RESUMEN
The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.
Asunto(s)
Proteínas Portadoras/metabolismo , Interferones/fisiología , Proteínas de Unión al ARN , Ribonucleoproteínas/metabolismo , eIF-2 Quinasa/metabolismo , Células 3T3 , Animales , Apoptosis , Activación Enzimática , RatonesRESUMEN
Catalysis of the formation of reactive oxygen species (RO2S) by low molecular weight complexes of iron has been implicated in several pathological conditions in the retina since photoreceptors and retinal pigment epithelial cells are likely to be especially sensitive to RO2S. Since protective proteins cannot cross the blood-retinal barrier, it is likely that the retina performs its own protective functions by synthesizing proteins that bind iron and nonprotein iron complexes, the major catalysts of RO2S generation. Investigations were carried out to determine whether pigment epithelial cells are themselves sensitive to iron-generated RO2S and whether apo-transferrin and apo-hemopexin, known to be made locally in the retina, can perform a protective function. In 51Cr release assays, the toxicity of exogenous RO2S including hydrogen peroxide or superoxide (generated by xanthine oxidase/hypoxanthine) to human retinal pigment epithelial cells was inhibited by the iron chelators, desferrioxamine and apo-transferrin. Free but not protein-bound ferric iron and heme exacerbated the toxic effect. The toxic effect of heme was abolished by the heme-scavenging, extracellular antioxidant, apo-hemopexin, and also by exogenous bovine serum albumin. In addition, heme toxicity was inhibited by a 3 h preincubation of cells with either heme, apo-hemopexin, or heme-hemopexin 24 h prior to the toxicity assay. It is concluded, first, that toxic effects of iron and heme can be prevented by apo-transferrin or apo-hemopexin and, second, that exposure of RPE cells to free heme or hemopexin sets in motion a series of biochemical events resulting in protection against oxidative stress. It is probable that these include heme oxygenase induction.