Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.
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PMID:A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity. 173 35

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.
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PMID:Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest. 840 Apr 61

Random RAS2 mutants of Saccharomyces cerevisiae were screened for activating traits. A total of 69 distinct mutations were identified, affecting 44 different amino acid residues. Many activated alleles do not bypass the requirement for the nucleotide exchange factor, CDC25, nor is the severity of RAS2 phenotypic traits strictly correlated with the capacity to bypass CDC25. In vivo interactions of mutant RAS2 proteins with RAS effectors (adenylate cyclase and RAF), CDC25 and GTPase activating proteins (IRA2 and NF1) were assayed to assess how the various amino acid substitutions influence interactions with regulatory and target proteins of RAS. Nearly all activated RAS2 proteins were observed to interact better with adenylate cyclase and RAF, although some distinct differences were found. Several amino acid substitutions that reduce the affinity of RAS2 for guanine nucleotides apparently elevate the fraction of nucleotide-free RAS2, which has greater CDC25 affinity. Amino acid alterations that reduce the affinity of RAS2 for GTPase activating proteins included substitutions both within the switch I/switch II domain and distinctly outside it. One mutant, RAS2-Y78F, bound a lower fraction of GTP in vivo than the wild-type protein. The Y78F substitution is localized to the switch II domain, a region of the RAS protein that undergoes guanine nucleotide-dependent conformational changes.
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PMID:Novel, activated RAS mutations alter protein-protein interactions. 880 95

Ras proteins play the role of molecular switches by conformational change between a GTP and a GDP-bound state. In the yeast Saccharomyces cerevisiae, they are encoded by two partially redundant genes RAS1 and RAS2 with a different pattern of gene expression. They are essential for growth because they are required for the activation of the adenylate cyclase and thus the protein kinase A pathway. Other possible biological functions remains to be established. To achieve their biological function, they need to be processed after their synthesis, they are modified farnesylated and palmitoylated at their C-terminal end at their CaaX box. Palmitoylation, involved in membrane localization, is not essential for growth but required for glucose signaling whereas farnesylation appears to participate in adenylate cyclase activation. In the GTP-bound state ras proteins interact through their conserved effector domain with the adenylate cyclase, the product of the CYR1/CDC35 gene. They also interact with GTPase activating proteins encoded by IRA1 and IRA2. These proteins are specific for yeast ras. It has been shown that Ira2p recognizes specific residues of yeast ras not shared by mammalian ras. The interaction with the guanine nucleotide exchange factor (GEF) of the CDC25 family is enhanced by dominant negative mutations such as RAS2ala22. Using the two hybrid approach, we have showed the key role of position 80 in Ras2p and confirmed the involvement of the a2 helix, the other switching part of ras, in this interaction and the induced effect. As a counterpart we have identified positions in HGRF55 conserved in other GEF involved in ras interaction. The triggering elements of ras activation: the GEF Cdc25p and Sdc25p are limiting components of the ras system. Cdc25p is part of a multimolecular complex associated with the membrane. We have shown that it can form homodimers and heterodimers with Sdc25p. It is an unstable protein containing a cyclin destruction box. Therefore its activity on ras could be regulated by controlling its cellular content.
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PMID:[Ras proteins in Saccharomyces cerevisiae, their partners and their activation]. 925 49

Neurofibromin (NF1) (the product of Nf1 gene) is a large cytosolic protein known as a negative regulator of Ras. A fragment of some 400 residues located at the center of the NF1 GAP-Related Domain (NF1-GRD) has strong identity with other molecules of the GAP family, which comprises, among others, the mammalian proteins NF1 and p120GAP, and the yeast proteins IRA1 and IRA2. GAP family members are known by their ability to promote the GTPase activity of Ras proteins, facilitating the transit of those proteins to their inactive state. Recent findings (Tong et al., 2002, Nat Neurosci 5:95-96) indicate that NF1 may be involved in the regulation of adenyl cyclase activity. Our results show that NF1-GRD cooperates with Ras in the anchorage-independent growth capacity of Ras-expressing fibroblasts, without affecting: (i) their ability to grow in low serum, (ii) their cellular adhesion capability, or (iii) the expression of key proteins involved in cell-cell and cell-matrix interactions. On the other hand, NF1 overexpression induces an increase in the expression levels of the focal adhesion kinase (FAK), and specific changes in the activation status of the mitogen-activated protein kinases (MAPKs). These results suggest the existence of a Ras-independent NF1-dependent pathway able to modify the levels of expression of FAK and the levels of activation of MAPKs. Because FAK and many proteins recently found to bind NF1 have a role in the cytoskeleton, this pathway may involve rearrangement of cytoskeletal components that facilitate anchorage independence.
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PMID:NF1 modulates the effects of Ras oncogenes: evidence of other NF1 function besides its GAP activity. 1450 61

The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha 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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PMID:Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae. 1533 5

Vacuolar (H+)-ATPases (V-ATPases) are ubiquitous, ATP-driven proton pumps that acidify organelles or the extracellular space. A rapid and effective mechanism for regulating V-ATPase activity involves reversible dissociation of the two functional domains of the pump, V1 and V0. This process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. To identify regulators that control this process in vivo, a genetic screen was performed in yeast to search for mutants that cannot disassemble their V-ATPases when grown in the absence of glucose. This screen identified IRA1 (inhibitory regulator of the Ras/cAMP pathway 1) and IRA2 as essential genes for regulating V-ATPase dissociation in vivo. IRA1 and IRA2 encode GTPase-activating proteins that negatively regulate Ras in nutrient-poor conditions. Down-regulation of Ras lowers cAMP levels by reducing adenylate cyclase activity. Decreased cAMP levels in turn lead to reduced activity of protein kinase A (PKA). Our results show that targeted deletion of IRA2 results in defective disassembly of the V-ATPase in response to glucose depletion, and reexpression of the gene rescues this phenotype. Glucose-dependent dissociation is also blocked in strains expressing the dominant active RAS2val19 allele or in strains deficient for the regulatory subunit of PKA, both of which lead to constitutively active PKA. These results reveal a role for PKA in controlling glucose-dependent V-ATPase assembly in yeast.
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PMID:The Ras/cAMP/protein kinase A pathway regulates glucose-dependent assembly of the vacuolar (H+)-ATPase in yeast. 1893 98