The murine double-stranded RNA-dependent protein kinase PKR is required for resistance to vesicular stomatitis virus

Interferon (IFN)-induced antiviral responses are mediated through a variety of proteins, including the double-stranded RNA-dependent protein kinase PKR. Here we show that fibroblasts derived from PKR(-/-) mice are more permissive for vesicular stomatitis virus (VSV) infection than are wild-type fibroblasts and demonstrate a deficiency in alpha/beta-IFN-mediated protection. We further show that mice lacking PKR are extremely susceptible to intranasal VSV infection, succumbing within days after instillation with as few as 50 infectious viral particles. Again, alpha/beta-IFN was unable to rescue PKR(-/-) mice from VSV infection. Surprisingly, intranasally infected PKR(-/-) mice died not from pathology of the central nervous system but rather from acute infection of the respiratory tract, demonstrating high virus titers in the lungs compared to similarly infected wild-type animals. These results confirm the role of PKR as the major component of IFN-mediated resistance to VSV infection. Since previous reports have shown PKR to be nonessential for survival in animals challenged with encephalomyocarditis virus, influenza virus, and vaccinia virus (N. Abraham et al., J. Biol. Chem. 274:5953-5962, 1999; Y. Yang et al., EMBO J. 14:6095-6106, 1995), our findings serve to highlight the premise that host dependence on the various mediators of IFN-induced antiviral defenses is pathogen specific.     

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.     

Putrescine transport in hypoxic rat main PASMCs is required for p38 MAP kinase activation

Hypoxic pulmonary vascular remodeling in rats is associated with increased polyamine transport in pulmonary artery smooth muscle cells (PASMCs). We therefore defined constitutive and hypoxia-induced polyamine transport properties of rat cultured PASMCs and determined the impact of polyamine transport blockade on hypoxia-induced accumulation of p38 MAP kinase. PASMCs exhibited polyamine transport pathways that were characterized by Michaelis-Menten kinetics. RNA synthesis inhibition attenuated while inhibition of protein synthesis increased polyamine uptake, thus suggesting regulation by ornithine decarboxylase-antizyme. The presence of two transporters with overlapping selectivities, one for putrescine and another for all three polyamines, was inferred by cross-competition studies and by findings that only putrescine uptake was sodium dependent and that hypoxia caused a selective, time-dependent induction of putrescine transport. The pathophysiological significance of augmented putrescine import was suggested by the observation that polyamine transport inhibition suppressed hypoxia-induced p38 MAP kinase phosphorylation. These results indicate that rat PASMCs express two polyamine transporters and that a specific increase in the putrescine uptake pathway is necessary for hypoxia-induced activation of p38 MAP kinase.     

The ferritin-like protein Frm is a target for the humoral immune response to Listeria monocytogenes and is required for efficient bacterial survival

The identity and role of listerial antigens recognized by antibodies following listerial infection is largely unknown. We identified the listerial ferritin-like protein Frm as a target of the humoral response following infection of mice with pathogenic Listeria monocytogenes. Specific antibodies to Frm are detected in antisera of mice infected with a pathogenic strain but not in antisera of mice infected with a non-pathogenic L. innocua strain. Antibodies raised to purified listerial ferritin allowed us to demonstrate that the expression of Frm is both growth phase- and temperature-dependent. Using an isogenic Δfrm mutant, we find that ferritin is essential for bacterial growth in chemically defined minimal media but not in complex media such as brain–heart infusion. Mutant bacteria also exhibit a defect in intracellular replication. The Δfrm strain is hypersensitive to hydrogen peroxide indicating that the Frm is required for protection against reactive oxygen intermediates under various growth conditions. Animal studies show that Frm contributes to pathogenesis in mice, in particular, at early time points following infection.     

Increased interaction between PACT molecules in response to stress signals is required for PKR activation

PKR (protein kinase, RNA activated) is an interferon (IFN)-induced serine-threonine protein kinase and is one of the key mediators in IFN's cellular actions. Although double-stranded (ds) RNA is the most relevant PKR activator during viral infections, PACT acts as a stress-modulated activator of PKR and is an important regulator of PKR dependent signaling pathways in the absence of viral infections. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In the present study, we have investigated the functional significance of PACT-PACT interaction in mediating PKR activation in response to cellular stress. Our results suggest that enhanced interaction between PACT molecules when PACT is phosphorylated in response to stress signals on serines 246 and 287 is essential for efficient PKR activation. Using a point mutant of PACT that is deficient in PACT-PACT interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.     

p38 mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation.

Basic fibroblast growth factor (FGF-2) is a member of a family of polypeptides that have roles in a wide range of biological processes. To determine why different cell types show distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF-2 (PC12 cells) was compared with that present on the surface of a cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a specific inhibitor of extracellular signal-regulated kinase activation blocks differentiation but has little effect on proliferation. In contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this kinase block proliferation but have no effect on differentiation. This effect on proliferation is specific for FGF-2 since the same concentrations of inhibitors have little or no effect on proliferation induced by serum.     

Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. aeruginosa.     

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.     

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.