As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2␣) at Ser 51 is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2␣ stress kinases. We used gene targeting to replace the wild-type Ser 51 allele with an Ala in the eIF2␣ gene to test the hypothesis that translational control through eIF2␣ phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2␣ mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-␣, and serum deprivation. TNF␣ treatment induced eIF2␣ phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2␣ kinase PKR. In addition, expression of a phospho-mimetic Ser 51 to Asp mutant eIF2␣-activated caspase 3, indicating that eIF2␣ phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2␣ phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2␣ kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2␣. We propose that eIF2␣ phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.

Both the rate of overall translation and the specific acceleration of proinsulin synthesis are known to be glucose-regulated processes in the beta-cell. In this study, we propose that glucose-induced stimulation of overall translation in beta-cells depends on a protein phosphatase-1-mediated decrease in serine-51 phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), a pivotal translation initiation factor. The decrease was rapid and detectable within 15 min and proportional to the range of glucose concentrations that also stimulate translation. Lowered net eIF2alpha phosphorylation was not associated with a detectable decrease in activity of any eIF2alpha kinase. Moreover, okadaic acid blocked glucose-induced eIF2alpha dephosphorylation, suggesting that the net effect was mediated by a protein phosphatase. Experiments with salubrinal on intact cells and nuclear inhibitor of protein phosphatase-1 (PP1) on cell extracts suggested that this phosphatase was PP1. The net effect contained, however, a component of glucose-induced folding load in the endoplasmic reticulum because coincubation with cycloheximide further amplified the effect of glucose on eIF2alpha dephosphorylation. Thus, the steady-state level of eIF2alpha phosphorylation in beta-cells is the result of a balance between folding-load-induced phosphorylation and PP1-dependent dephosphorylation. Because defects in the pancreatic endoplasmic reticulum kinase-eIF2alpha signaling system lead to beta-cell failure and diabetes, deregulation of the PP1 system could likewise lead to cellular dysfunction and disease.

The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the beta-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection.