RESUMEN
Ste11 is the mitogen-activated protein kinase (MAPK) kinase kinase in the MAPK cascades that mediate mating, high osmolarity glycerol, and filamentous growth responses in Saccharomyces cerevisiae. We show stimulation of the mating pathway by pheromone promotes an accelerated turnover of Ste11 through a MAPK feedback and ubiquitin-dependent mechanism. This degradation is pathway specific, because Ste11 is stable during activation of the high osmolarity glycerol pathway. Because the steady-state amount of Ste11 does not change significantly during pheromone induction, we infer that maintenance of MAPK activation involves repeated cycles in which naive Ste11 is activated and then targeted for degradation. This model predicts that elimination of active Ste11 would rapidly curtail MAPK activation upon attenuation of the upstream signal. This prediction is confirmed by the finding that blocking ubiquitin-dependent Ste11 degradation during pheromone induction abolishes the characteristic attenuation profile for MAPK activation.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Feromonas/biosíntesis , Ubiquitina/metabolismo , Estabilidad de Enzimas , Retroalimentación , Glicerol/metabolismo , Modelos Biológicos , Concentración Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome. Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo. As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety. Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12. Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7. These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK.
Asunto(s)
Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Feromonas/metabolismo , Proteínas Quinasas/biosíntesis , Saccharomyces cerevisiae , Alineación de SecuenciaRESUMEN
Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Unión al Calcio/genética , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transducción de Señal , Secuencia de Aminoácidos , Supervivencia Celular , Pared Celular/metabolismo , Eliminación de Gen , Glicosilación , Proteínas Fluorescentes Verdes , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes/metabolismo , Manosidasas/farmacología , Glicoproteínas de Membrana , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis , Feromonas/farmacología , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Temperatura , Factores de Tiempo , alfa-ManosidasaRESUMEN
Ash1 (for asymmetric synthesis of HO) was first uncovered in genetic screens that revealed its role in mating-type switching. Ash1 prevents HO expression in daughter cells. Because Ash1 has a zinc finger-like domain related to that of the GATA family of transcription factors, it presumably acts by repressing HO transcription. Nonswitching diploid cells also express Ash1, suggesting it could have functions in addition to regulation of HO expression. We show here that Ash1 has an essential function for pseudohyphal growth. Our epistasis analyses are consistent with the deduction that Ash1 acts separately from the mitogen-activated protein kinase cascade and Ste12. Similarly to the case in yeast form cells, Ash1 is asymmetrically localized to the nuclei of daughter cells during pseudohyphal growth. This asymmetric localization reveals that there is a previously unsuspected daughter cell-specific function necessary for pseudohyphal growth.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Factores de Transcripción/metabolismo , Dedos de Zinc , Compartimento Celular , Diferenciación Celular , Epistasis Genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Morfogénesis/genética , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner. The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1. In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis. Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade. Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation. Both proteins also affect the timing and extent of Mpk1 activation. This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation. Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2. Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1. This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Microfilamentos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Feromonas/farmacología , Proteína Quinasa C , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Transducción de Señal , Diferenciación Celular/fisiología , Proteínas del Citoesqueleto , Activación Enzimática , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Modelos Biológicos , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Reproducción/fisiología , Factores de Tiempo , Transcripción GenéticaRESUMEN
The Ste12p transcription factor controls the expression of Ty1 transposable element insertion mutations and genes whose products are required for mating in Saccharomyces cerevisiae. The binding site for Ste12p is a consensus DNA sequence known as a pheromone response element (PRE). Upstream activating sequences (UASs) derived from known Ste12p-dependent genes have previously been characterized to require either multiple PREs or a single PRE coupled to a binding site for a second protein. The Ste12p-dependent UAS from Ty1, called a sterile response element (SRE), is of the second type and is comprised of a PRE and an adjacent TEA (TEF-1, Tec1, and AbaA motif) DNA consensus sequence (TCS). In this report, we show by UV cross-linking analysis that two proteins, Ste12p and a protein with an apparent size of 72 kDa, directly contact the Ty1 SRE. Other experiments show that Tec1p is required for formation of the Ty1 SRE protein-DNA complex and is physically present in the complex. These results establish a direct role for Tec1p in the Ty1 SRE and yet another set of combinatorial interactions that achieve a qualitatively distinct mode of transcriptional regulation with Ste12p.
Asunto(s)
Elementos Transponibles de ADN/fisiología , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Bases , Secuencia de Consenso , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/química , Regulación Fúngica de la Expresión Génica/genética , Datos de Secuencia Molecular , Peso Molecular , Saccharomyces cerevisiae/genética , Activación Transcripcional/genética , Rayos UltravioletaRESUMEN
Ste7 is a MEK (MAPK/ERK kinase) family member that functions in the pheromone induced mating response pathway of Saccharomyces cerevisiae. We analysed the catalytic competence and in vivo function of Ste7 variants that have alterations of stimulatory and feedback phosphorylation sites. These analyses led us to unanticipated insights into two separate feedback mechanisms that impede the output of the mating response MAPK activation pathway.
Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Retroalimentación/fisiología , Levaduras/enzimologíaRESUMEN
In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MAPK activation cascade mediating this signal is made up of Ste11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades. Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway. Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Saccharomyces cerevisiae/enzimología , Transducción de Señal , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , MutaciónRESUMEN
Ste7p and Mkk1p are MEK (MAPK/ERK kinase) family members that function in the mating and cell integrity signal transduction pathways in Saccharomyces cerevisiae. We selected STE7 and MKK1 mutations that stimulated their respective pathways in the absence of an inductive signal. Strikingly, serine-to-proline substitutions at analogous positions in Ste7p (position 368) and Mkk1p (position 386) were recovered by independent genetic screens. Such an outcome suggests that this substitution in other MEKs would exhibit similar properties. The Ste7p-P368 variant has higher basal enzymatic activity than Ste7p but still requires induction to reach full activation. The higher activity associated with Ste7p-P368 allows it to compensate for defects in the cell integrity pathway, but it does so only when it is overproduced or when Ste5p is missing. This behavior suggests that Ste5p, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7p specificity.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/fisiología , Transducción de Señal , Alelos , Secuencia de Aminoácidos , Cruzamientos Genéticos , Genes Fúngicos , Variación Genética , Genotipo , Histidina/metabolismo , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Mitogen-activated protein kinases function in at least five, physiologically distinct, signaling pathways in yeast. These include pathways that mediate response to mating pheromone, pseudohyphal development and invasive growth, cell integrity, sporulation, and response to high extracellular osmolarity. These kinases and their upstream activating kinases comprise signaling modules that, in at least some cases, exist as multiprotein complexes. Studies during the past year have revealed that the Ste5 protein of the mating pheromone response pathway serves as a scaffold to promote interactions among the protein kinases in this pathway, and to prevent their interaction with kinases of other modules.
Asunto(s)
Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Transducción de Señal/fisiología , Activación Enzimática , Glicerol/metabolismo , Mitógenos , Concentración Osmolar , Proteína Quinasa C/fisiologíaRESUMEN
Intracellular signaling from receptor tyrosine kinases in mammalian cells results in activation of a signal cascade that includes the guanine nucleotide-binding protein Ras and the protein kinases Raf, MEK [mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase], and MAPK. MAPK activation that is dependent on the coupling of Ras and Raf was reconstituted in yeast. Yeast genes were isolated that, when overexpressed, enhanced the function of Raf. One of them is identical to BMH1, which encodes a protein similar to members of the mammalian 14-3-3 family. Bacterially synthesized mammalian 14-3-3 protein stimulated the activity of Raf prepared from yeast cells expressing c-Raf-1. Thus, the 14-3-3 protein may participate in or be required for activation of Raf.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Tirosina 3-Monooxigenasa , Proteínas ras , Proteínas 14-3-3 , Secuencia de Aminoácidos , Activación Enzimática , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-raf , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiaeRESUMEN
Pheromones induce haploid cells of Saccharomyces cerevisiae to differentiate into a mating-competent state. Ste11p is one of several protein kinases required to transmit the pheromone-induced signal and to maintain basal expression of certain mating-specific genes in the absence of pheromone stimulation. To identify potential regulators of Ste11p, we screened for suppressors that restored mating and basal transcriptional competence to a strain with a conditionally functional Ste11p. This screen uncovered a novel gene we call MOT2, for modulator of transcription. A mot2 deletion mutation leads to modest increases in the basal amounts of mRNA for several pheromone-responsive genes. Yet mot2 deletion does not affect the signal transmission activity of the pathway in either the presence or absence of pheromone stimulation. Therefore, we propose that Mot2p, directly or indirectly, represses basal transcription of certain mating-specific genes. Because mot2 deletion mutants also have a conditional cell lysis phenotype, we expect that Mot2p regulatory effects may be more global than for mating-specific gene expression.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Reguladores , Feromonas/farmacología , Regiones Promotoras Genéticas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Ubiquitina-Proteína Ligasas , Dedos de Zinc/genéticaRESUMEN
Pheromone-stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone-induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas Fúngicas/genética , Feromonas/fisiología , Proteínas Tirosina Fosfatasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN de Hongos , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Plásmidos , Proteínas Tirosina Fosfatasas/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Clonación Molecular , Secuencia de Consenso , Activación Enzimática , MAP Quinasa Quinasa 2 , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Relación Estructura-Actividad , Xenopus laevis/embriologíaRESUMEN
Mitogen-activated protein kinases are regulators of proliferation and differentiation in many eukaryotes. Studies during the last year have revealed that functionally distinct signal pathways in yeast use related protein kinase cascades for mitogen-activated protein kinase activation. These cascades act as intracellular signaling modules that are likely to be conserved from yeast to mammals.
