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Enzo Martegani - One of the best experts on this subject based on the ideXlab platform.
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evidence for adenylate cyclase as a scaffold protein for Ras2 ira interaction in saccharomyces cerevisie
Cellular Signalling, 2014Co-Authors: Sonia Colombo, Chiara Paiardi, Katrien Pardons, Joris Winderickx, Enzo MarteganiAbstract:Abstract Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.
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nuclear Ras2 gtp controls invasive growth in saccharomyces cerevisiae
PLOS ONE, 2013Co-Authors: Serena Broggi, Enzo Martegani, Sonia ColomboAbstract:Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.
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activation state of the Ras2 protein and glucose induced signaling in saccharomyces cerevisiae
Journal of Biological Chemistry, 2004Co-Authors: Sonia Colombo, Joris Winderickx, Daniela Ronchetti, Johan M Thevelein, Enzo MarteganiAbstract:Abstract The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Gα protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of Ras2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.
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properties of the catalytic domain of cdc25 a saccharomyces cerevisiae gdp gtp exchange factor comparison of its activity on full length and c terminal truncated Ras2 proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: E Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the Ras2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length Ras2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on Ras2. Comparison of the activities of Ras2-42/37kDa and Ras2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. Ras2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25 Mm .
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Properties of the Catalytic Domain of CDC25, a Saccharomyces cerevisiae GDP/GTP Exchange Factor: Comparison of Its Activity on Full-Length and C-Terminal Truncated Ras2 Proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: E Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the Ras2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length Ras2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on Ras2. Comparison of the activities of Ras2-42/37kDa and Ras2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. Ras2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25 Mm .
Andrea Parmeggiani - One of the best experts on this subject based on the ideXlab platform.
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properties of the catalytic domain of cdc25 a saccharomyces cerevisiae gdp gtp exchange factor comparison of its activity on full length and c terminal truncated Ras2 proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: E Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the Ras2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length Ras2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on Ras2. Comparison of the activities of Ras2-42/37kDa and Ras2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. Ras2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25 Mm .
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Properties of the Catalytic Domain of CDC25, a Saccharomyces cerevisiae GDP/GTP Exchange Factor: Comparison of Its Activity on Full-Length and C-Terminal Truncated Ras2 Proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: E Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the Ras2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length Ras2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on Ras2. Comparison of the activities of Ras2-42/37kDa and Ras2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. Ras2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25 Mm .
Sonia Colombo - One of the best experts on this subject based on the ideXlab platform.
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evidence for adenylate cyclase as a scaffold protein for Ras2 ira interaction in saccharomyces cerevisie
Cellular Signalling, 2014Co-Authors: Sonia Colombo, Chiara Paiardi, Katrien Pardons, Joris Winderickx, Enzo MarteganiAbstract:Abstract Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.
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nuclear Ras2 gtp controls invasive growth in saccharomyces cerevisiae
PLOS ONE, 2013Co-Authors: Serena Broggi, Enzo Martegani, Sonia ColomboAbstract:Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.
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activation state of the Ras2 protein and glucose induced signaling in saccharomyces cerevisiae
Journal of Biological Chemistry, 2004Co-Authors: Sonia Colombo, Joris Winderickx, Daniela Ronchetti, Johan M Thevelein, Enzo MarteganiAbstract:Abstract The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Gα protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of Ras2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.
Alexander Levitzki - One of the best experts on this subject based on the ideXlab platform.
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differential activation of yeast adenylyl cyclase by ras1 and Ras2 depends on the conserved n terminus
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Naama Hurwitz, Marisa Segal, Irit Marbach, Alexander LevitzkiAbstract:Abstract Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-Ras2 or Ras2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 Ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.
