Ras-specific Guanine Nucleotide Exchange Factor Research Articles

Autopromotion of K-Ras4B Feedback Activation Through an SOS-Mediated Long-Range Allosteric Effect.

The Ras-specific guanine nucleotide exchange factors Son of Sevenless (SOS) regulates Ras activation by converting inactive GDP-bound to active GTP-bound states. The catalytic activity of Ras is further allosterically regulated by GTP−Ras bound to a distal site through a positive feedback loop. To address the mechanism underlying the long-range allosteric activation of the catalytic K-Ras4B by an additional allosteric GTP–Ras through SOS, we employed molecular dynamics simulation of the K-Ras4BG13D•SOScat complex with and without an allosteric GTP-bound K-Ras4BG13D. We found that the binding of an allosteric GTP−K-Ras4BG13D enhanced the affinity between the catalytic K-Ras4BG13D and SOScat, forming a more stable conformational state. The peeling away of the switch I from the nucleotide binding site facilitated the dissociation of GDP, thereby contributing to the increased nucleotide exchange rate. The community networks further showed stronger edge connection upon allosteric GTP−K-Ras4BG13D binding, which represented an increased interaction between catalytic K-Ras4BG13D and SOScat. Moreover, GTP−K-Ras4BG13D binding transmitted allosteric signaling pathways though the Cdc25 domain of SOS that enhanced the allosteric regulatory from the K-Ras4BG13D allosteric site to the catalytic site. This study may provide an in-depth mechanism for abnormal activation and allosteric regulation of K-Ras4BG13D.

SOS1 interacts with Grb2 through regions that induce closed nSH3 conformations.

Grb2 is an adaptor protein connecting the epidermal growth factor receptor and the downstream Son of sevenless 1 (SOS1), a Ras-specific guanine nucleotide exchange factor (RasGEF), which exchanges GDP by GTP. Grb2 contains three SH domains: N-terminal SH3 (nSH3), SH2, and C-terminal SH3 (cSH3). The C-terminal proline-rich (PR) domain of SOS1 regulates nSH3 open/closed conformations. Earlier, several nSH3 binding motifs were identified in the PR domain. More recently, we characterized by nuclear magnetic resonance and replica exchange simulations possible cSH3 binding regions. Among them, we discovered a cSH3-specific binding region. However, how PR binding at these sites regulates the nSH3/cSH3 conformation has been unclear. Here, we explore the nSH3/cSH3 interaction with linked and truncated PR segments using molecular dynamics simulations. Our 248 μs simulations include 620 distinct trajectories, each 400 ns. We construct the effective free energy landscape to validate the nSH3/cSH3 binding sites. The nSH3/cSH3-SOS1 peptide complex models indicate that strong peptide binders attract the flexible nSH3 n-Src loop, inducing a closed conformation of nSH3; by contrast, the cSH3 conformation remains unchanged. Inhibitors that disrupt the Ras-SOS1 interaction have been designed; the conformational details uncovered here may assist in the design of polypeptides inhibiting Grb2-SOS1 interaction, thus SOS1 recruitment to the membrane where Ras resides.

High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1.

Grb2 is an adaptor protein that recruits Ras-specific guanine nucleotide exchange factor, Son of Sevenless 1 (SOS1), to the plasma membrane. SOS1 exchanges GDP by GTP, activating Ras. Grb2 consists of an SH2 domain flanked by N- and C-terminal SH3 domains (nSH3/cSH3). Grb2 nSH3/cSH3 domains have strong binding affinity for the SOS1 proline-rich (PR) domain that mediates the Grb2-SOS1 interaction. The nSH3/cSH3 domains have distinct preferred binding motifs: PxxPxR for nSH3 and PxxxRxxKP for cSH3 (x represents any natural amino acid). Several nSH3-binding motifs have been identified in the SOS1 PR domain but none specific for cSH3 binding. Even though both nSH3 and cSH3 exhibit the strongest binding to the SOS1 peptide PVPPPVPPRRRP, this mutually exclusive binding combined with other potential nSH3/cSH3 binding regions in SOS1 makes understanding the Grb2-SOS1 interaction challenging. To identify the SOS1-cSH3 binding sites, we selected seven potential binding segments in SOS1. The synthesized peptides were tested for their binding to nSH3/cSH3. Our NMR data reveal that the PKLPPKTYKREH peptide has strong binding affinity for cSH3, but very weak for nSH3. The binding specificity suggests that the most likely Grb2-SOS1 binding mode is through nSH3-PVPPPVPPRRRP and cSH3-PKLPPKTYKREH interactions, which is supported by replica-exchange simulations for the Grb2-SOS1 complex models. We propose that nSH3/cSH3 binding peptides, which effectively interrupt Grb2-SOS1 association, can serve as tumor suppressors. The Grb2-SOS1 mechanism outlined here offers new venues for future therapeutic strategies for upstream mutations in cancer, such as in EGFR.

