Residues crucial for Ras interaction with GDP-GTP exchangers (original) (raw)
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The EMBO Journal
In the yeast Saccharomyces cerevisiae genetic and biochemical evidence indicates that the product of the CDC25 gene activates the RAS/adenylyl cyclase/protein kinase A pathway by acting as a guanine nucleotide protein. Here we report the isolation of a mouse brain cDNA homologous to CDC25. The mouse cDNA, called CDC25Mm, complements specifically point mutations and deletion/disruptions of the CDC25 gene. In addition, it restores the cAMP levels and CDC25-dependent glucoseinduced cAMP signalling in a yeast strain bearing a disruption of the CDC25 gene. The CDC25Mm-encoded protein is 34% identical with the catalytic carboxy terminal part of the CDC25 protein and shares significant homology with other proteins belonging to the same family. The protein encoded by CDC2S m, prepared as a glutathione S-transferase fusion in Escherichia coli cells, activates adenylyl cyclase in yeast membranes in a RAS2-dependent manner. Northern blot analysis of mouse brain poly(A) + RNA reveals two major transcripts of -1700 and 5200 nucleotides. Transcripts were found also in mouse heart and at a lower level in liver and spleen.
Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2001
The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition`in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role. ß 0167-4889 / 01 / $^see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 8 9 ( 0 1 ) 0 0 0 6 7 -2
FEMS Yeast Research, 2007
The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Rasguanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucoseinduced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.
The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway
Cell, 1987
The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RASprl9 gene, but not by high copy plasmids expressing a normal RAS2 or RASl gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RASladenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.
Proceedings of the National Academy of Sciences, 1995
Although both Rasl 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 RASJ-RAS2 orRAS2-RAS] genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts rasi 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 Ras2A, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than RaslA, in which the C-terminal domain of Rasl 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 Rasl, Rasl-Ras2, RaslA, Ras2, and Ras2-Rasl strains throughout the growth curve. This was found to result from the higher expression of Rasl and Rasl-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Rasl and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent Cterminal domains are responsible for these differences, through interaction with other regulatory elements.
Two Distinct Regions of Ras Participate in Functional Interaction with GDP-GTP Exchangers
European Journal of Biochemistry, 1995
We have previously implemented a combined genetichiochemical approach, for analysis of insertiondeletion mutants, to identify sites of Harvey-Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger. We showed that positions 101-106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction. Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitutions had no effect on coupling to adenylyl cyclase. Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch I1 region (amino acids 69-78). In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cyclase activation. These findings establish the crucial role of the 102-105 region in the catalyzed exchange reaction and suggest that the 69-74 area would be required for the functional interaction with both exchangers and effector molecules.
Proceedings of the National Academy of Sciences, 1990
A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase...
A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae
Molecular and cellular biology, 1988
In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25. We isolated several dominant extragenic suppressors of the yeast cdc25 mutation. They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35). One of these suppressors was a mutated RAS2 gene in which the transition C/G----T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein. The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L.A. Feig, B. Pan, T.M. Roberts, and G.M. Cooper, Proc. Natl. Acad. Sci. USA 83:4607-4611, 1986). These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.
Journal of Biological Chemistry, 1999
Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25 Mm , are described. In vitro, the mutant GEF W1056E and GEF T1184E proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21 ras , and strongly reduce affinity of the nucleotide-free ras⅐GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras⅐GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF W1056E mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.