RESUMEN
Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1 but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins, whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1 and also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.
Asunto(s)
Arsenicales/metabolismo , Arsenitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Modelos BiológicosRESUMEN
The cell wall integrity (CWI) signaling pathway is best known for its roles in cell wall biogenesis. However, it is also thought to participate in the response to genotoxic stress. The stress-activated protein kinase Mpk1 (Slt2, is activated by DNA damaging agents through an intracellular mechanism that does not involve the activation of upstream components of the CWI pathway. Additional observations suggest that protein kinase C (Pkc1), the top kinase in the CWI signaling cascade, also has a role in the response to genotoxic stress that is independent of its recognized function in the activation of Mpk1. Pkc1 undergoes hyper-phosphorylation specifically in response to genotoxic stress; we have found that this requires the DNA damage checkpoint kinases Mec1 (Mitosis Entry Checkpoint) and Tel1 (TELomere maintenance), but not their effector kinases. We demonstrate that the casein kinase 1 (CK1) ortholog, Hrr25 (HO and Radiation Repair), previously implicated in the DNA damage transcriptional response, associates with Pkc1 under conditions of genotoxic stress. We also found that the induced association of Hrr25 with Pkc1 requires Mec1 and Tel1, and that Hrr25 catalytic activity is required for Pkc1-hyperphosphorylation, thereby delineating a pathway from the checkpoint kinases to Pkc1. We used SILAC mass spectrometry to identify three residues within Pkc1 the phosphorylation of which was stimulated by genotoxic stress. We mutated these residues as well as a collection of 13 phosphorylation sites within the regulatory domain of Pkc1 that fit the consensus for CK1 sites. Mutation of the 13 Pkc1 phosphorylation sites blocked hyper-phosphorylation and diminished RNR3 (RiboNucleotide Reductase) basal expression and induction by genotoxic stress, suggesting that Pkc1 plays a role in the DNA damage transcriptional response.
RESUMEN
Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.
Asunto(s)
Hongos , Estrés Fisiológico , Brasil , Hongos/fisiologíaRESUMEN
Two stress-activated MAP kinase (SAPK) pathways in Saccharomyces cerevisiae respond to osmotic imbalances. The High Osmolarity Glycerol (HOG) pathway is activated in response to hyper-osmotic stress, whereas the Cell Wall Integrity (CWI) pathway is activated in response to hypo-osmotic stress. However, there is also evidence of complex interplay and crosstalk between the two pathways. For example, treatment with zymolyase, a mixture of cell wall degrading enzymes, is known to activate the SAPK Hog1 of the HOG pathway and the SAPK Mpk1 of the CWI pathway sequentially, with Mpk1 activation dependent upon Hog1. Additionally, the PTP2- and PTP3-encoded tyrosine-specific protein phosphatases play a key role in down-regulation of Hog1, but may also down-regulate Mpk1. In this study, we show that hyperactivation of Mpk1 in a ptp2 ptp3 null mutant is an indirect consequence of Hog1 hyperactivation, which induces accumulation of intracellular glycerol and an attendant hypo-osmotic stress. Mpk1 hyperactivity in the absence of PTP2 and PTP3 was suppressed by a hog1 null mutation, or by restoration of osmotic balance with a constitutive form of the glycerol channel Fps1. We found similarly that activation of Mpk1 in response to zymolyase treatment is partly a consequence of Hog1-driven glycerol accumulation. Thus, we have identified two conditions in which glycerol serves as a mediator of crosstalk between the HOG and CWI pathways.
Asunto(s)
Glicerol , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Saccharomyces cerevisiae , Activación Enzimática , Glicerol/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Presión Osmótica , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP's CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP's CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies.IMPORTANCE Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.
Asunto(s)
Caspofungina/farmacología , Pared Celular/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , Sesquiterpenos/farmacología , Xantonas/farmacología , Antifúngicos/farmacología , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Simulación del Acoplamiento Molecular , Saccharomyces cerevisiae/efectos de los fármacos , Análisis de Secuencia de ARNRESUMEN
The yeast high-osmolarity glycerol (HOG) stress-activated protein kinase Hog1 is activated in response to hyperosmotic stress, inducing the production and retention of glycerol to restore osmotic balance. Hog1 promotes retention of glycerol through closure of the plasma-membrane glycerol channel Fps1. Treatment of yeast with the toxic trivalent metalloid arsenite (As(III)) also activates Hog1 as part of a protective response in which Hog1 closes Fps1, the main entry port for As(III). In this study, we investigated how cells treated with As(III) avoid creating a new stress caused by the accumulation of glycerol in the absence of hyperosmotic stress conditions. We found that As(III) treatment did not induce glycerol accumulation and, in fact, blocked the accumulation of glycerol induced by constitutive Hog1 activity. We demonstrated that As(III) blocks glycerol production indirectly after its metabolic activation to methylarsenite (MAs(III)), which is a potent inhibitor of glycerol-3-phosphate dehydrogenase. Finally, we used a biotinylated arsenic probe to establish that Cys306 of yeast Gpd1, a highly conserved residue within the active site, is the key target of MAs(III). Conservative mutations at this residue greatly diminished Gpd1 activity. This study offers insight into mechanisms by which SAPK outputs are tailored to specific stressors.
