RESUMEN
We have cloned from a chicken intestinal cDNA library Cmdr1, the first avian P-glycoprotein. Cmdr1 is 67% and 69% identical to proteins encoded by the human MDR1 and MDR2 genes, respectively. Functional expression of Cmdr1 in both mouse NIH 3T3 and yeast cells demonstrated that Cmdr1 represents the avian ortholog of human Mdr1, since it confers resistance to several anticancer drugs and the fluorescent dye rhodamine 6G. Northern and immunoblot analysis showed that CMDR1 is highly expressed throughout the intestine and in the liver, and to a considerable extent in kidney, brain, lung, heart, eye and follicles. In situ hybridization revealed a cell type-specific expression of CMDR1 in the intestinal epithelium, with high levels in the villi of the small and large intestine as well as crypt cells. These data suggest that Cmdr1 could play a role in intestinal detoxification. Most interestingly, immunoblotting showed that Cmdr1 is also expressed in ovarian tissues, particularly in theca cells, the major site for ovarian estrogen production in birds. Indeed, competition experiments indicated that Cmdr1 interacts with estradiol, since rhodamine 6G efflux was efficiently blocked by estradiol in NIH 3T3 cells expressing Cmdr1. Rhodamine efflux was also blocked by PSC-833, a specific inhibitor of steroid-transporting P-glycoproteins from mammalian cells. We propose that Cmdr1 in ovarian cells could be involved in the cell type-specific transport or release of estrogen that is essential for avian follicular development.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Estradiol/metabolismo , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Mucosa Intestinal , Ratones , Datos de Secuencia Molecular , Ovario/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We show here using synchronized Swiss mouse 3T3 fibroblasts that p70 S6 kinase (p70S6k) and mitogen-activated protein kinases (p42mapk/p44mapk) are not only activated at the G0/G1 boundary, but also in cells progressing from M into G1. p70S6k activity increases 20-fold in G1 cells released from G0. Throughout G1, S, and G2 it decreases constantly, so that during M phase low kinase activity is measured. The kinase is reactivated 10-fold when cells released from a nocodazole-induced metaphase block enter G1 of the next cell cycle. p42mapk/p44mapk in G0 cells are activated transiently early in G1 and are reactivated late in mitosis after nocodazole release. p70S6k activity is dependent on permanent signaling from growth factors at all stages of the cell cycle. Immunofluorescence studies showed that p70S6k and its isoform p85S6k become concentrated in localized spots in the nucleus at certain stages in the cell cycle. Cell cycle-dependent changes in p70S6k activity are associated with alterations in the phosphorylation state of the protein. However, examination of the regulation of a p70S6k mutant in which the four carboxyl-terminal phosphorylation sites are changed to acidic amino acids suggests that a mechanism independent of these phosphorylation sites controls the activity of the enzyme during the cell cycle.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Regulación Enzimológica de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas S6 RibosómicasRESUMEN
Treatment of Swiss mouse 3T3 fibroblasts with certain cyclic nucleotide phosphodiesterase inhibitors (theophylline, SQ 20,006, and MY-5445) prevents the activation of the M(r) 70,000 S6 kinase (p70S6k) induced by a variety of external stimuli. Concentrations giving half-maximal inhibition were 800, 50, and 25 microM, respectively. Western blot analysis and immunocomplex kinase assays showed that these compounds inhibit the phosphorylation and activation of p70S6k without affecting the erk-encoded mitogen-activated protein (MAP) kinases or the rsk-encoded S6 kinase (p90rsk). A distinct collection of cAMP and cGMP agonists and analogues did not suppress p70S6k activation, indicating that 1) high intracellular cyclic nucleotide concentrations do not antagonize the p70S6k pathway and 2) phosphodiesterase inhibitors block p70S6k activation by a mechanism that is independent of cAMP or cGMP production. The effect of theophylline and SQ 20,006, but not MY-5445, on p70S6k signaling may be due in part to the inhibition of a phosphatidylinositol 3-kinase that acts upstream of p70S6k. Finally, in contrast to many other cell types, cAMP and cGMP were also found to have no inhibitory effect on the MAP kinase/p90rsk signaling pathway in Swiss 3T3 fibroblasts.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Alprostadil/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Relación Dosis-Respuesta a Droga , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Cinética , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Ácidos Nicotínicos/farmacología , Fosfatidilinositol 3-Quinasas , Inhibidores de Fosfodiesterasa/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ftalazinas/farmacología , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Teofilina/farmacologíaRESUMEN
Treatment of fibroblasts with wortmannin or demethoxyviridin, two potent inhibitors of phosphatidylinositol 3-kinase, prevents the activation of ribosomal protein S6 kinase, which is induced by a variety of external stimuli. Concentrations giving 50% inhibition of 45 nM (wortmannin) and 400 nM (demethoxyviridin) were obtained when epidermal growth factor was used as an S6 kinase activator; with platelet-derived growth factor, the concentrations giving 50% inhibition were about three-times higher. Western-blot analysis and immunocomplex kinase assays showed that wortmannin and demethoxyviridin specifically block the phosphorylation and activation of p70 S6 kinase without affecting the M(r) 90,000 ribosomal S6 kinase (p90rsk) or mitogen-activated protein kinases. Consistent with the irreversible nature of the inhibition of phosphatidylinositol 3-kinase by these compounds, treatment of cells with wortmannin, followed by washing out of the inhibitor, still led to inhibition of p70 S6 kinase activation. Several S6 kinase agonists not previously known to activate phosphatidylinositol 3-kinase (A23187, bombesin and phorbol 12-myristate 13-acetate) were found to increase the production of phosphatidylinositol 3,4,5-trisphosphate in a wortmannin-sensitive manner. These results support a model in which phosphatidylinositol 3-kinase acts upstream of p70 S6 kinase in a mitogenic signalling pathway; the existence of a phosphatidylinositol 3-kinase-independent pathway is also evident.