Abstract 5145: A new discovering motif on SOS1 PR domain clues Grb2-SOS1 association

Abstract Grb2 is an adaptor protein that recruits Ras-specific guanine nucleotide exchange factor (GEF), such as Son of sevenless 1 (SOS1), to the plasma membrane. SOS1 exchanges GDP by GTP, activating Ras. Grb2 consists of an SH2 domain in the middle and flanks nSH3/cSH3 domains at both ends. Both nSH3/cSH3 domains bind to SOS1’s C-terminal proline rich (PR) domain and have the preference for interacting with the PXXPXR motif. Our NMR data suggest that the arginine residue, followed by prolines in the PXXPXR motif, is responsible for the SH3-SOS1 association, yielding large chemical shift perturbations (CSPs) on the binding interface of SH3. The SOS1’s PR motif, PVPPPVPPRRRP, exhibits the strongest binding affinity to both SH3 domains. Using comprehensive techniques involving molecular dynamics (MD) simulation and NMR measurement, we uncover a new SOS1’s PR motifs, PKLPPKTYKREH, which shows strong binding to cSH3 but exhibits very weak affinity to nSH3. Unlike the arginine residue in the PXXPXR motif, the lysine residue in the PXXPPKXXKR motif induces weak interaction with cSH3. However, high affinity interaction is mainly derived from the hydrophobic interaction between the PXXP motif and cSH3, indicating the different binding modes of interaction. Our results suggest that Grb2 nSH3/cSH3 bind to SOS1 at distinct locations and provide atomic detailed models of Grb2-SOS1 association. Recent Grb2 targeted therapy reveals that peptide inhibitors, which interrupt Grb2-SOS1 interaction, effectively prevent the migration of breast cancer cell. The mechanism of Grb2-SOS1 interaction may enlighten the promising drug treatment development. Citation Format: Tsung-Jen Liao, Hyunbum Jang, Ruth Nussinov, David Fushman. A new discovering motif on SOS1 PR domain clues Grb2-SOS1 association [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5145.

The Interdependent Activation of Son-of-Sevenless and Ras

The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guanosine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

CIIA negatively regulates the Ras–Erk1/2 signaling pathway through inhibiting the Ras-specific GEF activity of SOS1

Son of sevenless 1 (SOS1) is a Ras-specific guanine-nucleotide-exchange factor (GEF) that mediates intracellular signaling processes induced by receptor tyrosine kinases. In this study, we show that CIIA (also known as VPS28) physically associates with SOS1 and thereby inhibits the GEF activity of SOS1 on Ras, which prevents the epidermal growth factor (EGF)-induced activation of the Ras-Erk1/2 pathway. Furthermore, CIIA inhibited cyclin D1 expression, as well as DNA, synthesis in response to EGF. Intriguingly, CIIA failed to inhibit the Ras-specific GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L and E846K). Taken together, our results suggest that CIIA functions as a negative modulator of the SOS1-Ras signaling events initiated by peptide growth factors including EGF.

Abstract 4399: Investigation of RasGRP3 expression and function in human breast cancers and breast-derived ductal adenocarcinoma cell lines.