Asunto(s)
Arsenitos/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Arsenitos/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Mutación , Presión Osmótica/fisiología , Fosforilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. Our recent findings have begun to address two important and related questions. First, do various stresses activate a SAPK through common pathways initiated at the cell surface, or through alternative, intracellular inputs? Second, how does an activated SAPK mount a specific response appropriate to the particular stress experienced? Our work has uncovered the mechanisms by which two stresses, arsenite treatment and DNA damage, stimulate the yeast SAPKs Hog1 and Mpk1, respectively. We found that these stresses activate the SAPKs through intracellular inputs that modulate their basal phosphorylation, rather than by activation of the protein kinase cascades known to stimulate them. Both stresses act through targeting, in different ways, the tyrosine-specific or dual-specificity protein phosphatases that normally maintain the SAPKs in a low-activity state. Previous work has demonstrated that basal signal flux through SAPK pathways is important for the sensitivity and dynamic response to external signals. Our work reveals that basal activity of SAPKs is additionally important to allow SAPK activation by intracellular inputs that modulate that activity. Additionally, because different stressors may activate SAPKs by modulation of basal signal through inputs at distinct nodes along the canonical activation pathway, stress-specific SAPK outputs may be controlled, in part, by the specific intracellular mechanisms of their activation. Thus, understanding the intracellular pathways through which various stressors activate SAPKs is likely to provide insight into how they elicit physiologically coherent responses to the specific stress experienced.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Estrés Fisiológico , Animales , Biomarcadores , Activación Enzimática , Humanos , Espacio Intracelular/metabolismoRESUMEN
Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. The Saccharomyces cerevisiae SAPK Mpk1 (Slt2) is a well-characterized component of the cell-wall integrity (CWI) signaling pathway, which responds to physical and chemical challenges to the cell wall. However, Mpk1 is also activated in response to genotoxic stress through an unknown pathway. We show that, in contrast to cell-wall stress, the pathway for Mpk1 activation by genotoxic stress does not involve the stimulation of the MAP kinase kinases (MEKs) that function immediately upstream of Mpk1. Instead, DNA damage activates Mpk1 through induction of proteasomal degradation of Msg5, the dual-specificity protein phosphatase principally responsible for maintaining Mpk1 in a low-activity state in the absence of stress. Blocking Msg5 degradation in response to genotoxic stress prevented Mpk1 activation. This work raises the possibility that other Mpk1-activating stressors act intracellularly at different points along the canonical Mpk1 activation pathway.
Asunto(s)
Daño del ADN/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Pared Celular/metabolismo , Citoplasma/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de SeñalRESUMEN
Stress-activated MAPKs (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. In this study, we delineate the intracellular signaling pathway by which the trivalent toxic metalloid arsenite [As(III)] activates the yeast SAPK Hog1. We demonstrate that, to activate Hog1, As(III) must enter the cell through the glycerol channel Fps1 and must be metabolized to methyl arsenite [MAs(III)] by the dimeric methyltransferase Mtq2:Trm112. We found that Mtq2:Trm1 displays SAM-dependent methyltransferase activity toward both As(III) and MAs(III). Additionally, we present genetic and biochemical evidence that MAs(III), but not As(III), is a potent inhibitor of the protein tyrosine phosphatases (Ptp2 and Ptp3) that normally maintain Hog1 in an inactive state. Inhibition of Ptp2 and Ptp3 by MAs(III) results in elevated Hog1 phosphorylation without activation of the protein kinases that act upstream of the SAPK and raises the possibility that other Hog1-activating stressors act intracellularly at different points along the canonical Hog1 activation pathway. Finally, we show that arsenate [As(V)], a pentavalent form of arsenic, also activates Hog1, but through a pathway that is distinct from that of As(III) and involves activation of the Hog1 MEK Pbs2.
