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At least 19 recordsLinked to original sources
PubMed1993

A murine CDC25/ras-GRF-related protein implicated in Ras regulation.

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development.

Amino Acid Sequence↗
PubMed1990

Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string.

We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25-22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.

Amino Acid Sequence↗
PubMed1990

In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties.

The attenuated GTP regulation adenylyl cyclase (CDC35) lysates or membranes prepared from cells of a cdc25ts strain is enhanced 2.5- to 6-fold by mixing these lysates or membranes with lysates or membranes from a cdc35ts strain harboring wild-type CDC25. The kinetics of activation of the Saccharomyces cerevisiae adenylyl cyclase in vitro is first order, as is the activation of mammalian adenylyl cyclase. The rate of enzyme activation in the presence of non-hydrolysable analogs of GTP increases with the number of CDC25 gene copies present in the cell. When GppNHp was used the rate of activation of the cyclase in a strain harboring a multicopy plasmid of CDC25 was 7.0-fold higher than the rate in an isogenic strain with the cdc25-2 mutation. The rate of adenylyl cyclase activation from a strain with a disrupted CDC25 gene is 14.7-fold lower than the rate in an isogenic strain containing the CDC25 gene on a multicopy plasmid. The reconstitution experiments described provide direct biochemical evidence for the role of the CDC25 protein in regulating the RAS dependent adenylyl cyclase in S.cerevisiae. The reconstitution experiments and the kinetic experiments may also provide a biochemical assay for the CDC25 protein and can form the basis for its characterization. In this study we also show that adenylyl cyclase activity in ras1ras2byc1 cells is found in the soluble fraction, whereas in wild-type strain it is found in the membrane fraction. Overexpression of the gene CDC25 in the ras1ras2bcy1 strain relocalizes adenylyl cyclase activity to the membrane fraction. This finding suggests a biochemical link between CDC25 and CDC35 in the absence of RAS, in addition to its role in regulating RAS dependent adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1992

Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.

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 glucose-induced 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 CDC25Mm, 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 approximately 1700 and 5200 nucleotides. Transcripts were found also in mouse heart and at a lower level in liver and spleen.

Adenylyl Cyclases↗
PubMed1992

Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division.

The Saccharomyces cerevisiae gene CDC6, whose protein product is required for DNA replication, is transcribed only in late G1 and S phases. We have discovered a critical reason why CDC6 expression is regulated in this fashion. Constitutive CDC6 transcription greatly delayed the initiation of M phase without effecting the G1-S transition or growth rate. This occurred in both fission and budding yeasts. The CDC6-induced M phase delay was dependent on the wee1/mik1 mitotic inhibitor kinases and was greatly accentuated in strains defective for the cdc25/MIH1 mitotic inducer phosphatases, indicating that CDC6 indirectly inhibits activation of the p34cdc2/CDC28 M phase kinase. Thus CDC6 appears to have an important and perhaps unique dual role in S phase, it is first required for the initiation of DNA replication and then actively participates in the suppression of nuclear division.

Base Sequence↗
PubMed1992

Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15.

We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.

Amino Acid Sequence↗
PubMed1992

Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24.

In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras. Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al. have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25. We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm. This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids. Three cDNAs are predicted to encode protein products of 666 or 667 amino acids. The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively. A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24. Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts. Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs. In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form. We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.

3T3 Cells↗
PubMed1992

Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.

Alleles↗
PubMed1995

Guanine nucleotide exchange factors: activators of Ras superfamily proteins.

Members of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP-complexed and inactive GDP-complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP-bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP-bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras-related Rho (Dbl and Vav) proteins. Growth factor-mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10-50 fold and 5-10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras. We have determined that two Ras-related proteins, designated R-Ras and R-Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R-Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R-Ras, transforming activities. Therefore, TC21, but not R-Ras, may be activated by the same extracellular signaling events that activate Ras proteins. Dbl family proteins are believed to function as GEFs and activators of the Ras-related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras-independent activation of MAP kinases and Rho family proteins.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of CIF1, a yeast gene necessary for growth on glucose.

The cif1 mutation of Saccharomyces cerevisiae (Navon et al., Biochemistry 18, 4487-4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of function and located it in a 2.75 kb SphI-BstEII fragment situated at ca. 18 kb centromere distal of LYS2 and ca. 80 kb centromere proximal of TYR1 on chromosome II. Southern analysis demonstrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1.8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increase. cAMP concentration increased in response to galactose or 2,4-dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1 background did not restore growth on glucose, suggesting that the absence of cAMP signal is not the primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.

