also previously demonstrated to occur in response to stress conditions, such as activation of the heat shock response, presence of unfolded protein in the endoplasmic reticulum, growth factor depletion, and increases in cytosolic calcium (23,(41)(42)(43). All of these stress responses are frequently accompanied by apoptosis.…”

“…PKR mediates phosphorylation of eIF-2␣ in response to calcium depletion from the endoplasmic reticulum mediated by ionophore (23,41), a treatment used to induce apoptosis. Furthermore, removal of the growth factor interleukin 3 from an interleukin 3-dependent cell line induces the autophosphorylation of PKR, which in turn FIG.…”

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

“…Depletion of endoplasmic reticulum (ER) calcium stores results in PKR activation (Prostko et al, 1995), and expression of a dominant-negative mutant of PKR interferes with the ability of ER calcium depletion to reduce translation rates (Srivastava et al, 1995). The discovery of the PKR-like ER kinase (PERK) (Harding et al, 1999) has cast doubts on the involvement of PKR in the inhibition of protein synthesis caused by depletion of ER calcium stores.…”

Mutation (D472Y) in the Type 3 Repeat Domain of Cartilage Oligomeric Matrix Protein Affects Its Early Vesicle Trafficking in Endoplasmic Reticulum and Induces Apoptosis

“…44 Phosphorylation of eIF2␣ occurs in response to various stressful stimuli, including nutrient deprivation, heat shock, viral infection, and treatment with compounds that deplete endoplasmic reticular calcium levels. 44,45 Therefore, we examined cells expressing mutated COMP to determine whether they exhibited ER stress using anti-phospho-eIF2␣ antibody.…”