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J Biol Chem, Vol. 273, Issue 52, 34737-34744, December 25, 1998


Raf-1 Is Involved in the Regulation of the Interaction between Guanine Nucleotide Exchange Factor and Ha-Ras
EVIDENCES FOR A FUNCTION OF Raf-1 AND PHOSPHATIDYLINOSITOL 3-KINASE UPSTREAM TO Ras*

Carmela Giglione and Andrea ParmeggianiDagger

From the Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras·Raf complex, showed that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras·GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras·GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha . Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The Ha-Ras p21 protein is a central element for the transmission of extracellular signals within the cell, interacting with a growing number of regulators and effectors (1, 2). These are proteins containing modular domains, whose interactions with other ligands are only partially known (3-5). The regulators enhance the two basic intrinsic activities of Ras: 1) the hydrolysis of GTP, and 2) the GDP/GTP exchange, two reactions stimulated by GTPase-activating proteins (GAPs)1 and the guanine nucleotide exchange factors (GEFs), respectively (6). The effectors transmit the downstream signal to pathways controlling growth, differentiation, and other fundamental processes of the cell. Despite a multitude of in vitro and in vivo studies, the coordination mechanisms by which the activity of Ha-Ras is regulated are only now beginning to be defined. It is important to mention here that the interaction of protein ligands with Ha-Ras bears a specific difference in that GAPs and effectors show a much higher affinity for Ha-Ras·GTP than for Ha-Ras·GDP, whereas GEFs act on Ras·GTP and Ras·GDP with nearly the same efficiency (3, 7-11). The common property of GAPs and Ras effectors to show a pronounced specificity for activated Ras is in line with the proposed role of p120-GAP not only as GTPase-activating protein but also as an effector of Ras (12). In line with this, the best investigated effector of Ha-Ras, c-Raf-1, competes with p120-GAP and neurofibromin for binding to Ras·GTP (2, 13-15). Another effector of Ha-Ras is RalGDS, a GDP/GTP exchange factor of RalA p24 and RalB p24. RalGDS interacts with Ras in a GTP-dependent manner and inhibits the binding of both Raf-1 and GAP to Ha-Ras (16).

In this context, we have recently described that in vitro p120-GAP protects the active state of p21 from unproductive exchanges of bound GTPgamma S after the accomplishment of the GEF-stimulated GDP/GTP exchange on Ras (17). It is possible that other protein ligands of Ras, such as Raf, may act similar to GAP to compete with GEF for binding to Ha-Ras·GTP and consequently to be involved not only downstream of p21 but also upstream on the activation mechanism leading to the GDP/GTP exchange on Ha-Ras p21. In agreement with this is the finding that in fibroblast cell lines, a mutant of a platelet-derived growth factor receptor lacking the ability to bind to phosphatidylinositol 3-kinase (PI3K), another putative effector of Ras p21, is unable to stimulate the GDP/GTP exchange on Ras p21. Moreover, when expressed in Xenopus oocytes, a constitutively active form of PI3K increased the amount of GTP bound to Ras p21 (18, 19).

Because no in vitro experiment demonstrating an interference between effectors such as Raf or PI3K and GEFs has yet been reported, in this work, we have analyzed the influence of Raf on the action of GEF to shed more light on the mechanism controlling the activation of Ha-Ras. We have found that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. Furthermore, we have provided evidence supporting the ability of PI3K to inhibit the GEF-stimulated exchange on the active state of Ha-Ras. The competition between GEF and Raf or PI3K is proposed to represent a mechanism to selectively promote the productive GDP/GTP exchange. This would confer a dual role to the effector as a transmitter of the downstream signal of Ras and as an upstream regulator of Ras activation.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression and Purification of Raf Proteins-- pGEX-KG encoding Raf residues 1-149 was provided by Dr. M. S. Marshall (Department of Medicine, Indiana University School of Medicine, Indianapolis, IN) and grown in DH5alpha cells. BL21 cells containing pGEX 2T encoding Raf residues 51-131 were provided by Dr. P. N. Lowe (Wellcome Research Laboratories, Beckenham, Kent, UK). The bacteria were grown at 37 °C in 400 ml of LB medium containing 50 µg/ml ampicillin. Expression of both GST-Raf proteins was induced overnight at 24 °C by adding 0.01 mM isopropyl-1-thio-beta -D-galactopyranoside at a cell density of 0.4 A600 units. The cell culture, which was chilled to 4 °C at 2.0 A600 units, was centrifuged at 4,000 × g for 10 min, and the pellet was resuspended in 35 ml of Buffer A (150 mM NaCl, 16 mM Na2HPO4, and 4 mM NaH2PO4, pH 7.3) containing 2 mM phenylmethylsulfonyl fluoride. After sonication and the addition of 1% Triton X-100, the suspension was centrifuged at 17,000 × g for 30 min, and the supernatant, which was mixed batchwise with 2 ml of glutathione-Sepharose 4B (Pharmacia), was gently shaken for 1 h at 4 °C. The resin was washed twice with Buffer A, and the protein eluted with glutathione (3 mg/ml) was concentrated to 3 ml with Aquacide II (Calbiochem) and dialyzed against 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 14 mM ME, and 50% glycerol. The protein solutions were stable for more than 1 year when stored at -20 °C.

