Differential activation of yeast adenylate cyclase by wild type and mutant RAS proteins

Broek, Daniel; Samiy, Nasrollah; Fasano, Ottavio; Fujiyama, Asao; Tamanoi, Fuyuhiko; Northup, John K.; Wigler, Michael

doi:10.1016/s0092-8674(85)80057-x

Cited by 366 publications

(243 citation statements)

References 36 publications

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“…The preparation of yeast membrane extracts has been previously described (3,42). RAS2 and RAS2va1-L9 were purified from E. coli isolates containing expression systems as previously described (3).…”

Section: Methodsmentioning

confidence: 99%

“…Adenylate cyclase assays were performed as described (3,28,42) except that an ATP-generating system was omitted. The legend to Fig.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously shown that yeast and mammalian Ras proteins purified from Escherichia colilvector expression systems are potent activators of the adenylate cyclase present in crude fractions of yeast membranes (3). Surprisingly, we observed that RAS2 protein bound to GDP appeared to activate adenylate cyclase although activation was only one half to one third that of RAS2 protein bound to a nonhydrolyzable GTP analog.…”

mentioning

confidence: 86%

“…They are structurally, biochemically, and functionally similar to the mammalian Ras proteins, and at least one of their effector systems is known (3,9,10,18,24,37,41,42). The yeast RAS genes were originally isolated by using mammalian ras genes as probes to screen libraries of S. cerevisiae genomic DNA.…”

mentioning

confidence: 99%

“…duced in the mutant RAS2val-19 protein which has an amino acid substitution analogous to one of the oncogenic forms of the mammalian Ras proteins (37, 41; adenylate cyclase, and this appears to be their major physiological effector (3,42).…”

mentioning

confidence: 99%

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Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

Field¹,

Broek²,

Kataoka³

et al. 1987

Mol. Cell. Biol.

111

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In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.ras genes are ubiquitous in eucaryotes (9,24,27,32). In mammals, they comprise a family of at least three members (H-, K-, and N-ras) which share structural and functional properties. They were first identified, in their mutant forms, as potent oncogenes (4,5,7,12,22,26,29,33,36,38,39). Although the normal ras genes are not oncogenic, they too can cause the malignant transformation of cells when expressed at abnormally high levels (7). In mammals, Ras proteins are 21,000-dalton molecules that are localized to the cytoplasmic side of the cell membrane (44). They bind GTP and GDP (30) and have an intrinsic GTPase activity (14,21,35). Moreover, some oncogenic forms of Ras protein are deficient in GTPase activity (14,21,35). Based on these properties, and reasoning by analogy to other known guanine nucleotide-binding proteins, the G proteins (reviewed in reference 15), most investigators think that Ras proteins act as transducers to convey extracellular signals to an intracellular effector pathway. According to this model, Ras protein bound to GTP activates its effector, and then shuts itself off through its intrinsic GTPase activity. Mutant Ras proteins which are defective in GTPase would thus cause abnormally prolonged stimulation of the effector system, explaining their oncogenicity.The RAS1 and RAS2 proteins of the yeast Saccharomyces cerevisiae provide an ideal system for testing aspects of this model. They are structurally, biochemically, and functionally similar to the mammalian Ras proteins, and at least one of their effector systems is known (3,9,10,18,24,37,41,42). The yeast RAS genes were originally isolated by using mammalian ras genes as probes to screen libraries of S. cerevisiae genomic DNA. They encode proteins which are highly homologous to the mammalian Ras proteins, particularly at their amino termini (9, 24). They undergo processing events very similar to those of their mammalian counterparts and localize to membrane fractions (8,13,25,31,44). Like the mammalian proteins, they bind guanine nucleotides; they have an intrinsic GTPase activity, and this activity is reduced in the mutant (17), and that has enabled us to create yeast strains which greatly overexpress that enzyme. As a result, we have been able to develop a more sensitive biochemical assay for Ras proteins. Using this system we have reexamined the guanine nucleotide dependence of RAS2 protein and conclude that RAS2 may b...

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Section: Methodsmentioning

confidence: 99%

“…Adenylate cyclase assays were performed as described (3,28,42) except that an ATP-generating system was omitted. The legend to Fig.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 86%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

Field¹,

Broek²,

Kataoka³

et al. 1987

Mol. Cell. Biol.

111

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Functional cloning of the adenylate cyclase gene ofCandida albicans inSaccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues ofCHS6 andSAP185

Mallet

Renault

Jacquet

2000

Yeast

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We have cloned a genomic fragment of Candida albicans by complementation of a Saccharomyces cerevisiae cyr1 mutant. This fragment contains the two‐thirds C‐terminal part of the adenylate cyclase CaCYR1. The complete gene has been sequenced from PCR fragments amplified from genomic DNA, and contains an ORF of 1690 amino acids closely related to other fungal adenylate cyclases. Adjacent to the adenylate cyclase gene, we have sequenced six other putative genes. CaCHS6, CaYNL191 and CaYJL098 are named on the basis of their close similarity with S. cerevisiae genes. ORFs CaYJL097a and CaYJL097b represent two repeated homologues of the S. cerevisiae YJL097w, which probably arose from an ancient duplication. The last one is a hypothetical ORF, CaYKR049, which presents only a very weak similarity with YKR049. The S. cerevisiae homologues of three of these genes are co‐localized on chromosome X but with a different order and orientation. The EMBL Accession No. of CaCYR1 is AJ250877 and the insert containing the other genes is No. AJ250310. Copyright © 2000 John Wiley & Sons, Ltd.

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Inhibition of an activated Ras protein with genetically selected peptide aptamers

Kurtz

Esposito

Tang

et al. 2003

Biotech & Bioengineering

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Mutant alleles of Ras maintain an activated, GTP-bound conformation and relay mitogenic signals that cannot be turned off. A genetic selection in Saccharomyces cerevisiae was used to identify peptide aptamers that suppress the growth arrest phenotype of an activated Ras allele. Peptide aptamers were expressed as C-terminal fusions to glutathione-S-transferase. Modifications that alter the coding capacity of the peptide aptamer indicate it is necessary for Ras2-Val19 suppression. Aptamer expression also reduces the elevated levels of cAMP and suppresses the heat shock sensitivity characteristic of Ras-activated yeast cells. The peptide aptamer retains suppressor activity when fused to thioredoxin. The peptide aptamer expression strategy described here indicates that aptamers presented as unconstrained peptides have functional capacity in vivo.

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Differential activation of yeast adenylate cyclase by wild type and mutant RAS proteins

Cited by 366 publications

References 36 publications

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

Functional cloning of the adenylate cyclase gene ofCandida albicans inSaccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues ofCHS6 andSAP185

Inhibition of an activated Ras protein with genetically selected peptide aptamers

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