Adenosine Triphosphate↗
PubMed1994

Isolation of a CDC25 family gene, MSI2/LTE1, as a multicopy suppressor of ira1.

We have identified MSI2 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold-sensitive phenotype. Under the non-permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.

Amino Acid Sequence↗
PubMed1994

LTE1 of Saccharomyces cerevisiae is a 1435 codon open reading frame that has sequence similarities to guanine nucleotide releasing factors.

The DNA sequence of the LTE1 gene on the left arm of chromosome I of Saccharomyces cerevisiae has been determined. The LTE1 open reading frame comprises 4305 bp that can be translated into 1435 amino acid residues. The position of this open reading frame corresponds well to that of a 4.7 kb transcript that has been mapped to this position. The derived amino acid sequence has significant similarities to the amino acid sequence of the guanine nucleotide releasing factor isolated from a rat brain library. The carboxy-terminus of the LTE1 protein also shows similarities to other guanine nucleotide exchange factors of the S. cerevisiae CDC25 family.

Amino Acid Sequence↗
PubMed1994

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.

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 CDC25Mm.

Adenylyl Cyclases↗
PubMed1995

The minimal active domain of the mouse ras exchange factor CDC25Mm.

The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.

Animals↗
PubMed1997

The production of soluble interleukin 4 receptors is preferentially regulated by the murine Th2 cell subset.

In order to understand how the endogenous production of soluble IL-4 receptors (sIL-4r) is regulated, the authors tested prototypic clones of Th1 and Th2 murine CD4+ T cell subsets for their ability to regulate their expression of sIL-4r. Results showed that although both types of clones produced low levels of sIL-4r under resting conditions, only the Th2 clones upregulated sIL-4r expression following antigenic stimulation. Inhibition of endogenous IL-4 with a neutralizing anti-IL-4 mAb had only a minor (approximately 20%) inhibitory effect on sIL-4r production by the Th2 cells, and addition of rIL-4 to Th1 cells resulted only in a modest two-fold increase in sIL-4r levels, suggesting that IL-4 is not the only factor that regulates sIL-4r production and that the ability of Th2 clones to upregulate sIL-4r expression can be relatively independent of IL-4. Indeed, the production of sIL-4r by Th2 cells was found to be regulated by cell contact and/or IL-1 mediated signals. Transcripts for both sIL-4r and mIL-4r were detected by RT-PCR on both resting and activated Th1 and Th2 cells, with the relative levels of expression being moderately higher in the Th2 clones. Moreover, the expression of sIL-4r-specific transcripts appeared to increase to a greater extent than those of mIL-4r after activation of Th2 cells with APCs, both in the presence and absence of antigen. Taken together, these results predict that increased sIL-4r production in vivo might be preferentially associated with Th2-type responses and indicate that even though the production of IL-4 and sIL-4r is mediated by the same cells (i.e. Th2 cells), the synthesis of sIL-4r can be regulated independently from that of IL-4 through alternative signals such as cell contact and/or IL-1. These properties may allow for changing ratios of sIL-4r to IL-4 and sIL-4r to mIL-4r during different phases of an immune response and are consistent with a regulatory role for sIL-4r on IL-4 activity in vivo.

Animals↗
PubMed1994

Expression of two different products of CDC25Mm, a mammalian Ras activator, during development of mouse brain.

The CDC25Mm gene codes for Ras-guanine nucleotide exchange factors. Four different full-length cDNA clones derived from the same gene and coding for proteins of different sizes that have in common the last 661 amino acids have been isolated from mouse brain. In order to investigate the expression of the products of this gene in different tissues we have prepared two polyclonal antibodies directed toward two different regions of the protein comprised in the last C-terminal 472 amino acids. While in most of the tested tissues we have been unable to definitely identify CDC25Mm products, in NIH3T3 fibroblasts we have found a poorly expressed 120-kDa protein. In the mouse brain we have identified two proteins of 140 and 58 kDa. While the former is expressed in the adult mouse, the latter is present in the embryo and persists for few days after birth. This finding suggests that differential expression of various forms of CDC25Mm may be involved in brain development.

3T3 Cells↗