Expression and Purification of C-Sdc25p-- The Escherichia coli strain SCS-1 was transformed with a pGEX vector containing the 3'-terminal region of the Saccharomyces cerevisiae SDC25 gene encoding the last 550 C-terminal residues and was grown at 24 °C in LB medium containing 50 µg/ml ampicillin. The procedure for the induction and purification of C-Sdc25p, as described by Poullet et al. (11), was used with the following modifications. The induction of C-Sdc25p was started at a cell density of 0.3 A600 unit using 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside for 12 h at 24 °C. The cells, which were harvested by centrifugation at 4,000 × g for 10 min, were washed with 150 mM NaCl. The pellet was then resuspended in 60 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 7 mM ME, 10% glycerol, 1 mM EDTA, 0.5 mM Pefabloc (Boehringer), 4 mg/ml lysozyme, and 100 µg/ml DNase 1. After sonication, the suspension was centrifuged twice at 17,000 × g for 30 min, and the supernatant was mixed batchwise for 20 min at 4 °C with 10 ml of glutathione-agarose (Sigma), equilibrated with Buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 7 mM ME, 10% glycerol, and 2.5 mM CaCl2). The glutathione-agarose suspension was washed four times with Buffer B. The GST-C-Sdc25p fusion protein bound to the resin was then cleaved with 5 units/ml human thrombin (Sigma) in 5 ml of Buffer B for 30 min at 30 °C. The supernatant was recovered, and the procedure was repeated once more. The solutions of GST-cleaved C-Sdc25p were concentrated with Aquacide II, dialyzed against 50 mM Tris-HCl, pH 7.5, 30 mM KCl, 7 mM ME, and 50% glycerol, and stored at -20 °C.

Expression and Purification of PI3K RBD-- Bacteria expressing the RBD (amino acid residues 133-314) of PI3K p110alpha were provided by Dr. J. Downward (Imperial Cancer Research Fund, London, UK). The cells were grown at 37 °C in 400 ml of LB medium containing 100 µg/ml ampicillin. The expression of GST-PI3K RBD protein was induced overnight at 24 °C by adding 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside at a cell density of 0.5 A600 units. The cell culture, which was chilled to 4 °C at 2.0 A600 units, was centrifuged at 4,000 × g for 15 min, and the pellet was resuspended in 35 ml of Buffer A containing 2 mM phenylmethylsulfonyl fluoride. After sonication and the addition of 1% Triton X-100, the suspension was centrifuged at 17,000 × g for 30 min, and the supernatant, which was mixed batchwise with 2 ml of glutathione-Sepharose 4B (Pharmacia), was gently shaken for 2 h at 4 °C. The resin was washed six times with Buffer A, and the protein eluted with glutathione (3 mg/ml) was concentrated to 1 ml with Aquacide II (Calbiochem), dialyzed overnight at 4 °C against 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 14 mM ME, and 50% glycerol, and stored at -20 °C.

