Sck1 negatively regulates Gpa2-mediated glucose signaling in Schizosaccharomyces pombe.
ABSTRACT: Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 G? binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2(K270E)-activated G? relative to Gpa2(+) G?. Deletion of sck1(+) or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2(+) or Gpa2(K270E), suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2(K270E) GDP-GTP exchange rate mutant, GTPase-defective Gpa2(R176H) weakly binds Sck1 in the two-hybrid screen and a deletion of sck1(+) in a Gpa2(R176H) strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1(+) in a gpa2? strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.
Project description: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.
Project description: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.
Project description: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.
Project description:The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.
Project description:Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction) slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Galpha subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Deltagit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.
Project description:Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2 The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.
Project description:Addition of glucose to starved yeast cells elicits a dramatic restructuring of the transcriptional and metabolic state of the cell. While many components of the signaling network responsible for this response have been identified, a comprehensive view of this network is lacking. We have used global analysis of gene expression to assess the roles of the small GTP-binding proteins, Ras2 and Gpa2, in mediating the transcriptional response to glucose. We find that 90% of the transcriptional changes in the cell attendant on glucose addition are recapitulated by activation of Ras2 or Gpa2. In addition, we find that protein kinase A (PKA) mediates all of the Ras2 and Gpa2 transcriptional effects. However, we also find that most of the transcriptional effects of glucose addition to wild-type cells are retained in strains containing a PKA unresponsive to changes in cAMP levels. Thus, most glucose-responsive genes are regulated redundantly by a Ras/PKA-dependent pathway and by one or more PKA-independent pathways. Computational analysis extracted RRPE/PAC as the major response element for Ras and glucose regulation and revealed additional response elements mediating glucose and Ras regulation. These studies provide a paradigm for extracting the topology of signal transduction pathways from expression data.
Project description:The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgtlp protein family. In budding yeast, Sgtlp associates with Skplp and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgtlp, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgtlp proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.
Project description:The cAMP-PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the G(alpha) protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as G(beta) mimics for Gpa2, based on their predicted seven-bladed beta-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved.
Project description:The Schizosaccharomyces pombe wis1(+) gene is essential for cell survival under stress conditions. The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition. We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo. Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal. Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues. Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.