RESUMEN
The fission yeast Sty1/Spc1 MAP kinase, like the mammalian JNK/SAPK and p38/CSBP1 kinases, is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, heat shock, UV light and the protein synthesis inhibitor anisomycin. Sty1 is activated by a single MAPKK, Wis1. We demonstrate that the conserved MAPKKK phosphorylation sites Ser 469 and Thr 473 in the catalytic domain of Wis1 are normally essential for Sty1 activation. However, when mildly overexpressed, a mutant Wis1 kinase lacking these conserved phosphorylation sites is able to support stress inducible gene expression and activation of the Sty1 MAP kinase in response to an oxidative or osmotic stress or to a mild heat shock. We show that phosphorylation and activation of Sty1 under these conditions is not due to inactivation of the Pyp1 MAP kinase phosphatase. These results reveal a novel MAPKKK-independent pathway by which the Wis1 MAPKK can activate the Sty1 MAPK in response to stress in fission yeast.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Secuencia Conservada , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Quinasas Quinasa Quinasa PAM , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Schizosaccharomyces/genéticaRESUMEN
The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Transducción de Señal/fisiología , Anisomicina/farmacología , Catalasa/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Activación Enzimática , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/fisiología , Genes Fúngicos/fisiología , Calor , Peróxido de Hidrógeno/farmacología , Quinasas Quinasa Quinasa PAM , Mitosis , Mutación , Cloruro de Potasio/farmacología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Fosfatasas/genética , ARN de Hongos/análisis , ARN Mensajero/análisis , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Tiamina/fisiologíaRESUMEN
The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK. We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , Quinasas Quinasa Quinasa PAM , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Proteína Quinasa CDC2 , Activación Enzimática , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Mitosis/fisiología , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Mapeo Restrictivo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
The atf1+ gene of Schizosaccharomyces pombe encodes a bZIP transcription factor with strong homology to the mammalian factor ATF-2. ATF-2 is regulated through phosphorylation in mammalian cells by the stress-activated mitogen-activated protein (MAP) kinases SAPK/JNK and p38. We show here that the fission yeast Atf1 factor is also regulated by a stress-activated kinase, Sty1. The Sty1 kinase is stimulated by a variety of different stress conditions including osmotic and oxidative stress and heat shock. Deletion of the atf1+ gene results in many, but not all, of the phenotypes associated with loss of Sty1, including sensitivity to environmental stress and inability to undergo sexual conjugation. Furthermore, we identify a number of target genes that are induced rapidly in a manner dependent upon both the Sty1 kinase and the Atf1 transcription factor. These genes include gpd1+, which is important for the response of cells to osmotic stress, the catalase gene lambda important for cells to combat oxidative stress, and pyp2+, which encodes a tyrosine-specific MAP kinase phosphatase. Induction of Pyp2 by Atf1 is direct in that it does not require de novo protein synthesis and results in a negative feedback loop that serves to control signaling through the Sty1/Wis1 pathway. We show that Atf1 associates stably and is phosphorylated by the Sty1 kinase in vitro. Taken together, these results indicate that the interaction between AM and Sty1 is direct. These findings highlight a remarkable level of conservation in transcriptional control by stress-activated MAP kinase pathways between fission yeast and mammalian cells.
