Exploring the scope of TOR/PKA co-regulation in MDN
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Ted Powers
PROVIDER: E-GEOD-3160 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae.
Chen Jenny C-Y JC Powers Ted T
Current genetics 20060106 5
The target of rapamycin (TOR) signaling pathway is an essential regulator of cell growth in eukaryotic cells. In Saccharomyces cerevisiae, TOR controls the expression of many genes involved in a wide array of distinct nutrient-responsive metabolic pathways. By exploring the TOR pathway under different growth conditions, we have identified novel TOR-regulated genes, including genes required for branched-chain amino acid biosynthesis as well as lysine biosynthesis (LYS genes). We show that TOR-dep ...[more]
Similar Datasets
Project description:To identify genes co-regulated by TOR and PKA during growth in MDN Keywords: Media, Wild type and mutant strains, and dose response
2005-08-19 | GSE3160 | GEO
Project description:The conserved cAMP-dependent protein kinase (PKA) holoenzyme is composed of two catalytic and two regulatory subunits. It plays critical roles in the regulation of many biological processes in eukaryotic organisms. In the human fungal pathogen Candida albicans, the PKA kinase has been extensively investigated for its importance in the regulation of morphological transitions and virulence. It has been long thought that the PKA catalytic subunit is essential for cell viability in C. albicans. Paradoxically, the single adenylyl cyclase-encoding gene, CRY1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. In this study, we successfully generated a null double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1 or t2t1), which encode two isoforms of the PKA catalytic subunit in C. albicans. We reevaluated the roles of the PKA catalytic subunit in cell growth and phenotypic transitions. Inactivation of the PKA catalytic subunit by deletion of both TPK1 and TPK2 blocked filamentation and dramatically attenuated the ability of white-to-opaque switching, but promoted sexual mating in C. albicans. Tpk2 plays a major role in these regulations, while Tpk1 generally functions as a negative regulator in morphological transitions and sexual mating. A comparative transcriptomic analysis demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared to the WT strain, the general transcriptional activity and expression of genes involved in metabolism, translation, biosynthesis, adhesion and filamentation are significantly decreased in both the t2t1 and cyr1/cyr1 mutants. And a portion of stress-response and cell wall-related genes were upregulated in these mutants, which is consistent with their increased ability of anti-stresses. To further explore the global regulatory role of the PKA kinase, we performed quantitative phosphoproteomics analysis. Combining with bioinformatics analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. Cell wall and membrane-related proteins (e.g. Ecm3, Bni1, and Smi1) were enriched in Tpk1-specific targets, while Tpk2-specific substrates include transporters, filamentation and cytoskeleton-related proteins (e.g. Smf3, Sep7, and Mhp1). There were also many Tpk1 and Tpk2 overlapped and coordinately regulated-substrates. Our study clarifies the essentiality of the PKA catalytic subunit and shed new insights into the global regulatory features of the cAMP/PKA pathway in C. ablicans. The t2t1 null mutant generated in this study would also be a new resource for the field to study this important pathway.
2017-08-18 | GSE92621 | GEO
Project description:Rapamycin-sensitive transgenic Arabidopsis lines (BP12) expressing yeast FK506 Binding Protein12 (FKBP12) were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf and shoot growth and development leading to poor nutrient uptake and light energy utilization. Genetic and physiological studies together with RNA-Seq and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and lifespan in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, ribosomal RNA and protein synthesis.
2012-12-18 | GSE42968 | GEO
Project description:Rapamycin-sensitive transgenic Arabidopsis lines (BP12) expressing yeast FK506 Binding Protein12 (FKBP12) were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf and shoot growth and development leading to poor nutrient uptake and light energy utilization. Genetic and physiological studies together with RNA-Seq and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and lifespan in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, ribosomal RNA and protein synthesis. Arabidopsis WT (Col)and BP12-2 (TOR knockdown line) seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5 MS medium to 0.5 MS+10 ug/ml rapamycin. Triplicate samples of rapamycin treated WT and BP12-2 seedlings were used for RNA-Seq analysis (Illumina Hiseq 2000). Paired-end alignments were obtained through aligning short reads onto the reference Arabidopsis Genome (TAIR9) using Bowtie. More than 80% of the reads mapped onto the genome. Htseq-count was used to count the reads from the Bowtie derived output files. Differential expressed genes were identified using edgeR. The FDR-corrected P value for differential expression was set to be <=0.05.
