Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways

Caffeine is a natural purine analogue that elicits pleiotropic effects leading ultimately to cell's death by a largely uncharacterized mechanism. Previous works have shown that this drug induces a rapid phosphorylation of the Mpk1p, the final mitogen‐activated protein (MAP) kinase of the Pkc1p‐mediated cell integrity pathway. In this work, we showed that this phosphorylation did not necessitate the main cell wall sensors Wsc1p and Mid2p, but was abolished upon deletion of ROM2 encoding a GDP/GTP exchange factor of Rho1p. We also showed that the caffeine‐induced phosphorylation of Mpk1p was accompanied by a negligible activation of its main downstream target, the Rlm1p transcription factor. This result was consolidated by the finding that the loss of RLM1 had no consequence on the increased resistance of caffeine‐treated cells to zymolyase, indicating that the cell wall modification caused by this drug is largely independent of transcriptional activation of Rlm1p‐regulated genes. Additionally, the transcriptional programme elicited by caffeine resembled that of rapamycin, a potent inhibitor of the TOR1/2 kinases. Consistent with this analysis, we found that the caffeine‐induced phosphorylation of Mpk1p was lost in a tor1Δ mutant. Moreover, a tor1Δ mutant was, like mutants defective in components of the Pkc1p‐Mpk1p cascade, highly sensitive to caffeine. However, the hypersensitivity of a tor1 null mutant to this drug was rescued neither by sorbitol nor by adenine, which was found to outcompete caffeine effects specially on mutants in the PKC pathway. Altogether, these data indicated that Tor1 kinase is a target of caffeine, whose inhibition incidentally activates the Pkc1p‐Mpk1p cascade, and that the caffeine‐dependent phenotypes are largely dependent on inhibition of Tor1p‐regulated cellular functions. Finally, we found that caffeine provoked, in a Rom2p‐dependent manner, a transient drop in intracellular levels of cAMP, that was followed by change in expression of genes implicated in Ras/cAMP pathway. This result may pose Rom2p as a mediator in the interplay between Tor1p and the Ras/cAMP pathway.

[1]  J. Heitman,et al.  Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast , 1991, Science.

[2]  I. Howald,et al.  TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. , 1998, Genetics.

[3]  Y. Lou,et al.  Stimulatory effect of topical application of caffeine on UVB-induced apoptosis in mouse skin. , 2002, Oncology research.

[4]  D. Botstein,et al.  Singular value decomposition for genome-wide expression data processing and modeling. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. François,et al.  A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol , 1997, Yeast.

[6]  Fulai Jin,et al.  Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Price,et al.  Caffeine inhibits the checkpoint kinase ATM , 1999, Current Biology.

[8]  J. Hoheisel,et al.  Genome-wide Analysis of the Response to Cell Wall Mutations in the Yeast Saccharomyces cerevisiae* , 2003, Journal of Biological Chemistry.

[9]  C. W. Moore,et al.  A spheroplast rate assay for determination of cell wall integrity in yeast , 1998, Yeast.

[10]  N. Yanagishima,et al.  Effect of cyclic AMP, theophylline and caffeine on the glucose repression of sporulation in Saccharomyces cerevisiae. , 1973, Archiv fur Mikrobiologie.

[11]  R. Newburgh,et al.  INHIBITION OF 5′‐NUCLEOTIDASE IN RAT BRAIN BY METHYLXANTHINES , 1975, Journal of neurochemistry.

[12]  J. François,et al.  Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift , 2004, Molecular Genetics and Genomics.

[13]  T. Cooper.  Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. , 2002, FEMS microbiology reviews.

[14]  J. Heitman,et al.  FKBP12‐rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol‐4 kinase activity. , 1995, The EMBO journal.

[15]  M. Brenner,et al.  Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum. , 1984, Developmental biology.

[16]  David E. Levin,et al.  Cell Wall Integrity Signaling in Saccharomyces cerevisiae , 2005, Microbiology and Molecular Biology Reviews.

[17]  Pooja Jain,et al.  The YEASTRACT database: a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae , 2005, Nucleic Acids Res..

[18]  J. François,et al.  Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae , 1999, Yeast.

[19]  T. Schmelzle,et al.  Activation of the RAS/Cyclic AMP Pathway Suppresses a TOR Deficiency in Yeast , 2004, Molecular and Cellular Biology.