Asunto(s)
Arsenitos/farmacología , Espacio Intracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Estrés Fisiológico , Metilación , Modelos Biológicos , Saccharomyces cerevisiae/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
The plasma membrane aquaglyceroporin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Fps1 functions as a homotetramer, and control of its channel activity in response to hyperosmotic shock involves a redundant pair of fungus-specific regulators, Rgc1 and Rgc2 (regulators of the glycerol channel), and the mitogen-activatd protein kinase (MAPK) Hog1 (high-osmolarity glycerol response). Rgc1 and Rgc2 maintain Fps1 in an open-channel state by binding to its C-terminal cytoplasmic domain. Phosphorylation of Rgc1 and Rgc2 by Hog1 induces their eviction from Fps1 and consequent channel closure. In the absence of Fps1 channel function, cells experience chronic cell wall stress, which may be exploited for antifungal drug development. We show here that Rgc1 and Rgc2 form homodimers and heterodimers with each other and that dimer formation of Rgc2 is mediated by its N-terminal domain. Mutations that prevent Rgc2 dimerization block its ability to open Fps1. Therefore, the Rgc-Rgc dimer interface might be an attractive drug target.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicerol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Immunoblotting , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Mutación/genética , Concentración Osmolar , Fosforilación , Multimerización de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
The aquaglyceroprin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Control of Fps1 channel activity in response to hyperosmotic shock involves a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain. This interaction involves a tripartite pleckstrin homology (PH) domain within Rgc2 and a partial PH domain within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the rapid eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Presión Osmótica/fisiología , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Many fungal species use glycerol as a compatible solute with which to maintain osmotic homeostasis in response to changes in external osmolarity. In Saccharomyces cerevisiae, intracellular glycerol concentrations are regulated largely by the high osmolarity glycerol (HOG) response pathway, both through induction of glycerol biosynthesis and control of its flux through the plasma membrane Fps1 glycerol channel. The channel activity of Fps1 is also controlled by a pair of positive regulators, Rgc1 and Rgc2. In this study, we demonstrate that Candida glabrata, a fungal pathogen that possesses two Fps1 orthologs and two Rgc1/-2 orthologs, accumulates glycerol in response to hyperosmotic stress. We present an initial characterization of mutants with deletions in the C. glabrata FPS1 (CAGL0C03267 [www.candidagenome.org]) and FPS2 (CAGL0E03894) genes and find that a double mutant accumulates glycerol, experiences constitutive cell wall stress, and is hypersensitive to treatment by caspofungin, an antifungal agent that targets the cell wall. This mutant is cleared more efficiently in mouse infections than is wild-type C. glabrata by caspofungin treatment. Finally, we demonstrate that one of the C. glabrata RGC orthologs complements an S. cerevisiae rgc1 rgc2 null mutant, supporting the conclusion that this regulatory assembly is conserved between these species.
Asunto(s)
Candida glabrata/metabolismo , Proteínas Fúngicas/metabolismo , Glicerol/metabolismo , Porinas/metabolismo , Estrés Fisiológico , Animales , Antifúngicos/farmacología , Candida glabrata/genética , Candida glabrata/patogenicidad , Caspofungina , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Equinocandinas/farmacología , Proteínas Fúngicas/genética , Lipopéptidos , Ratones/microbiología , Mutación , Concentración Osmolar , Porinas/genéticaRESUMEN
The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.
Asunto(s)
Pared Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estructura MolecularRESUMEN
The Saccharomyces cerevisiae Fps1 glycerol channel is a member of the major intrinsic protein (MIP) family of plasma membrane channel proteins that functions in osmoregulatory pathways to transport glycerol passively out of the cell. The MIP family is subdivided into members that are selectively permeable to water (aquaporins) and those permeated by glycerol (aquaglyceroporins or glycerol facilitators). Although aquaporins function as homo-tetramers with each monomer possessing its own channel, previous studies have suggested that aquaglyceroporins may function as monomers. Here we provide both genetic and biochemical evidence that Fps1 functions as a homotetramer to regulate glycerol transport in yeast.
