RESUMO
Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Delta cells were defective in their expression. Consistently, smp1Delta cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Delta cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição MEF2 , Proteínas de Membrana Transportadoras/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Fatores de Regulação Miogênica , Pressão Osmótica , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
BACKGROUND & AIMS: Chromosomal instability, a hallmark of most colorectal cancers, has been related to altered chromosome segregation and the consequent deficit in genetic integrity. A role for the tumor suppressor gene APC has been proposed in colorectal cancer that leads to compromised chromosome segregation even though the molecular mechanism is not yet understood. Here, we tackled the genetic basis for the contribution of APC to chromosomal instability in familial adenomatous polyposis and sporadic colorectal cancer. METHODS: We have used video-microscopy of primary cultures and molecular genetic methods to address these issues in human samples and in genetically defined mouse models that either recapitulate the familial adenomatous polyposis syndrome (Apc(1638N)), or develop tumors in the absence of APC mutations (pvillin-KRASV12G). RESULTS: Mutations in APC were associated with an increased incidence in cell cycle defects during the completion of cytokinesis. Transcriptome analysis performed on mouse models indicated a significant up-regulation of genes that regulate accurate mitosis. Notably, we identified up-regulated expression of BUB1B and MAD2L1, 2 genes that are involved in the mitotic checkpoint, but have so far not been implicated in chromosomal instability induced by APC loss of function. In vitro modulation of APC expression suggested a causal association for this upregulation, which was consistently found in sporadic and familial adenomatous polyposis lesions, as an early event in colorectal tumorigenesis. CONCLUSIONS: In addition to the known function of APC during correct spindle assembly and positioning, we propose a concomitant involvement of APC in the surveillance mechanism of accurate mitosis.
Assuntos
Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inativação Gênica , Genes APC , Proteínas Quinases/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular , Instabilidade Cromossômica , Neoplasias Colorretais/metabolismo , Citocinese , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos , Mitose/genética , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Estatmina/genética , Estatmina/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Expression of the HXT1 gene, which encodes a low affinity glucose transporter in Saccharomyces cerevisiae, is regulated positively in response to glucose by the general glucose induction pathway, involving the Snf3/Rgt2 membrane glucose sensors, the SCF-Grr1 ubiquitination complex and the Rgt1 transcription factor. In this study we show that, in addition to the glucose signaling pathway, regulation of HXT1 expression also requires the HOG pathway. Deletion of components in the glucose signaling pathway or in the HOG pathway results in impaired HXT1 expression. Genetic analyses showed that, whereas the glucose signaling pathway regulates HXT1 through modulation of the Rgt1 transcription factor, the HOG pathway modulates HXT1 through regulation of the Sko1-Tup1-Ssn6 complex. Coordinated regulation of the two signaling pathways is required for expression of HXT1 by glucose and in response to osmostress.