The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.

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.

Depletion of endoplasmic reticulum (ER) Ca

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2 alpha (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostko et al. J. Biol. Chem. 267:16751-16754, 1992). Alterations in eIF-2 alpha phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2 alpha in a manner blocked by actinomycin D. Phosphorylation of eIF-2 alpha in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2 alpha phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2 alpha occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2 alpha and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2 alpha in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2 alpha phosphorylation and translational repression.