Asunto(s)
Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Activación Enzimática/efectos de los fármacos , Mitógenos/farmacología , Proteínas Quinasas/genética , Schizosaccharomyces/enzimología , Transducción de SeñalRESUMEN
Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells. Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases. FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism. Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form. This modification of STE7 requires the STE11 kinase, which is proposed to act before STE7 during signal transmission. Surprisingly, STE7 hyperphosphorylation also requires a functional FUS3 (or KSS1) kinase. Using in vitro assays for FUS3 phosphorylation, we show that pheromone activates STE7 even in the absence of FUS3 and KSS1. Therefore, STE7 activation must precede modification of FUS3 (and KSS1). These findings suggest that STE7 hyperphosphorylation is a consequence of its activation but not the determining event.
Asunto(s)
Quinasas Quinasa Quinasa PAM , Proteínas Quinasas Activadas por Mitógenos , Péptidos/fisiología , Feromonas/fisiología , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , Proteínas Fúngicas/metabolismo , Factor de Apareamiento , Quinasas de Proteína Quinasa Activadas por Mitógenos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Pheromone-stimulated haploid yeast cells undergo a differentiation process that allows them to mate. Transmission of the intracellular signal involves threonine and tyrosine phosphorylation of the redundant FUS3 and KSS1 kinases, which are members of the MAP kinase family. FUS3/KSS1 phosphorylation depends on two additional kinases, STE11 and STE7 (refs 2, 5, 6). Genetic analyses predict an ordered pathway where STE11 acts before STE7 and FUS3/KSS1 (refs 2, 7). Here we report that STE7 is a dual-specificity kinase that modifies FUS3 at the appropriate sites and stimulates its catalytic activity in vitro. From these data and previous genetic results, we argue that STE7 is the physiological activator of FUS3. Recent indications that MAP kinase activators are related to STE7 suggest that signal transduction pathways in many, if not all, eukaryotic organisms use homologous kinase cascades.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Quinasas de Proteína Quinasa Activadas por Mitógenos , Peso Molecular , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Some Ty1 transposable-element insertion mutations of Saccharomyces cerevisiae activate adjacent-gene expression. These Ty1-activated genes are regulated similarly to certain mating genes. This report shows that the MCM1 protein, which binds to several mating genes, also binds to a transcriptional regulatory sequence in Ty1. The binding of MCM1 to Ty1 correlates with the ability of its binding site to function as a component of the Ty1 transcriptional activator. This correlation supports the idea that MCM1 is important for Ty1-activated gene expression. At mating-gene promoters, MCM1 binds with coactivators or repressors such as STE12, alpha 1, or alpha 2. In contrast, MCM1 binds without these associated DNA-binding proteins at its site in Ty1. This finding suggests that its role in Ty1-mediated transcription is different from that at mating genes.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Secuencia de Consenso , ADN de Hongos/metabolismo , Proteína 1 de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Transcripción GenéticaRESUMEN
STE4 encodes the beta-subunit of a heterotrimeric guanine nucleotide-binding protein (G protein) that is an early and essential component of the pheromone signal transduction pathway. From a ste4 deletion strain we have isolated both dominant and recessive suppressors that show increased transcription of pheromone responsive genes and have regained the ability to mate, albeit at a low level. Each of these suppressor mutations suppresses ste4 and ste5 deletions but not deletions in STE7, STE11, or STE12. Among the dominant mutations, we have identified two alleles of STE11, a gene that encodes a protein kinase activity essential for mating. One allele contains an alteration in the putative regulatory domain of the protein kinase; the second allele has an alteration in the catalytic site. In strains carrying these mutations, a second protein kinase required for mating, STE7, becomes hyperphosphorylated, just as it does in wild-type cells treated with pheromone. Thus, a protein kinase cascade appears to be an essential feature of the response pathway and probably connects the receptor/G protein to an identified transcription factor, STE12.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas de Unión al GTP/metabolismo , Quinasas Quinasa Quinasa PAM , Péptidos/metabolismo , Feromonas/metabolismo , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Levaduras/enzimología , Alelos , Clonación Molecular , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Factor de Apareamiento , Fenotipo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Supresión Genética , Transcripción Genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
The STE11 gene of Saccharomyces cerevisiae is one of several genes required for mating between two haploid cell types of this yeast. Its product is required for response to a signal that causes arrest of the mitotic cell cycle in the G1 phase and induction of mating-type-specific genes. The nucleotide sequence of the STE11 gene was determined. The predicted amino acid sequence shows homology to the protein kinase family. We demonstrate that the STE11 product has kinase catalytic activity and that this activity is required for its in vivo functions.