Fuyuhiko Tamanoi - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of yeast Ras2 mutants reveals a new region of ras protein involved in the interaction with gtpase activating proteins
Journal of Biological Chemistry, 1994Co-Authors: D R Wood, P Poullet, B A Wilson, M Khalil, K Tanaka, J F Cannon, Fuyuhiko TamanoiAbstract:Abstract We report biochemical characterization of two recently identified mutants of yeast Ras2, Ras2-E99K and Ras2-E130K. These mutants exhibit dominant activating phenotypes in yeast. Characterization of their intrinsic GTPase and GDP dissociation as well as their ability to stimulate adenylate cyclase showed that these activities of Ras2-E99K mutant protein were similar to those of the wild type protein. Ras2-E130K protein, on the other hand, differed from the wild type protein with a fast GDP dissociation rate and 2-fold higher activation of adenylate cyclase. When the sensitivity to GTPase-activating protein (GAP) was examined, we found that the Ras2-E99K protein was approximately 1200-fold less sensitive to NF1-GAP activity. In addition, the affinity for NF1 as revealed by competition binding experiments was reduced more than 150-fold with Ras2-E99K protein. Thus, the Ras2-E99K mutation affects interaction with GAP proteins. This mutation is particularly interesting because it is the first mutation identified in the alpha 3 region of ras protein that affects GAP interaction. The alpha 3 region appears to be directly involved in interaction with NF1, since peptides containing the sequence encompassing residue 99 of Ras2 inhibit NF1-GAP activity. These results suggest that the interaction between ras and GAP involves a larger region within ras than previously recognized.
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new activated Ras2 mutations identified in saccharomyces cerevisiae
Oncogene, 1993Co-Authors: B A Wilson, Fuyuhiko Tamanoi, M Khalil, J F CannonAbstract:: Activating mutations in RAS proto-oncogenes encode proteins with greater GTP binding. Such mutant proteins are responsible for many human cancers. Six new amino acids were discovered that can yield an activated Saccharomyces cerevisiae Ras2 protein when they are altered. These new Ras2 alleles were found among a collection of 35 random mutations that exhibit a dominant reduction of glycogen accumulation. The Ras2-P41S and Ras2-E99K alleles encode proteins that have lost responsiveness to GTPase activating proteins. They affect amino acids in loop 2 and helix 3 respectively and illustrate that GTPase activating proteins recognize a larger portion of the RAS structure than previously realized. Ras2 mutations E130K, S153F, A154T, and A157S alter amino acids proximal to the guanine binding site and probably influence nucleotide binding either directly or indirectly.
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s farnesylation and methyl esterification of c terminal domain of yeast Ras2 protein prior to fatty acid acylation
Journal of Biological Chemistry, 1991Co-Authors: A Fujiyama, Fuyuhiko Tamanoi, S Tsunasawa, F SakiyamaAbstract:Abstract Posttranslational processing/modification is required for membrane localization and activation of ras proteins. In the case of yeast Ras2 protein, we have reported that the process starts with the removal of the initiator methionine followed by polyisoprenylation, removal of 3 amino acid residues from the C terminus, methyl esterification, and fatty acid acylation (Fujiyama, A., and Tamanoi, F. (1990) J. Biol. Chem. 265, 3362-3368). In this study, we demonstrate that polyisoprenylation and methyl esterification of the cysteine residue in the C-terminal domain of the Ras2 protein are involved in the conversion process from precursor form to intermediate form. The polyisoprenoid moiety attached to the Ras2 protein was identified as a 15-carbon farnesyl group through two independent experiments: the release of S-farnesylcysteine with carboxypeptidase Y from the Ras2 protein, and the recovery of radioactive farnesol through methyliodide treatment of the Ras2 protein purified from yeast cells labeled with [3H]mevalonic acid. The farnesyl group attached to the Ras2 protein was detected predominantly in the C-terminal peptide, SGSGGCC, both in the intermediate and in the fatty acid acylated Ras2 protein. The C-terminal cysteine of the intermediate protein is also modified by methyl esterification in a nearly stoichiometric manner.