2012-12-18 | E-GEOD-42968 | biostudies-arrayexpress
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action. This SuperSeries is composed of the SubSeries listed below.
2011-06-30 | GSE22272 | GEO
Project description:The yeast cyclic-AMP dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the catalytic subunit, tpk1, reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ regulator (nej1), co-function in the same pathway. Chromatin immunoprecipitation and resection data suggest that the recruitment of repair proteins and DNA resection is influenced by tpk1 deletion. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair, in addition to nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (the human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.
2021-09-10 | PXD024695 | Pride
Project description:The yeast cyclic-AMP dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the catalytic subunit, tpk1, reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ regulator (nej1), co-function in the same pathway. Chromatin immunoprecipitation and resection data suggest that the recruitment of repair proteins and DNA resection is influenced by tpk1 deletion. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair, in addition to nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (the human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.
2021-09-10 | PXD024768 | Pride
Project description:The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that is activated by varying nutrient and environmental signals and governs a plethora of eukaryotic biological processes, but its role in Cryptococcus neoformans remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in C. neoformans. We successfully deleted TLK1, but not TOR1. TLK1 deletion did not result in any evident in vitro phenotypes, suggesting that Tlk1 is mainly dispensable for the growth of C. neoformans. We further demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To analyze the functions of Tor1 in more detail, we constructed constitutive TOR1 overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of C. neoformans. We also found that TOR1 overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, autophagy, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of signaling pathways, including the Hog1 pathway. In conclusion, our study demonstrates that a single Tor1 kinase plays a variety of roles in the fungal meningitis pathogen C. neoformans.
2019-04-03 | GSE129227 | GEO
Project description:All organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how they are integrated to produce global changes in a cell. Past work from our lab, focused on engineering the budding yeast Saccharomyces cerevisiae for fermentation of the non-native pentose sugar xylose, discovered that hyperactivation of the RAS/Protein Kinase A (PKA) pathway was needed for rapid anaerobic xylose fermentation. Interestingly, the mechanism of PKA hyperactivation has a dramatic impact on growth and metabolism on xylose; deletion of the RAS inhibitor IRA2 permits rapid growth and fermentation, while deletion of the PKA regulatory subunit BCY1 allows for fermentation without growth on xylose. To understand how a single deletion in the PKA pathway can decouple growth and metabolism, we performed transcriptomic analysis of these strains, predicting that altered PKA activity would impact global gene expression and identify pathways important for growth and metabolism coordination. Notably, we found enriched differential expression of lipid metabolism genes, targets of the phospholipid biosynthetic gene transcription factor Ino4, and genes containing the Aft1/2 consensus motif. These results suggested that dysfunctional lipid homeostasis may be responsible for decoupling growth and metabolism in the bcy1∆ strain. In parallel work, we also directly evolved the bcy1∆ strain to grow anaerobically on xylose and found point mutations in TPK1, OPI1, RIM8, and TOA1 permitted growth. Interestingly, Opi1 is the inhibitor of Ino4, further supporting the role of lipid homeostasis in growth and metabolism coordination. This work shows that a single genetic change can have dramatic impacts on multiple aspects of cellular physiology.
2023-05-01 | GSE220465 | GEO
Project description:The Tor kinase is one of the major regulatory nodes in a eukaryotic cell and until now, little is known about this kinase in filamentous fungi. Here, we analysed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans and the main causative agent of invasive aspergillosis. Because deletion of the single tor gene of A. fumigatus was apparently lethal, we generated a conditional lethal tor mutant. This was achieved by fusing the tor gene with the inducible xylose promoter and replacing the endogenous tor gene by the inducible xylp-tor gene cassette. The generated transgenic strain opened up the possibility to activate or silence the tor gene under controlled conditions. Since we did not observe any effect of rapamycin on Tor of A. fumigatus, the generation of the inducible Tor gene was even more valuable for an in-depth analysis of the Tor kinase. By LC-MS/MS analyses of mycelial proteins we identified the regulatory network of Tor. Tor controls a variety of genes and proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation. Tor is a major kinase responsible for protein import into the mitochondria, and consequently responsible for the correct function of most processes occurring into these organelles, e.g. respiration and ornithine metabolism. Regulation of iron acquisition by Tor was found to be independent of the HapX transcription factor.
2022-02-28 | PXD001601 | Pride