[20]  R. Upadhya,et al.  Maf1 is an essential mediator of diverse signals that repress RNA polymerase III transcription. , 2002, Molecular cell.

[21]  M. Wigler,et al.  cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae , 1988, Cell.

[22]  P. Tortora,et al.  Effect of caffeine on glucose-induced inactivation of gluconeogenetic enzymes in Saccharomyces cerevisiae. A possible role of cyclic AMP. , 1982, European journal of biochemistry.

[23]  Stuart L. Schreiber,et al.  Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins , 2000, Current Biology.

[24]  A. Caminade,et al.  Dendrimeric coating of glass slides for sensitive DNA microarrays analysis. , 2003, Nucleic acids research.

[25]  R. Deschenes,et al.  Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[26]  U. Jung,et al.  Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase , 2002, Molecular microbiology.

[27]  Troy Ketela,et al.  Saccharomyces cerevisiae Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of thePKC1-MPK1 Cell Integrity Pathway , 1999, Journal of bacteriology.

[28]  M. Wigler,et al.  Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  L. Bardwell,et al.  Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[31]  M. Gabriel,et al.  The influence of Congo red on the cell wall and (1 → 3)-β-d-glucan microfibril biogenesis in Saccharomyces cerevisiae , 1992, Archives of Microbiology.

[32]  Michael N. Hall,et al.  TOR signalling in bugs, brain and brawn , 2003, Nature Reviews Molecular Cell Biology.

[33]  M. A. de la Torre-Ruiz,et al.  Regulation of the Cell Integrity Pathway by Rapamycin-sensitive TOR Function in Budding Yeast* , 2002, The Journal of Biological Chemistry.

[34]  M. Jafari,et al.  Dose and time dependent effects of caffeine on superoxide release, cell survival and DNA fragmentation of alveolar macrophages from rat lung. , 2000, Toxicology.

[35]  Stanley Brul,et al.  Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae , 2004, Yeast.

[36]  M. Cárdenas,et al.  Tor and Cyclic AMP-Protein Kinase A: Two Parallel Pathways Regulating Expression of Genes Required for Cell Growth , 2005, Eukaryotic Cell.

[37]  A. Sentenac,et al.  Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[38]  S. Schreiber,et al.  Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Gerald R. Fink,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1987 .

[40]  Evelyn Mack Truitt.  On Screen , 1974 .

[41]  J. Thorner,et al.  Adenosine 3',5'-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae , 1981, Journal of bacteriology.

[42]  M. Bard,et al.  Caffeine resistance of Saccharomyces cerevisiae , 1980, Journal of bacteriology.

[43]  M. Molina,et al.  Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C. , 1993, Molecular & general genetics : MGG.

[44]  J. Heinisch,et al.  A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene , 1998, Molecular and General Genetics MGG.

[45]  Ted Powers,et al.  TOR Complex 1 Includes a Novel Component, Tco89p (YPL180w), and Cooperates with Ssd1p to Maintain Cellular Integrity in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[46]  I. Stansfield,et al.  An MBoC Favorite: TOR controls translation initiation and early G1 progression in yeast , 2012, Molecular biology of the cell.

[47]  J. François,et al.  A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation , 2003, Letters in applied microbiology.

[48]  S. Powers,et al.  Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function , 1991, Molecular and cellular biology.

[49]  U. Jung,et al.  The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. , 1995, Genes & development.

[50]  R. D. Gietz,et al.  High-efficiency transformation of plasmid DNA into yeast. , 2001, Methods in molecular biology.

[51]  W. Sabbagh,et al.  Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. , 2001, Molecular cell.

[52]  C. Smythe,et al.  ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK , 1999, Oncogene.

[53]  Ted Powers,et al.  Mechanism of Metabolic Control , 2000, The Journal of cell biology.

[54]  M. Molina,et al.  Regulatory Mechanisms for Modulation of Signaling through the Cell Integrity Slt2-mediated Pathway in Saccharomyces cerevisiae * , 2000, The Journal of Biological Chemistry.

[55]  M. Snyder,et al.  SBF Cell Cycle Regulator as a Target of the Yeast PKC-MAP Kinase Pathway , 1997, Science.

[56]  U. Jung,et al.  Yeast Rpi1 Is a Putative Transcriptional Regulator That Contributes to Preparation for Stationary Phase , 2002, Eukaryotic Cell.

[57]  Reciprocal Regulation between Slt2 MAPK and Isoforms of Msg5 Dual-specificity Protein Phosphatase Modulates the Yeast Cell Integrity Pathway* , 2004, Journal of Biological Chemistry.

