Biomedical subjects

C S Hoffman

Publications and source records attributed to C S Hoffman.

At least 19 recordsLinked to original sources

PubMed2005

Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe primarily detects glucose via a cAMP-signalling pathway. Components of this pathway include the Git3 G-protein-coupled receptor and a heterotrimeric G-protein, from which the Gpa2 Galpha subunit activates adenylate cyclase (Git2/Cyr1). Three additional proteins, Git1, Git7 and Git10 are required to generate a cAMP response even in a strain expressing an activated form of Gpa2, which is capable of bypassing the loss of the GPCR and Gbetagamma dimer. Therefore, Git1, Git7 and Git10 either act in a G-protein-independent manner or are required to stabilize or assemble a functional signalling complex. Although prior data suggested that the Cgs2 cAMP phosphodiesterase (PDE) does not regulate the cAMP response, we now have evidence that along with adenylate cyclase regulation, PDE activation is important for limiting the response to glucose. Finally, regulation of protein kinase A activation appears to involve both traditional post-translational regulation of the function of the components of the cAMP pathway and glucose-dependent transcriptional regulation of some of these cAMP pathway genes.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

The phospholipase B homolog Plb1 is a mediator of osmotic stress response and of nutrient-dependent repression of sexual differentiation in the fission yeast Schizosaccharomyces pombe.

Although phospholipase B (PLB) enzymes have been described in eukaryotes from yeasts to mammals, their biological functions are poorly understood. Here we describe the characterization of plb1, one of five genes predicted to encode PLB homologs in the fission yeast, Schizosaccharomyces pombe. The plb1 gene is dispensable under normal growth conditions but required for viability in high-osmolarity media and for normal osmotic stress-induced gene expression. Unlike mutants defective in function for the stress-activated MAP kinase Spc1, plb1Delta cells are not hypersensitive to oxidative or temperature stresses, nor do they undergo a G2-specific arrest in response to osmotic stress. In addition to defects in osmotic stress response, plb1Delta cells exhibit a cold-sensitive defect in nutrient-mediated mating repression, a phenotype reminiscent of mutants in the cyclic AMP (cAMP) pathway. We show that, like plb1Delta cells, mutants in the cAMP pathway are defective for growth in high-osmolarity media, demonstrating a previously unrecognized role for the cAMP pathway in osmotic stress response. Furthermore, we show that gain-of function in the cAMP pathway can rescue the osmosensitive growth defect of plb1Delta cells, suggesting that the cAMP pathway is a potential downstream target of the actions of Plb1 in S. pombe.

Amino Acid Sequence↗

PubMed2001

The git5 Gbeta and git11 Ggamma form an atypical Gbetagamma dimer acting in the fission yeast glucose/cAMP pathway.

Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Galpha and git5 Gbeta are both required for glucose-triggered cAMP signaling. The git5 Gbeta is a unique member of the Gbeta family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gbeta folding and interaction with Ggamma subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Ggamma gene. Co-immunoprecipitation studies confirm the composition of this Gbetagamma dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Galpha or the git5 Gbeta. Overexpression of the gpa2 Galpha partially suppresses loss of either the git5 Gbeta or the git11 Ggamma, while mutational activation of the Galpha fully suppresses loss of either Gbeta or Ggamma. Deletion of gpa2 (Galpha), git5 (Gbeta), or git11 (Ggamma) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Galpha subunit remains partially active in the absence of the Gbetagamma dimer and that the git5 Gbeta subunit remains partially active in the absence of the git11 Ggamma subunit. The addition of the CAAX box from the git11 Ggamma to the carboxy-terminus of the git5 Gbeta partially suppresses the loss of the Ggamma. Thus the Ggamma in this system is presumably required for localization of the Gbetagamma dimer but not for folding of the Gbeta subunit. In mammalian cells, the essential roles of the Gbeta amino-terminal coiled-coil domains and Ggamma partners in Gbeta folding may therefore reflect a mechanism used by cells that express multiple forms of both Gbeta and Ggamma subunits to regulate the composition and activity of its G proteins.

Amino Acid Sequence↗

PubMed2001

Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

Amino Acid Sequence↗

PubMed2000

The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation.

Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gbeta subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway. While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet the gene possesses some of the coding capacity for this structure 5' to its ORF. Although both gpa2 (Galpha) and git5 (Gbeta) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels. Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains. Multicopy gpa2 partially suppresses the loss of git5, while the converse is not true. These data suggest that Gbeta is required for activation of adenylate cyclase either by promoting the activation of Galpha or by independently activating adenylate cyclase subsequent to Galpha stimulation as seen in type II mammalian adenylate cyclase activation.

Adenylyl Cyclases↗

PubMed2000

Glucose monitoring in fission yeast via the Gpa2 galpha, the git5 Gbeta and the git3 putative glucose receptor.

The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six "upstream" genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Galpha) while git5 encodes a Gbeta subunit. Multicopy suppression studies with gpa2(+) previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Galpha followed by Gbetagamma. We show here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2(R176H) allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.

Adenylyl Cyclases↗

PubMed2000

Protein kinase A and mitogen-activated protein kinase pathways antagonistically regulate fission yeast fbp1 transcription by employing different modes of action at two upstream activation sites.

A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site.

Activating Transcription Factor 1↗

PubMed2000

Mutagenesis and gene cloning in Schizosaccharomyces pombe using nonhomologous plasmid integration and rescue.

Genes are commonly cloned in yeasts and bacteria by plasmid complementation, where the introduction of the gene of interest into a host strain carrying a recessive mutation in that gene suppresses the host's mutant phenotype. However, a lack of low copy cloning vectors in the fission yeast Schizosaccharomyces pombe can complicate this approach especially when overexpression of one gene may suppress a defect in another gene or when overexpression of the desired gene is detrimental, if not lethal, to the cell. We describe here a method of identifying mutations in S. pombe that allows for the rapid and direct cloning of the defective gene. This involves the nonhomologous integration of a marked plasmid into the yeast genome and its subsequent rescue into Escherichia coli, so that DNA at the site of insertion is incorporated into the recovered plasmid. As two of three insertions obtained in this study occurred outside of the affected gene's open reading frame, this method should be applicable to cloning both essential genes and nonessential genes.

Cloning, Molecular↗

PubMed1999

GABAergic regulation of lordosis: influence of gonadal hormones on turnover of GABA and interaction of GABA with 5-HT.

The role of GABAergic neurons in activating female sexual behavior and possible mechanisms for GABAergic effects on behavior were examined in female rats. First, effects of the ovarian hormones estrogen and progesterone (P), at doses which promote lordosis, on levels and turnover/activity of GABA, were examined in brain areas which regulate lordosis. Utilizing AOAA, an inhibitor of GABA degradation, the accumulation rate of GABA (turnover/activity) was assessed in ovariectomized (Ovx), Ovx + estrogen and Ovx + estrogen + P-treated rats. Estradiol increased GABA accumulation rates in the arcuate-median eminence and in the area dorsal to and surrounding the VMN (VMN-S). P administration following estrogen priming enhanced GABA turnover in the medial preoptic area (mPOA) and further increased turnover in the VMN-S while GABA turnover decreased in the dorsomedial nucleus. No effects of hormones were noted in the VMN itself or in the dorsal midbrain central gray. Reverse dialysis of the GABAA antagonist bicuculline into the basomedial hypothalamus was associated with a time-dependent inhibition of lordosis and a 300% increase in 5-HT release in the basomedial hypothalamus as measured by in vivo dialysis. These results provide additional evidence that GABAergic neurons mediate the physiological regulation of female sexual behavior and suggest that such mediation may involve an interaction with 5-HT containing neurons.

Animals↗

PubMed1995

sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase.

Schizosaccharomyces pombe regulates intracellular cAMP levels, and thus cAMP-dependent protein kinase (PKA) activity, in response to changes in nutrient conditions. Mutations in any of eight git genes inhibit glucose repression of fbp1 transcription, alter the cell morphology, and cause a reduction in the growth rate. The eight git genes encode components of an adenylate cyclase activation pathway, adenylate cyclase itself, and the catalytic subunit of PKA. Three of these genes have been identified in other studies as regulators of meiosis. Here we show that the sck1 gene, cloned as a high copy number suppressor of a mutation in git3, is able to suppress the defects conferred by a mutation in any of these git genes. Sequence analysis suggests that sck1 encodes a protein most closely related to the Saccharomyces cerevisiae SCH9 protein kinase that had previously been identified as a high copy number suppressor of mutations in S. cerevisiae that reduce or eliminate PKA activity. Disruption of the sck1 gene causes a significant delay in exit from stationary phase when combined with a disruption of the pka1 (git6) gene encoding the catalytic subunit of PKA. However, the sck1 disruption by itself has little or no effect upon fbp1 transcription, meiosis, or exit from stationary phase, and does not enhance the constitutive fbp1 transcription observed in a pka1 mutant. Therefore, sck1 appears to function in a redundant fashion to pka1, but to varying degrees, in the pathways regulated by pka1.