Asunto(s)
Glicerol/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Transporte Biológico , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Concentración Osmolar , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Mpk1 MAPK of the yeast cell wall integrity pathway uses a noncatalytic mechanism to activate transcription of stress-induced genes by recruitment of initiation factors to target promoters. We show here that Mpk1 additionally serves a function in transcription elongation that is also independent of its catalytic activity. This function is mediated by an interaction between Mpk1 and the Paf1 subunit of the Paf1C elongation complex. A mutation in Paf1 that blocks this interaction causes a specific defect in transcription elongation of an Mpk1-induced gene, which results from Sen1-dependent premature termination through a Nab3-binding site within the promoter-proximal region of the gene. Our findings reveal a regulatory mechanism in which Mpk1 overcomes transcriptional attenuation by blocking recruitment of the Sen1-Nrd1-Nab3 termination complex to the elongating polymerase. Finally, we demonstrate that this mechanism is conserved in an interaction between the human ERK5 MAPK and human Paf1.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Glucosiltransferasas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas , Factores de TranscripciónRESUMEN
The Mpk1 MAP kinase of the cell wall integrity (CWI) signalling pathway induces transcription of the FKS2 gene in response to cell wall stress through a non-catalytic mechanism that involves stable association of Mpk1 with the Swi4 transcription factor. This dimeric complex binds to a Swi4 recognition site in the FKS2 promoter. The Swi6 transcription factor is also required to bind this ternary complex for transcription initiation to ensue. In this context, the Mlp1 pseudokinase serves a redundant function with Mpk1. We have identified three additional genes, CHA1, YLR042c and YKR013w, that are induced by cell wall stress through the same mechanism. We report on the behaviour of several promoter-lacZ reporter plasmids designed to detect cell wall stress transcription through this pathway.
Asunto(s)
Pared Celular/fisiología , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo , Fusión Artificial Génica , Pared Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Glucosiltransferasas/biosíntesis , Glucosiltransferasas/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Plásmidos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas de Unión al ARN , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 mitogen-activated protein kinase of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1-Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1-Swi4 complex for transcriptional activation of FKS2. Here, we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1-Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 beta-importin binds the Mpk1-regulated Swi6 NLS but not the Cdc28-regulated NLS.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Pared Celular/enzimología , Pared Celular/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Carioferinas/genética , Carioferinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Señales de Localización Nuclear/genética , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina/metabolismo , Factores de Transcripción/genéticaRESUMEN
The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Delta mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Delta mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.
Asunto(s)
Proteínas Portadoras/fisiología , Glicerol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/fisiología , Transporte Biológico , Proteínas Portadoras/genética , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos , Presión Osmótica , Fosforilación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity signaling pathway uses a noncatalytic mechanism to activate the SBF (Swi4/Swi6) transcription factor. Active Mpk1 forms a complex with Swi4, the DNA-binding subunit of SBF, conferring the ability to bind DNA. Because SBF activation is independent of Mpk1 catalytic activity but requires Mpk1 to be in an active conformation, we sought to understand how Mpk1 interacts with Swi4. Mutational analysis revealed that binding and activation of Swi4 by Mpk1 requires an intact D-motif-binding site, a docking surface common to MAPKs that resides distal to the phosphorylation loop but does not require the substrate-binding site, revealing a novel mechanism for MAPK target regulation. Additionally, we found that Mpk1 binds near the autoinhibitory C terminus of Swi4, suggesting an activation mechanism in which Mpk1 substitutes for Swi6 in promoting Swi4 DNA binding. Finally, we show that caffeine is an atypical activator of cell wall integrity signaling, because it induces phosphorylation of the Mpk1 C-terminal extension at Ser423 and Ser428. These phosphorylations were dependent on the DNA damage checkpoint kinases, Mec1/Tel1 and Rad53. Phosphorylation of Ser423 specifically blocked SBF activation by preventing Mpk1 association with Swi4, revealing a novel mechanism for regulating MAPK target specificity.
Asunto(s)
Cafeína/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Fosfodiesterasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Alelos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pared Celular/metabolismo , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/genética , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/genéticaRESUMEN
The cell wall integrity mitogen-activated protein kinase (MAPK) cascade of Saccharomyces cerevisiae drives changes in gene expression in response to cell wall stress. We show that the MAPK of this pathway (Mpk1) and its pseudokinase paralog (Mlp1) use a noncatalytic mechanism to activate transcription of the FKS2 gene. Transcriptional activation of FKS2 was dependent on the Swi4/Swi6 (SBF) transcription factor and on an activating signal to Mpk1 but not on protein kinase activity. Activated (phosphorylated) Mpk1 and Mlp1 were detected in a complex with Swi4 and Swi6 at the FKS2 promoter. Mpk1 association with Swi4 in vivo required phosphorylation of Mpk1. Promoter association of Mpk1 and the Swi4 DNA-binding subunit of SBF were codependent but did not require Swi6, indicating that the MAPK confers DNA-binding ability to Swi4. Based on these data, we propose a model in which phosphorylated Mpk1 or Mlp1 forms a dimeric complex with Swi4 that is competent to associate with the FKS2 promoter. This complex then recruits Swi6 to activate transcription. Finally, we show that human ERK5, a functional ortholog of Mpk1, is similarly capable of driving FKS2 expression in the absence of protein kinase activity, suggesting that this mammalian MAPK may also have a noncatalytic function in vivo.