RESUMEN
The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H(2)O(2) stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability.
Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Factores de Transcripción Activadores/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Factor de Transcripción Activador 1/genética , Factores de Transcripción Activadores/genética , Dimerización , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Peróxido de Hidrógeno/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Presión Osmótica/efectos de los fármacos , Oxidantes/farmacología , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Eliminación de Secuencia , Sorbitol/farmacología , Edulcorantes/farmacologíaRESUMEN
There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.
Asunto(s)
Campos Electromagnéticos , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/efectos de la radiación , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Proteómica , Proteínas de Schizosaccharomyces pombe/química , Electricidad EstáticaRESUMEN
Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress- and Sty1-dependent patterns of regulation.