Amino Acid Sequence↗

PubMed1994

Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8).

In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed1993

Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies.

We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker. From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.

Cloning, Molecular↗

PubMed1993

Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe.

An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these 'upstream' git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these 'upstream' git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

The fission yeast genes pyp1+ and pyp2+ encode protein tyrosine phosphatases that negatively regulate mitosis.

We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway.

Amino Acid Sequence↗

PubMed1991

A fission-yeast gene encoding a protein with features of protein-tyrosine-phosphatases.

Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosine-phosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene, designated pyp1+, was identified in a cDNA library with this PCR probe, cloned, and sequenced. The sequence of the gene predicted a 550-amino acid protein with Mr 61,586, which includes amino acid sequences that are highly conserved in mammalian PTPases. Disruption of the pyp1+ gene resulted in viable cells. Overexpression of the pyp1+ gene in S. pombe permitted detection of a protein of apparent Mr 63,000.

Amino Acid Sequence↗

PubMed1991

Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway.

Transcription of the fbp1 gene, encoding fructose-1,6-bisphosphatase, of Schizosaccharomyces pombe is subject to glucose repression. Previous work has demonstrated that several genes (git genes) are required for this repression. In this report we demonstrate that one of these genes, git2, is the same as the cyr1 gene, which encodes adenylate cyclase, and that loss-of-function mutations in git2 cause constitutive fbp1 transcription. Addition of cAMP to the growth medium suppresses the transcriptional defect in git2 mutants as well as in strains that carry mutations in any of six additional git genes. Similarly, exogenous cAMP represses fbp1 transcription in wild-type cells grown on a derepressing carbon source. Different levels of adenylate cyclase activity in different git2 mutants, coupled with the result that some git2 mutants display intragenic complementation, strongly suggest that adenylate cyclase acts as a multimer and that different git2 mutations alter distinct activities of adenylate cyclase, including catalytic activity and response to glucose. Additional experiments demonstrate that this cAMP signaling pathway is independent of the S. pombe ras1 gene and works by activation of cAMP-dependent protein kinase.

Cloning, Molecular↗

PubMed1990

Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe.

Transcription of the fbp1 gene of Schizosaccharomyces pombe, encoding fructose-1,6-bisphosphatase, is glucose repressible. We have constructed two hybrid genes, containing the fbp1 promoter, that allow selection for mutations that alter transcriptional regulation of fbp1. Strains carrying fbp1-ura4 and fbp1-lacZ fusions are phenotypically Ura-, resistant to 5-fluoro-orotic acid, and express a low level of beta-galactosidase activity when grown under repressing conditions (8% glucose). By selecting for Ura+ strains grown under repressing conditions, we have isolated 187 independent mutants that constitutively express the fbp1-ura4 fusion. These mutants identify ten complementation groups that represent ten unlinked git (glucose insensitive transcription) genes. The git gene products are required in trans for glucose repression of expression from the fbp1 promoter since these mutations also alter expression of the fbp1-lacZ fusion. We have shown that transcription of the wild type fbp1 gene in most git mutants is elevated to a level consistent with the increased expression of the fbp1-lacZ hybrid gene. Mutations in some git genes confer additional phenotypes such as slow growth, temperature-sensitive lethality and reduced spore viability. Therefore, some of these genes are likely to encode factors that are of general importance for S. pombe transcription.

Alleles↗

PubMed1989

A transcriptionally regulated expression vector for the fission yeast Schizosaccharomyces pombe.

An expression vector for the fission yeast Schizosaccharomyces pombe is described. The vector is designed to facilitate the construction of transcriptional fusions to the promoter of the S. pombe fructose bisphosphatase gene. Transcription from this promoter is regulated by glucose repression over a range of greater than 100-fold. The tight regulation by this promoter should allow for the maintenance of genes whose products are lethal to S. pombe and for the high level production of their protein or RNA products. Intermediate levels of expression can also be achieved by growth on different carbon sources.

Base Sequence↗