Activation of the Ras-GRF/CDC25Mm exchange factor by lysophosphatidic acid.

The Ras-GRF exchange factor can activate Ras-dependent responses following the activation of heterotrimeric G-protein and calcium signalling. In stable lines of NIH-3T3 fibroblasts that express Ras-GRF, the agonist lysophosphatidic acid (LPA) increases the phosphorylation state and activity of Ras-GRF. The stimulation of Ras-GRF can be demonstrated in vitro, in an assay using recombinant Ras substrate, and in situ, by a selective increase in the ability of LPA to stimulate mitogen-activated protein (MAP) kinase. The increase in Ras-GRF phosphorylation state, which occurs on serine residues, and the increase in exchange factor activity are blocked by pretreatment with pertussis toxin. Activation of Ras-GRF by LPA can also be inhibited by chelation of intracellular calcium and treatment of the Ras-GRF with protein phosphatase 1 (PP1), supporting a model in which Ras-GRF serves to integrate signals from multiple transduction pathways.     

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-gamma and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-gamma or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.     

Sites of phosphorylation by protein kinase A in CDC25Mm/GRF1, a guanine nucleotide exchange factor for Ras

Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser745 and Ser822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.     

Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.     

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.     

The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors

Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.     

Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.

The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.     

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.     

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.     

Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein.

smg p25A is a ras p21-like small GTP-binding protein which is implicated in the regulated secretory processes. We have recently found that bovine brain smg p25A is geranylgeranylated at its C-terminal region. In this study, we examined the function(s) of the C-terminal region of smg p25A. Limited proteolysis of bovine brain smg p25A with Achromobacter protease I produced an N-terminal fragment and a C-terminal tail. The Mrs of intact smg p25A, the N-terminal fragment, and the C-terminal tail were estimated to be about 24,000, 20,000, and less than 2,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal fragment contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities and showed these activities with kinetic properties similar to those of the intact protein but did not bind to plasma membranes or phosphatidylserine-linked Affigel under conditions in which the intact protein bound to them. The C-terminal tail neither contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities nor bound to plasma membranes or phosphatidylserine-linked Affigel. The GDP/GTP exchange protein specific for smg p25A, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of the intact smg p25A at a molar ratio of 1:1 and thereby inhibited its GDP/GTP exchange reaction but neither made a complex with the N-terminal fragment or the C-terminal tail nor affected the GDP/GTP exchange reaction of the N-terminal fragment. We expressed smg p25A in Escherichia coli and purified it to near homogeneity. This bacterial protein was not geranylgeranylated. Bacterial smg p25A did not bind to plasma membranes or phosphatidylserine-linked Affigel. smg p25A GDI neither made a complex with bacterial smg p25A nor affected its GDP/GTP exchange reaction. These results suggest that the N-terminal region of smg p25A has GDP/GTP-binding and GTPase activities but lacks the ability to interact with membranes and smg p25A GDI, that the C-terminal region of smg p25A plays important roles in its interaction with membranes and smg p25A GDI, and that some modifications of the C-terminal region, such as geranylgeranylation, which are absent in bacterial smg p25A, are important for these interactions.     

F-actin-dependent translocation of the Rap1 GDP/GTP exchange factor RasGRP2

RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors.     

A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.     

Mechanism of the guanine nucleotide exchange reaction of Ras GTPase--evidence for a GTP/GDP displacement model

Ras GTPases function as binary switches in the signaling pathways controlling cell growth and differentiation by cycling between the inactive GDP-bound and the active GTP-bound states. They are activated through interaction with guanine nucleotide exchange factors (GEFs) that catalyze the exchange of bound GDP with cytosolic GTP. In a conventional scheme, the biochemical roles of GEFs are postulated as stimulating the release of the bound GDP and stabilizing a nucleotide-free transition state of Ras. Herein we have examined in detail the catalyzed GDP/GTP exchange reaction mechanism by a Ras specific GEF, GRF1. In the absence of free nucleotide, GRF1 could not efficiently stimulate GDP dissociation from Ras. The release of the Ras-bound GDP was dependent upon the concentration and the structure of the incoming nucleotide, in particular, the hydrophobicity of the beta and gamma phosphate groups, suggesting that the GTP binding step is a prerequisite for GDP dissociation, is the rate-limiting step in the GEF reaction, or both. Using a pair of fluorescent guanine nucleotides (N-methylanthraniloyl GDP and 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP) as donor and acceptor probes, we were able to detect fluorescence resonance energy transfer between the incoming GTP and the departing GDP on Ras under controlled kinetic conditions, providing evidence that there may exist a novel intermediate of the GEF-Ras complex that transiently binds to two nucleotides simultaneously. Furthermore, we found that Ras was capable of binding pyrophosphate (PPi) with a dissociation constant of 26 microM and that PPi and GMP, but neither alone, synergistically potentiated the GRF1-stimulated GDP dissociation from Ras. These results strongly support a GEF reaction mechanism by which nucleotide exchange occurs on Ras through a direct GTP/GDP displacement model.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3

Activation of Rho/Rac GTPases during cell signaling requires the participation of GDP/GTP exchange factors of the Dbl family. Although the structure of the catalytic core of Dbl proteins has been established recently, the molecular changes that the full-length proteins experience during normal or oncogenic conditions of stimulation are still unknown. Here, we have used single-particle electron microscopy to solve the structures of the inactive (unphosphorylated), active (phosphorylated), and constitutively active (N-terminally deleted) versions of the exchange factor Vav3. Comparison of these forms has revealed the interdomain interactions maintaining the inactive Vav3 state and the dynamic changes that the overall Vav3 structure undergoes upon tyrosine phosphorylation. We have also found that the conformations of phosphorylated Vav3 and N-terminally deleted Vav3 are distinct, indicating that the acquisition of constitutive activity by exchange factors is structurally more complex than the mere elimination of inhibitory interactions between structural domains.     

Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1(G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins. Disruption of ROM2 resulted in a temperature-sensitive growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of deltarom1deltarom2 cells were similar to those of deltarho1 cells, including growth arrest with a small bud and cell lysis. Moreover, the temperature-sensitive growth phenotype of deltarom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rholp-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor. These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.     

Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.     

Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.