Suppressors of an Adenylate Cyclase Deletion in the Fission Yeast Schizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe utilizes two opposing signaling pathways to sense and respond to its nutritional environment. Glucose detection triggers a cyclic AMP signal to activate protein kinase A (PKA), while glucose or nitrogen starvation activates the Spc1/Sty1 stress-activated protein kinase (SAPK). One process controlled by these pathways is fbp1 ؉ transcription, which is glucose repressed. In this study, we isolated strains carrying mutations that reduce high-level fbp1 ؉ transcription conferred by the loss of adenylate cyclase (git2⌬), including both wis1 ؊ (SAPK kinase) and spc1 ؊ (SAPK) mutants. While characterizing the git2⌬ suppressor strains, we found that the git2⌬ parental strains are KCl sensitive, though not osmotically sensitive. Of 102 git2⌬ suppressor strains, 17 strains display KCl-resistant growth and comprise a single linkage group, carrying mutations in the cgs1 ؉ PKA regulatory subunit gene. Surprisingly, some of these mutants are mostly wild type for mating and stationary-phase viability, unlike the previously characterized cgs1-1 mutant, while showing a significant defect in fbp1-lacZ expression. Thus, certain cgs1 ؊ mutant alleles dramatically affect some PKAregulated processes while having little effect on others. We demonstrate that the PKA and SAPK pathways regulate both cgs1 ؉ and pka1 ؉ transcription, providing a mechanism for cross talk between these two antagonistically acting pathways and feedback regulation of the PKA pathway. Finally, strains defective in both the PKA and SAPK pathways display transcriptional regulation of cgs1 ؉ and pka1 ؉ , suggesting the presence of a third glucose-responsive signaling pathway.Eukaryotic cells employ a variety of signal transduction pathways to regulate growth and developmental responses to changes in their environment. Often, more than one signal transduction pathway regulates a given biological process. Signals generated by one pathway may moderate the function of other signaling pathways, a phenomenon referred to as cross talk. Alternatively, signaling pathways may act independently to produce a regulatory outcome.The fission yeast Schizosaccharomyces pombe regulates many growth and developmental processes in response to nutrient conditions through the action of two opposing signal transduction pathways. Glucose triggers the activation of adenylate cyclase, resulting in a cyclic AMP (cAMP) signal, which activates the cAMP-dependent protein kinase A (PKA) (3,15,24). Nutrient starvation activates a stress-activated protein kinase (SAPK) pathway, which is also activated by osmotic, oxidative, and heat stress (8,10,31,34). Cells lacking adenylate cyclase (git2 ϩ /cyr1 ϩ ) display characteristics of glucose-starved cells even while growing in nutrient-rich conditions in that they efficiently mate and sporulate and they actively transcribe the fbp1 ϩ gene, which is normally subject to glucose repression (15, 23). Cells carrying mutations affecting the Wis1 SAPK kinase (SAPKK) or the Spc1/Sty1 SAPK fail to respond to stress resulting in tempera...

Opposite Effects of Tor1 and Tor2 on Nitrogen Starvation Responses in Fission Yeast

“…No meiosis was observed in isogenic wild-type cells, as expected. The ability of cells to enter sexual development on rich medium characterizes mutants of the cAMP pathway, such as cells lacking the adenylate cyclase, Cyr1/Git2, or the protein kinase A, Pka1 (Stiefel et al 2004;Hoffman 2005). While the cAMP-dependent pathway is thought to regulate growth and developmental decisions mainly in response to glucose sensing, the nitrogen-starved phenotype of Tor2 depletion suggests that tor2 1 regulates growth and development in response to nitrogen availability.…”

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

“…Thus, zygomycetes seem to be the only class of fungi that have more than one isoform for the R subunit (1,2,8,9,11,14,26,46,50,51,52,54). The corresponding mutants with mutations in the genes encoding the PKA regulatory subunit have been obtained from several of these fungi, and there does not seem to be a unique way in how fungi respond to the deletion of the R subunit, since some of them, such as A. niger (46), have the same level of PKA activity as in the wild-type cell, whereas in others, such as C. neoformans or A. fumigatus (11,54), the PKA activity is increased.…”

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

“…Meanwhile, glucose starvation activates the Spc1/Sty1 stress-activated mitogen activated protein kinase pathway required for fbp1 + transcription. [13][14][15] The fbp1-ura4 reporter places uracil biosynthesis under the control of the glucose/cAMP pathway, such that cells with high cAMP levels due to glucose signaling repress reporter expression and cannot grow in medium lacking uracil (SC-ura) but do grow in medium containing the pyrimidine analog 5-fluoro-orotic acid (5FOA; Fig. 1A).…”