Abstract RasGRP3 is a member of the Ras guanine nucleotide releasing protein (RasGRP) family of the Ras-specific guanine nucleotide exchange factors. These proteins play role in the regulation of the activity of Ras signaling pathway which constitutive activation is demonstrated in many cancer types. The RasGRP3 protein has potential oncogenic effect, the amplification of the gen of the protein is observed in many malignant cancer types. Recent investigations have highlighted that RasGRP3 plays a role in tumorigenesis, hence it exerts effect on the proliferation, migration, survival and tumorigenecity of prostate adenocarcinoma-derived and melanoma cells. In light of this potential oncogenic effect we have examined the change of expression and potencial function of RasGRP3 in breast-derived ductal adenocarcinoma. The RasGRP3 and phosphoRasGRP3 expressions were examined in human ductal adenocarcinoma-derived samples from different grades and in 1 primary ductal adenocarcinoma-derived cell line called BT-474 and 5 different metastatic: JIMT-1, MCF7, SK-BR-3, MDA-MB-453 és T-47D cell lines both in mRNA (Q-PCR) and protein levels (Western blot; immunhisto-and cytochemistry). To explore the biological function of the protein RasGRP3 knockdown cultures were created on MCF7 and T-47D cell lines. To examine the role of RasGRP3 in the viability of cells annexin-V/PI staining was performed. To clarify the function of the protein in cell proliferation and in the developement of resistance against chemotherapeutic drugs Tamoxifen and Herceptin CyQuant assay was performed. To observe the RasGRP3 function in tumor formation and maintance SCID mouse model was used. According to our results the expression of RasGRP3 and the active phosphoRasGRP3 were elevated at protein level on the human ductal adenocarcinoma samples. This expression was increased in the tumor samples compared to the normal. RasGRP3 was found typically in the cytoplasm of the cells, while the phosphoRasGRP3 showed strong nuclear reaction. The RasGRP3 expression of the BT-474 cell line was lower then in the metastatics. The downregulation of RasGRP3 induced apoptosis, inhibited cell proliferation and sensitizied the T-47D cells to killing by Tamoxifen and Herceptin. In vivo tumor growth in mouse xenografts of both cell lines were decreased. Suppression of RasGRP3 expression inhibited downstream RasGRP3 responses AKT and ERK1/2 phosphorylation were reduced. Our results suggest the RasGRP3 may have an importante role in the regulation of cell growth, chemotherapeutic resistance, and tumor formation in breast cancer. Citation Format: Zsuzsanna Nagy, Edina Orosz, Ilona Kovács, Miklós Török, DezsŹ Tóth, Tamás Bíró, Peter M. Blumberg, Gabriella Czifra. Investigation of RasGRP3 expression and function in human breast cancers and breast-derived ductal adenocarcinoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4399. doi:10.1158/1538-7445.AM2013-4399

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

Quantitative model of Ras–phosphoinositide 3-kinase signalling cross-talk based on co-operative molecular assembly

In growth-factor-stimulated signal transduction, cell-surface receptors recruit PI3Ks (phosphoinositide 3-kinases) and Ras-specific GEFs (guanine nucleotide-exchange factors) to the plasma membrane, where they produce 3'-phosphorylated phosphoinositide lipids and Ras-GTP respectively. As a direct example of pathway networking, Ras-GTP also recruits and activates PI3Ks. To refine the mechanism of Ras-PI3K cross-talk and analyse its quantitative implications, we offer a theoretical model describing the assembly of complexes involving receptors, PI3K and Ras-GTP. While the model poses the possibility that a ternary receptor-PI3K-Ras complex forms in two steps, it also encompasses the possibility that receptor-PI3K and Ras-PI3K interactions are competitive. In support of this analysis, experiments with platelet-derived growth factor-stimulated fibroblasts revealed that Ras apparently enhances the affinity of PI3K for receptors; in the context of the model, this suggests that a ternary complex does indeed form, with the second step greatly enhanced through membrane localization and possibly allosteric effects. The apparent contribution of Ras to PI3K activation depends strongly on the quantities and binding affinities of the interacting molecules, which vary across different cell types and stimuli, and thus the model could be used to predict conditions under which PI3K signalling is sensitive to interventions targeting Ras.

The isolated catalytic hairpin of the Ras-specific guanine nucleotide exchange factor Cdc25 Mm retains nucleotide dissociation activity but has impaired nucleotide exchange activity

Cdc25 Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25 MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation – final steps in the GEF catalytic cycle – require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.

Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects

We investigated the role of the Ras/extracellular-regulated kinase (ERK) pathway in the development of tolerance to Delta(9)-tetrahydrocannabinol (THC)-induced reduction in spontaneous locomotor activity by a genetic (Ras-specific guanine nucleotide exchange factor (Ras-GRF1) knock-out mice) and pharmacological approach. Pre-treatment of wild-type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC-induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down-regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid-stimulated [(35)S]GTPgammaS binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras-GRF1 knock-out mice and SL327 pre-treated wild-type mice, CB1 receptor down-regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC-induced hypomotility.

Mechanism of the Guanine Nucleotide Exchange Reaction of Ras GTPaseEvidence 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.