[58]  B. Errede,et al.  Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[59]  Martin Vingron,et al.  Processing and quality control of DNA array hybridization data , 2000, Bioinform..

[60]  J. Crespo,et al.  Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. , 2002, Molecular cell.

[61]  J. Heitman,et al.  The TOR signaling cascade regulates gene expression in response to nutrients. , 1999, Genes & development.

[62]  H. Ashida,et al.  3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole Induces Apoptosis and Necrosis with Activation of Different Caspases in Rat Splenocytes , 2004, Bioscience, biotechnology, and biochemistry.

[63]  V. Potter,et al.  Control of the adenosine 3',5'-monophosphate-adenyl cyclase system in the livers of developing rats. , 1972, Cancer research.

[64]  F. Klis,et al.  Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance. , 2000, Microbiology.

[65]  N. Yanagishima,et al.  Effect of cyclic AMP, theophylline and caffeine on the glucose repression of sporulation inSaccharomyces cerevisiae , 1973, Archiv für Mikrobiologie.

[66]  D. Lew,et al.  Stress-specific Activation Mechanisms for the “Cell Integrity” MAPK Pathway* , 2004, Journal of Biological Chemistry.

[67]  D. E. Levin,et al.  A Second Osmosensing Signal Transduction Pathway in Yeast , 1995, The Journal of Biological Chemistry.

[68]  D. Wallace,et al.  The effect of caffeine on renal epithelial cells from patients with autosomal dominant polycystic kidney disease. , 2002, Journal of the American Society of Nephrology : JASN.

[69]  Rupert De Wachter,et al.  Classification of all putative permeases and other membrane multispanners of the Major Facilitator Superfamily encoded by the complete genome of Saccharomyces cerevisiae , 1997, German Conference on Bioinformatics.

[70]  J. François,et al.  A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae , 1998, Yeast.

[71]  Á. Durán,et al.  Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization , 1985, Journal of bacteriology.

[72]  E. Naderali,et al.  Effects of caffeine and theophylline on prostaglandin production by guinea-pig endometrium. , 1997, Prostaglandins, leukotrienes, and essential fatty acids.

[73]  Matthew D. W. Piper,et al.  Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae , 2022 .

[74]  S. Haggarty,et al.  Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[75]  M. Wigler,et al.  Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. , 1987, Genes & development.

[76]  D. E. Levin,et al.  Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1 , 2001, Molecular and Cellular Biology.

[77]  A. Marchler-Bauer,et al.  The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). , 1996, The EMBO journal.

[78]  R. Santen,et al.  Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex. , 2005, Molecular endocrinology.

[79]  J. Hoheisel,et al.  Correspondence analysis applied to microarray data , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[80]  S G Oliver,et al.  Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway. , 2003, Physiological genomics.

[81]  F. Klis,et al.  Differential regulation of cell wall biogenesis during growth and development in yeast. , 2001, Microbiology.

[82]  J. François,et al.  The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae , 2003, Molecular microbiology.

[83]  L. Johnston,et al.  Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. , 1996, The EMBO journal.

[84]  S. Snyder,et al.  Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[85]  J. Kunz,et al.  Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression , 1993, Cell.

[86]  G. Carignani,et al.  Large‐scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes , 2001, Yeast.

[87]  P. Russell,et al.  Mechanism of Caffeine-Induced Checkpoint Override in Fission Yeast , 2000, Molecular and Cellular Biology.

[88]  D. Botstein,et al.  Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[89]  R. Sentandreu,et al.  Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. , 1983, Journal of general microbiology.

[90]  Javier Arroyo,et al.  The Global Transcriptional Response to Transient Cell Wall Damage in Saccharomyces cerevisiae and Its Regulation by the Cell Integrity Signaling Pathway* , 2004, Journal of Biological Chemistry.

[91]  T. Powers,et al.  Yeast TOR signaling: a mechanism for metabolic regulation. , 2004, Current topics in microbiology and immunology.

[92]  Jong-In Park,et al.  Rom2p, the Rho1 GTP/GDP Exchange Factor of Saccharomyces cerevisiae, Can Mediate Stress Responses via the Ras-cAMP Pathway* , 2005, Journal of Biological Chemistry.

[93]  M. Hall,et al.  The expanding TOR signaling network. , 2005, Current opinion in cell biology.