Asunto(s)
Proteínas de Unión al ADN , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Factores de Transcripción/genética , Factor de Transcripción Activador 1 , Animales , Sitios de Unión , Catalasa/genética , Catalasa/metabolismo , Activación Enzimática , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Genes Supresores , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Mamíferos/fisiología , Meiosis , Mutación , Ósmosis , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero , Transducción de Señal , Estrés Fisiológico , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with G1 cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional G1 cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Candida albicans/genética , Ciclinas/genética , Glicoproteínas de Membrana , Chaperonas Moleculares , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular , Clonación Molecular , Proteínas Fúngicas/genética , Genes Fúngicos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The effects, in women, of heel lifts on EMG activity of the medial gastrocnemius and tibialis anterior muscles were analyzed to see whether they were different from those of men. Six women who wear high-heeled shoes regularly walked on a level floor with heel heights of zero, 2.5cm, 5.0cm, and 7.5cm. Results showed that as heel height increased, there were significant and progressive decreases in gastrocnemius and tibialis anterior EMG activity during walking. This decrease in gastrocnemius EMG activity in women is the same as in men, but the decrease in tibialis anterior EMG activity in women is opposite to that seen in men. The difference in responses of the tibialis anterior muscle between men and women may be related to the habitual use of high-heeled shoes by women. Since raising the heel decreases gastrocnemius muscle activity, thereby reducing tension in the Achilles tendon in both men and women, it was felt that heel lifts may provide a useful adjunct in the management of tendoachilles bursitis, tenosynovitis of the Achilles tendon, and postoperative management of a ruptured Achilles tendon.
Asunto(s)
Pierna/fisiología , Músculos/fisiología , Zapatos , Adulto , Electromiografía , Femenino , Humanos , Masculino , Factores SexualesRESUMEN
The interhormonal relationship within the pancreatic islets have been studied by previous investigators, but the cellular interplay and the sequence of events in the islet cell's response to stimulators has remained unclear. In the present study, pancreatic islets were isolated by collagenase digestion from normal and streptozotocin-diabetic hamsters the latter being maintained with insulin treatment. The diabetic animals were used to provide A- and B-cell enriched islets. The islets from normal and diabetic hamsters were cultured in medium 199 plus 10% fetal calf serum with 0.8 or 5 mg/ml glucose. The cultures were maintained for up to seven days with medium changes every third day. At specified intervals, media were collected and assayed for insulin, glucagon and somatostatin. Our results showed the expected increased insulin secretion by the B-cells in response to high glucose. However, after two days of culture accumulative insulin secretory response was reduced and at the end of seven days was less than the insulin produced in low glucose medium. Glucagon secretion by the A-cells was similar for low and high glucose media for the entire culture period. Somatostatin secretion by D-cells was stimulated by high glucose but was attenuated after 2 days. No correlation could be found between the concentration of hormone in the media and a possible effect on a specific islet secretion. However, the fact that insulin secretion by islets cultured in high glucose was decreased after two days may indicate a refractoriness produced by persistent hyperglycemia. Islets isolated from diabetic animals secreted more glucagon and less insulin than control islets. Somatostatin secretion was the same in both groups. It was concluded that paracrine relationships were relatively insignificant in the regulation of islet secretion in a prolonged culture environment and persistent high glucose reduced the B-cell response to glucose stimulation.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Glucagón/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Somatostatina/metabolismo , Animales , Células Cultivadas , Cricetinae , Femenino , Secreción de Insulina , MesocricetusRESUMEN
The covalent binding of photoactivated aflatoxin B1 ((AFB1) to DNA and its effect on template activity have been investigated. AFB1 in its ground state complexes more preferentially with denatured DNA than with native DNA. The covalent linkage between 3H-AFB1 and DNA under near-ultraviolet light irradiation shows 1 AFB1 per 529 Pi of calf thymus DNA. The photoaddition also induces a conformational change of the DNA, as detected by circular dichroism. Because the photobinding of AFB2 to DNA is negligible (1 AFB2 per 5485 Pi), we suggest that the 8,9-C=C bond is the major binding site of AFB1. The AFB1-DNA adduct shows a substantial inhibition of its template activity for DNA synthesis (by 52%) and RNA transcription (by 74%) in vitro. Since both AFB1 and AFB2 themselves had little inhibitory effect on the activities of DNA and RNA polymerases, the alteration of DNA by photoactivated AFB1 and AFB2 is responsible for the dramatic reduction of template activity in DNA and RNA synthesis. Short-chain polynucleotides retained by type VS membrane filter (0.025-micrometers pore size) indicate that premature chain termination occurred in transcribing the AFB1-DNA and AFB2-DNA adducts as the result of disrupted movement of the enzymes along the DNA chain.