RESUMEN
PURPOSE: To characterize the cellular distribution and DNA binding activity of the nuclear factor kappaB (NF-KappaB) in a model of radiation-induced lung damage in the rat. MATERIAL AND METHODS: The right lung of Fischer rats was irradiated with a single dose of 20 Gy. The cellular distributions of NF-KappaB proteins and mRNA were detected with immunohistochemistry and in-situ hybridization respectively. The DNA binding activity of NF-KappaB, nuclear and cytoplasmic levels of NF-KappaB proteins, and kinase activity towards IkappaBalpha (IKappaBAlpha) were determined using electrophoretic mobility shift assays (EMSA), Western blots and kinase assays, respectively. The mRNA level of interleukin 6 (IL-6) was determined using quantitative room temperature polymerase chain reaction. RESULTS: There was a continuous elevation of NF-KappaB DNA binding activity in the rat lung after ionizing irradiation over 6 months. The irradiated lung tissue exhibited an increased kinase activity towards IKappaBAlpha and a selective loss of nuclear IKappaBAlpha. The NF-KappaB-DNA binding complex switched from p50-p65 heterodimers in normal lung tissue to p50 homodimers in irradiated lung tissue. The increased level of IL-6 mRNA suggests transcriptional activation of NF-KappaB-dependent genes in the irradiated rat lung. CONCLUSIONS: The DNA binding activity of NF-KappaB is continuously activated after irradiation of the rat lung by loss of nuclear IKappaBAlpha. This might play a role in sustaining chronic inflammation and hyperproliferation of mesenchymal cells after irradiation.
Asunto(s)
ADN/metabolismo , Proteínas I-kappa B/metabolismo , Pulmón/efectos de la radiación , FN-kappa B/metabolismo , Animales , Western Blotting , Bronquios/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Relación Dosis-Respuesta en la Radiación , Femenino , Fibrosis , Vectores Genéticos , Inmunohistoquímica , Hibridación in Situ , Interleucina-6/metabolismo , Pulmón/metabolismo , Pulmón/ultraestructura , Lesión Pulmonar , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Inhibidor NF-kappaB alfa , FN-kappa B/biosíntesis , Subunidad p50 de NF-kappa B , Unión Proteica , Alveolos Pulmonares/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factor de Transcripción ReIARESUMEN
Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae. The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration [Vojtek, A. B., and Fraenkel, D. G. (1990) Eur. J. Biochem. 190, 371-375]; however, a physiological role of the modification causing the dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate exchange at position 14; Behlke et al. (1998) Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation. The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzyme concentrations. The kinetic data indicated that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et al. (1986) Eur. J. Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was detected at the high concentration of hexokinase 2 presumed to occur in the cell. Pseudophosphorylation by glutamate substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2. The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.
Asunto(s)
Hexoquinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Serina/metabolismo , Sustitución de Aminoácidos/genética , Dimerización , Activación Enzimática/genética , Estabilidad de Enzimas/genética , Ácido Glutámico/genética , Hexoquinasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/genética , Serina/genética , Especificidad por Sustrato/genéticaRESUMEN
Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominently labeled from 32P-inorganic phosphate after a shift of cells to medium with low glucose concentration [Vojtek, A. B., & Fraenkel D. G. (1990) Eur. J. Biochem, 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observation of a single tryptic peptide difference, its sequencing and size determination by mass spectrometry, and by mutation to alanine, which prevents phosphorylation in vivo. Although protein kinase A was unlikely to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro phosphorylation by protein kinase A at serine-15 could be shown by labeling and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.