RESUMEN
Oxidative stress is associated with cardiovascular and neurodegenerative diseases, diabetes, cancer, psychiatric disorders and aging. In order to counteract, eliminate and/or adapt to the sources of stress, cells possess elaborate stress-response mechanisms, which also operate at the level of regulating transcription. Interestingly, it is becoming apparent that the metabolic state of the cell and certain metabolites can directly control the epigenetic information and gene expression. In the fission yeast Schizosaccharomyces pombe, the conserved Sty1 stress-activated protein kinase cascade is the main pathway responding to most types of stresses, and regulates the transcription of hundreds of genes via the Atf1 transcription factor. Here we report that fission yeast cells defective in fatty acid synthesis (cbf11, mga2 and ACC/cut6 mutants; FAS inhibition) show increased expression of a subset of stress-response genes. This altered gene expression depends on Sty1-Atf1, the Pap1 transcription factor, and the Gcn5 and Mst1 histone acetyltransferases, is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters, and results in increased cellular resistance to oxidative stress. We propose that changes in lipid metabolism can regulate the chromatin and transcription of specific stress-response genes, which in turn might help cells to maintain redox homeostasis.
Asunto(s)
Cromatina , Metabolismo de los Lípidos , Estrés Oxidativo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Acetiltransferasas/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Cromatina/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Factores de Transcripción/genéticaRESUMEN
General stress responses, which sense environmental or endogenous signals, aim at promoting cell survival and fitness during adverse conditions. In eukaryotes, mitogen-activated protein (MAP) kinase-driven cascades trigger a shift in the cell's gene expression program as a cellular adaptation to stress. Here, we review another aspect of activated MAP kinase cascades reported in fission yeast: the transient inhibition of cell polarity in response to oxidative stress. The phosphorylation by a stress-activated MAP kinase of regulators of the GTPase cell division cycle 42 (Cdc42) causes a transient inhibition of polarized cell growth. The formation of growth sites depends on limiting and essential polarity components. We summarize here some processes in which inhibition of Cdc42 may be a general mechanism to regulate polarized growth also under physiological conditions.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Schizosaccharomyces , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , GTP Fosfohidrolasas/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Polaridad CelularRESUMEN
Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1-kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation.
Asunto(s)
Polaridad Celular , Proliferación Celular , Proteínas Activadoras de GTPasa/metabolismo , Estrés Oxidativo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Activadoras de GTPasa/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Transducción de Señal , Factores de Tiempo , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Stress-dependent activation of signaling cascades is often mediated by phosphorylation events, but the exact nature and role of these phosphorelays are frequently poorly understood. Here, we review which are the consequences of the stress-dependent phosphorylation of a transcription factor on gene activation. In fission yeast, the MAP kinase Sty1 is activated upon several environmental hazards and promotes cell adaptation and survival, greatly through activation of a gene program mediated by the transcription factor Atf1. Although described decades ago, the role of the phosphorylation of Atf1 by Sty1 is still a matter of debate. We present here a brief review of recent data, obtained through the characterization of several phosphorylation mutant derivatives of Atf1, demonstrating that Atf1 phosphorylation does not stabilize the factor nor stimulates its binding to DNA. Rather, it provides a structural platform of interaction with the transcriptional machinery. Based on these findings, future work will establish how this phosphorylated trans-activation domain promotes the massive gene expression shift allowing cellular adaptation to stress.
Asunto(s)
Factor de Transcripción Activador 1/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosfoproteínas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Activación Transcripcional , Factor de Transcripción Activador 1/metabolismo , Estrés Oxidativo , Fosfoproteínas/metabolismo , Fosforilación , Estrés Fisiológico/genéticaRESUMEN
Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast (Schizosaccharomyces pombe), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation.
Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Activación Transcripcional , Factor de Transcripción Activador 1/química , Factor de Transcripción Activador 1/genética , Sustitución de Aminoácidos , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Viabilidad Microbiana , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Estrés Oxidativo , Proteínas Asociadas a Pancreatitis , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Regiones Promotoras Genéticas , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismoRESUMEN
Gap junctions allow intercellular communication. Their structural subunits are four-transmembrane proteins named connexins (Cxs), which can be post-transcriptionally regulated by developmental and cellular signalling cues. Cx translation and mRNA stability is regulated by miRNAs and RNA-binding proteins (RBPs) such as human antigen R (HuR). In addition, several Cxs have also been suggested to contain 5' internal ribosome entry site (IRES) elements that are thought to allow cap-independent translation in situations such as mitosis, stress and senescence. Furthermore, several recent reports have documented internal translation of Cx mRNAs that result in N-terminally truncated protein isoforms that may have unique gap junction-independent functions [Ul-Hussain et al. (2008) BMC Mol. Biol. 9, 52; Smyth and Shaw (2013) Cell Rep. 5, 611-618; Salat-Canela et al. (2014) Cell Commun. Signal. 12, 31; Ul-Hussain et al. (2014) J. Biol. Chem. 289, 20979-20990]. This review covers the emerging field of the post-transcriptional regulation of Cxs, with particular focus on the translational control of Cx 43 and its possible functional consequences.
Asunto(s)
Comunicación Celular/genética , Conexinas/genética , Uniones Comunicantes/genética , Biosíntesis de Proteínas , Conexinas/química , Uniones Comunicantes/química , Regulación de la Expresión Génica , Humanos , Estabilidad del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribosomas/genéticaRESUMEN
BACKGROUND: Connexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation. FINDINGS: Here, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform. CONCLUSIONS: Internal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.
Asunto(s)
Conexina 43/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Línea Celular , Conexina 43/genética , Células HeLa , Humanos , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Serina-Treonina Quinasas TORRESUMEN
DNA interaction with scorpiand azamacrocycles has been achieved through modulation of their binding affinities. Studies performed with different experimental techniques provided evidence that pH or metal-driven molecular reorganizations of these ligands regulate their ability to interact with calf thymus DNA (ctDNA) through an intercalative mode. Interestingly enough, metal-driven molecular reorganizations serve to increase or decrease the biological activities of these compounds significantly.