Mechanisms of Guanine Nucleotide Exchange and Rac-mediated Signaling Revealed by a Dominant Negative Trio Mutant

Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis. Dominant negative mutants of Rho GTPases, such as T17NRac1, that block the endogenous Rho protein activation by sequestering upstream guanine nucleotide exchange factors (GEFs) have been widely used to implicate specific members of the Rho family in various signaling pathways. We show here that such an approach could produce potentially misleading results since many Rho GEFs can interact with multiple Rho proteins promiscuously, and overexpression of one dominant negative Rho protein mutant may affect the activity of other members of the Rho family. Based on the available structural information, we have identified the highly conserved amino acid pairing of Asn(1406)Trio-Asp(65)Rac1 of the GEF-Rho GTPase interaction as the critical catalytic machinery required for the Rac1 GDP/GTP exchange reaction. The N1406A/D1407A mutant of Trio acted dominant negatively in vitro by retaining Rac1 binding activity but losing GEF catalytic activity and competitively inhibited Rac1 activation by wild type Trio. It readily blocked the platelet-derived growth factor (PDGF)-induced lamellipodia formation and inhibited the wild type Trio-induced serum response factor activation. Moreover the mutant was able to selectively inhibit Dbl-induced Rac1 activation without affecting RhoA activity in cells. In contrast to the non-discriminative inhibitory effect displayed by T17NRac1, the Trio mutant was ineffective in inhibiting PDGF-stimulated DNA synthesis and Dbl-induced transformation, revealing the Rac-independent functions of PDGF and Dbl. These studies identify a conserved pair of amino acid residues of the Trio-Rac interaction that is likely to be essential to the GEF catalysis of Rho family GTPases and demonstrate that a dominant negative mutant derived from a Rho GTPase regulator constitutes a new generation of specific inhibitors of Rho GTPase signaling pathways.

Structure Determination and Dynamics of Peptides Overlapping the Catalytic Hairpin of the Ras-Specific GEF Cdc25Mm

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.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Characterization and Properties of Dominant-negative Mutants of the Ras-specific Guanine Nucleotide Exchange Factor CDC25Mm

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.

G protein beta gamma subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm).

Ras-GRF1 has been implicated as a Ras-specific guanine nucleotide exchange factor (GEF), which mediates calcium- and muscarinic receptor-triggered signals in the brain. Although a Dbl homology domain known as a motif conserved among GEFs that target Rho family GTP-binding proteins exists in Ras-GRF1, GEF activity toward Rho family proteins has not been observed. Here we show that Ras-GRF1 exhibits Rac1-specific GEF activity when recovered from cells overexpressing G protein beta gamma subunits (Gbeta gamma). Substitution of conserved amino acids within the Dbl homology domain abolished this activity. Activation of the Rac pathway in the cell was further evidenced by synergistic activation of the stress kinase JNK1 by Ras-GRF1 and Gbeta gamma, which is sensitive to inhibitory action of dominant-negative Rac1(17N). In addition, association of Ras-GRF1 with Rac1(17N) was demonstrated by coimmunoprecipitation. Evidence for the involvement of tyrosine kinase(s) in Gbeta gamma-mediated induction of Rac1-specific GEF activity was provided by the use of specific inhibitors. These results suggest a role of Ras-GRF1 for regulating Rac-dependent as well as Ras-dependent signaling pathways, particularly in the brain functions.

Interaction between Pleckstrin homology domains and G protein betagamma-subunits: analyses of kinetic parameters by a biosensor-based method.

Pleckstrin homology (PH) domains, comprised of rather weakly conserved sequences of about 100 amino acid residues, are a protein motif found in many signaling and cytoskeletal proteins. PH domains have been shown to bind to the betagamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma), but the affinity of PH domains for Gbetagamma has not been quantitatively estimated in detail. To characterize the nature of the interaction between PH domains and Gbetagamma its kinetic parameters were analyzed using a BIAcore instrument. All PH domains tested (PH domains of ras-specific guanine nucleotide exchange factor (ras-GRF), phospholipase (PLC) gamma1, and Son of sevenless protein (Sos)) appeared to bind to Gbeta1gamma2 with affinity constants K(D) of 0.108, 0.318, and 0.208 microM, respectively. The binding of PH domains to Gbetagamma was inhibited by preincubation of Gbetagamma with the GDP-bound but not the GTP-bound form of Gialpha. This study showed a high affinity interaction between PH domains and Gbetagamma, and suggests a potential role of PH domains in Gbetagamma-mediated signal transduction in intact cells.

Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25 Mm originate both loss-of-function and gain-of-function proteins

The role of two residues within the catalytic domain of CDC25 Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25 Mm E1048K and CDC25 Mm S1122V were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25 Mm S1122A showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.