Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae

Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.     

Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase

Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF.     

PKA-dependent regulation of Cdc25 RasGEF localization in budding yeast

In Saccharomyces cerevisiae the Cdc25/Ras/cAMP pathway is involved in cell growth and proliferation regulation. Ras proteins are regulated by Ira1/2 GTPase activating proteins (GAPs) and Cdc25/Sdc25 guanine nucleotide exchange factors (GEFs).Most of cytosolic Cdc25 protein was found on internal membranes in exponentially growing cells, while upon incubation in a buffer with no nutrients it is re-localized to plasma membrane. The overexpression of Tpk1 PKA catalytic subunit also induces Cdc25 export from the nucleus, involving two serine residues near the Nuclear Localization Site (NLS): mutation of Ser⁸²⁵ and Ser⁸²⁶ to glutamate is sufficient to exclude physiologically expressed Cdc25 from the nucleus, mimicking Tpk1 overproduction effect. Mutation of these Ser residues to Ala abolishes the effect of nuclear export induced by Tpk1 overexpression on a Cdc25eGFP fusion. Moreover, mutation of these residues affects PKA-related phenotypes such as heat shock resistance, glycogen content and cell volume.     

Cloning and characterization of the S. pombe gene efc25+, a new putative guanine nucleotide exchange factor

We report the cloning and characterization of a new S. pombe gene, efc25⁺, for `exchange factor Cdc25-like'. The C-terminal region of the predicted product of this gene displays high sequence homology with a number of guanine nucleotide exchange factors for Ras. These include Cdc25 of Saccharomyces cerevisiae, Cdc25 of Saccharomyces kluyveri, Csc25 of Candida albicans, Sdc25 of S. cerevisiae and Ste6 of Schizosaccharomyces pombe. Disruption of efc25⁺ resulted in cells with a spherical shape reminiscent of the abnormal morphological phenotype of ras1 deletion mutants. However, unlike ras1 null mutants, strains deleted for efc25⁺ were proficient for mating and sporulation. This differs from the only other Ras1 exchange factor characterized so far in S. pombe, the Ste6 protein, whose deletion results in defects in mating and sporulation but not in cell shape. We hypothesize that Efc25 is an exchange factor for Ras1 and that it is involved in a signaling pathway different from that involving Ste6.     

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.     

Feedback regulation of Ras2 guanine nucleotide exchange factor (Ras2-GEF) activity of Cdc25p by Cdc25p phosphorylation in the yeast Saccharomyces cerevisiae

Ras-GEF Cdc25p has been found to be hyperphosphorylated upon glucose addition. This work provides evidence indicating that PKA activity positively regulates the degree of Cdc25p phosphorylation, and that the intracellular association of Cdc25p and Ras2p is independent of PKA activity. In vitro experiments revealed that the Ras2-GEF activity of Cdc25p is inhibited by Cdc25p phosphorylation. These data suggest a negative feedback mechanism by which intracellular cAMP synthesis is inhibited by PKA through Cdc25p phosphorylation.

Structured summary

MINT-8053016: CDC25p (uniprotkb:P04821) physically interacts (MI:0915) with ras2p (uniprotkb:P01120) by anti tag co-immunoprecipitation (MI:0007)MINT-8053030: ras2p (uniprotkb:P01120) physically interacts (MI:0915) with CDC25p (uniprotkb:P04821) by anti bait co-immunoprecipitation (MI:0006)     

Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm)

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 ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF 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.     

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

A role for the noncatalytic N terminus in the function of Cdc25, a Saccharomyces cerevisiae Ras-guanine nucleotide exchange factor

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor (GEF) for Ras proteins. Its catalytic domain is highly homologous to Ras-GEFs from all eukaryotes. Even though Cdc25 is the first Ras-GEF identified in any organism, we still know very little about how its function is regulated in yeast. In this work we provide evidence for the involvement of the N terminus of Cdc25 in the regulation of its activity. A truncated CDC25 lacking the noncatalytic C-terminal coding sequence was identified in a screen of high-copy suppressors of the heat-shock-sensitive phenotype of strains in which the Ras pathway is hyper-activated. The truncated gene acts as a dominant-negative mutant because it only suppresses the heat-shock sensitivity of strains that require the function of CDC25. Our two-hybrid assays and immunoprecipitation analyses show interactions between the N terminus of Cdc25 and itself, the C terminus, and the full-length protein. These results suggest that the dominant-negative effect may be a result of oligomerization with endogenous Cdc25. Further evidence of the role of the N terminus of Cdc25 in the regulation of its activity is provided by the mapping of the activating mutation of CDC25HS20 to the serine residue at position 365 in the noncatalytic N-terminal domain. This mutation induces a phenotype similar to activating mutants of other genes in the Ras pathway in yeast. Hence, the N terminus may exert a negative control on the catalytic activity of the protein. Taken together these results suggest that the N terminus plays a crucial role in regulating Cdc25 and consequently Ras activity, which in S. cerevisiae is essential for cell cycle progression.     

Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant Bud5 and Lte1 proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-ras but inactive in promoting exchange on the Ras-related proteins Ypt1 and Rsr1. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP- or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.     

Structure determination and dynamics of peptides overlapping the catalytic hairpin of the Ras-specific GEF Cdc25(Mm)

Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-like. To date, structural data are available only for Cdc25(Mm). We designed and synthesized Cdc25(Mm)-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25(Mm)(1178-1222) peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSos1, while residues corresponding to helix H in hSos1 show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25(Mm)(1178-1222) peptide is able to bind nucleotide-free Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties.     

Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.

Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1364 to 1383 of CDC25GEF inhibit interaction between GEFs and H-Ras. We propose that residues 1374 of CDC25GEF and 63 of H-Ras form an ion pair and that when this ion pair is reversed, functional interaction can still occur.     

A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development

BACKGROUND: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins. RESULTS: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins. CONCLUSIONS: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.     

Two ras pathways in fission yeast are differentially regulated by two ras guanine nucleotide exchange factors

How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity. Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs. Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating. So far there is only one established Ras1 GEF, Ste6. Paradoxically, ste6 null (ste6 Delta) mutants are sterile but normal in cell morphology. This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway. We thereby characterized a potential GEF, Efc25. Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway. Like ras1 Delta and scd1 Delta, efc25 Delta is synthetically lethal with a deletion in tea1, a critical element for cell polarity control. Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus. We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway. While ste6 expression is induced during mating, efc25 expression is constitutive. Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1. This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2. Reciprocally, Ste6 overexpression can block Scd1 activation. We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.