Schizosaccharomyces pombe Adenylate Cyclase Suppressor Mutations Suggest a Role for cAMP Phosphodiesterase Regulation in Feedback Control of Glucose/cAMP Signaling
Abstract:Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs2 1 were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git2 1 /cyr1 1 ), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git2 ÿ mutant alleles used in the suppressor selection and mutations in git1, and git11 1 , which are all required for adenylate cyclase activation. Notably, these… Show more
“…For examples, there is a predicted transcriptional regulation complex including DNA binding factor (Tbf1 and Spt2) (Cockell et al 2009), transcription factor TFIIH complex subunit (Rad15) (Murray et al 1992), histone acetyltransferase (Mst2) (Gómez et al 2005) and Jmjc domain chromatin-associated protein (Epe1) (Trewick et al 2007). There is also a potential signalling module associated with protein kinase and secondary messenger cAMP, which consists of cAMP-dependent protein kinase catalytic subunit (Pka1) (Matsuo et al 2008), serine/threonine protein kinase Ppk18 (Beltrao et al 2009) and a G-protein coupled receptor, Git3, which can activate the adenylate cyclase activity (Wang et al 2005). 10.1080/21501203.2016.1221862-F0003Figure 3.Network analysis of honokiol up-regulated gene sets indicates extensive protein–protein interactions are existed and form biofunctionally important modules.…”
The fast growing evidences have shown that the plant-derived compound honokiol is a promising candidate for treating multiple human diseases, such as inflammation and cancer. However, the mode-of-action (MoA) of honokiol remains largely unclear. Here, we studied the antifungal activity of honokiol in fission yeast model, with the goal of understanding the honokiol’s mechanism of action from the molecular level. We found that honokiol can inhibit the yeast growth at a dose-dependent way. Microarray analysis showed that honokiol has wide impacts on the fission yeast transcription levels (in total, 512 genes are up-regulated, and 42 genes are down-regulated). Gene set enrichment analysis indicated that over 45% up-regulated genes belong to the core environmental stress responses category. Moreover, network analysis suggested that there are extensive gene–gene interactions amongst the co-expression gene lists, which can assemble several biofunctionally important modules. It is noteworthy that several key components of central carbon metabolism, such as glucose transporters and metabolic enzymes of glycolysis, are involved in honokiol’s MoA. The complexity of the honokiol’s MoA displayed in previous studies and this work demonstrates that multiple omics approaches and bioinformatics tools should be applied together to achieve the complete scenario of honokiol’s antifungal function.
“…For examples, there is a predicted transcriptional regulation complex including DNA binding factor (Tbf1 and Spt2) (Cockell et al 2009), transcription factor TFIIH complex subunit (Rad15) (Murray et al 1992), histone acetyltransferase (Mst2) (Gómez et al 2005) and Jmjc domain chromatin-associated protein (Epe1) (Trewick et al 2007). There is also a potential signalling module associated with protein kinase and secondary messenger cAMP, which consists of cAMP-dependent protein kinase catalytic subunit (Pka1) (Matsuo et al 2008), serine/threonine protein kinase Ppk18 (Beltrao et al 2009) and a G-protein coupled receptor, Git3, which can activate the adenylate cyclase activity (Wang et al 2005). 10.1080/21501203.2016.1221862-F0003Figure 3.Network analysis of honokiol up-regulated gene sets indicates extensive protein–protein interactions are existed and form biofunctionally important modules.…”
The fast growing evidences have shown that the plant-derived compound honokiol is a promising candidate for treating multiple human diseases, such as inflammation and cancer. However, the mode-of-action (MoA) of honokiol remains largely unclear. Here, we studied the antifungal activity of honokiol in fission yeast model, with the goal of understanding the honokiol’s mechanism of action from the molecular level. We found that honokiol can inhibit the yeast growth at a dose-dependent way. Microarray analysis showed that honokiol has wide impacts on the fission yeast transcription levels (in total, 512 genes are up-regulated, and 42 genes are down-regulated). Gene set enrichment analysis indicated that over 45% up-regulated genes belong to the core environmental stress responses category. Moreover, network analysis suggested that there are extensive gene–gene interactions amongst the co-expression gene lists, which can assemble several biofunctionally important modules. It is noteworthy that several key components of central carbon metabolism, such as glucose transporters and metabolic enzymes of glycolysis, are involved in honokiol’s MoA. The complexity of the honokiol’s MoA displayed in previous studies and this work demonstrates that multiple omics approaches and bioinformatics tools should be applied together to achieve the complete scenario of honokiol’s antifungal function.
“…In S. pombe, cAMP levels are controlled by the regulation of both adenylyl cyclase and the Pde1 homolog, Cgs2 (21). Studies with S. pombe suggest that shortly after the glucose-stimulated activation of adenylyl cyclase, the Cgs2 phosphodiesterase is also activated, resulting in a modulation of cAMP levels (45). Sitedirected mutagenesis of the sites in the C. neoformans Cac1 and Pkr1 proteins should elucidate whether these candidate proteins are targets of Pka1 and if they have roles in the negative feedback loop that regulates cAMP levels in C. neoformans.…”
The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low-and highaffinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the G␣ subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1⌬ pka1⌬ mutant strain exhibits cAMP levels that are dramatically increased (ϳ15-fold) relative to those in a pka1⌬ single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels.
“…11,[24][25][26] The relative level of reporter expression reflects PDE activity. β-Galactosidase activity in the gpa2 -mutant strains, as compared with similar strains expressing either the wild-type S. pombe Cgs2 + PDE or the frame-shifted, and presumably inactive, Cgs2-2 truncated PDE, 16 demonstrate that all 4 murine PDEs are active in S. pombe ( Table 2; vehicle values). The relative level of PDE activity, as reflected by the degree to which β-galactosidase activity is elevated by the reduction in cAMP levels, is Cgs2 + > PDE4A > PDE2A ≥ PDE4B ≥ PDE8A > Cgs2-2.…”
Section: Construction Of Fission Yeast Strains Expressing Mammalian Pdesmentioning
confidence: 91%
“…Methods for yeast growth and transformations have been previously described. 16 Murine PDE genes were amplified by PCR using oligonucleotides containing approximately 60 nt of sequence flanking the S. pombe cgs2 + gene to direct homologous recombination to this locus.…”
Section: Strains Media and General Techniquesmentioning
Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3′,5′-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens. (Journal of Biomolecular Screening 2008:62-71)
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