Dissociation Rate Constants-- Dissociation of the p21·nucleotide complexes was determined at 30 °C by the nitrocellulose binding assay. The p21·[3H]GDP, p21·[gamma -35S]GTPgamma S, and p21·[3H]GTP complexes were obtained by incubating Ha-Ras p21·GDP with a 10-fold excess of [3H]GDP (340 Bq·pmol-1), [gamma -35S]GTPgamma S (4140 Bq·pmol-1), and [3H]GTP (270 Bq·pmol-1), respectively, for 10 min at 30 °C in Buffer C (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 3 mM EDTA, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin). 3 mM MgCl2 was then added to stabilize the p21·nucleotide complex. The dissociation was started by the addition of a 500-fold excess of unlabeled nucleotide with or without GEF, Raf, and PI3K. For details, see the figure legends. At various time intervals, aliquots (10 µl) were withdrawn and passed on nitrocellulose filters (0.45 µm of Sartorius SM 11 306), and the retained radioactivity was measured in a liquid scintillation counter (model Wallac 1410; Pharmacia). The rate constants were calculated as described previously (10, 20).

Scintillation Proximity Assay-- The interaction between Raf and Ras was monitored by assessing the ability of GEF to compete with GST-Raf for binding to p21·[3H]GTP using the scintillation proximity assay (SPA). The procedure described by Skinner et al. (21) and Gorman et al. (22) was used with the following modifications. The p21·[3H]GTP complexes were obtained by incubating Ha-Ras p21 with a 5-fold excess of [3H]GTP (360 Bq·pmol-1) for 10 min at 30 °C in Buffer C. MgCl2 (3 mM) was then added to stabilize the p21·nucleotide complex. The standard assay was performed in Eppendorf test tubes as follows: a solution (25 µl) containing different concentrations of C-Sdc25p, full-length CDC25Mm, or C-CDC25Mm-285 in 50 mM Tris-HCl, pH 7.5, was prepared; 2 mM dithiothreitol was added to each sample, followed by 175 µl of a mixture of p21·[3H]GTP (0.03 µM), anti-GST IgG (Molecular Probes; 0.02 mg/ml), GST-Raf (51-131) (0.05 µM), and protein A SPA beads (4.2 mg/ml) in 50 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, and 1 mM MgCl2. Control assays in which either GST-Raf (51-131) or GEF was omitted were also done. Bovine serum albumin was routinely included in the assays to a final concentration of 0.2 mg/ml. The Eppendorf test tubes were sealed, shaken for 1 h at 4 °C, and centrifuged at 760 × g for 2 min, and after equilibration at room temperature for 5 min, the radioactivity was counted in a Wallac 1410 scintillation counter. After subtraction of the blanks, the SPA signal was expressed as a percentage of the Ras/GST-Raf binding relative to the control value obtained in the absence of GEF proteins.

Other Assays-- Full-length CDC25Mm (p140-GRF), its catalytic domain C-CDC25Mm-285, and Ha-Ras were produced and purified as described previously (23). SDS-polyacrylamide gel electrophoresis was carried out using a 12% acrylamide gel (24). Protein concentration was measured by the Bio-Rad protein assay using bovine serum albumin as the standard, and, in the case of p21, it was also checked by [3H]GDP binding as determined by the nitrocellulose filter assay.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Raf RBDs Inhibit the GEF-dependent GTP/GTP Exchange on Ha-Ras p21-- Because both GEF and Raf act on the active state of Ha-Ras, we have searched for competition phenomena between the activities of these two proteins. For a proper analysis of the effect of Raf on the GEF activity on Ras, we first examined the influence of Raf on the intrinsic exchange activity of Ha-Ras, a parameter that has already been investigated by Herrmann et al. (15) using a fluorescence anisotropy assay and mantGpp(NH)p. The two Raf RBDs used, Raf (51-131) and Raf (1-149), were purified as GST-fusions, and their effects on the [gamma -35S]GTPgamma S dissociation from the Ha-Ras·[gamma -35S]GTPgamma S complex were determined. Both fragments behaved as nucleotide dissociation inhibitors, because the dissociation of GTPgamma S from Ha-Ras decreased after the addition of Raf proteins. Fig. 1A illustrates the apparent dissociation rate constant (k'-1) of the Ha-Ras·[gamma -35S]GTPgamma S complex as a function of increasing concentrations of GST-Raf (51-131). GST-Raf (51-131) almost completely inhibited the dissociation of [gamma -35S]GTPgamma S from Ha-Ras (Fig. 1B), in agreement with the findings of Herrmann et al. (15), who reported a dissociation constant of 18 nM for the affinity between GST-Raf (51-131) and Ras. From the experiments shown in the inset of Fig. 1A, a comparable K'd value (12 nM) was obtained. Under the same experimental conditions used for GST-Raf (51-131), an inhibitory effect was also observed in the presence of GST-Raf (1-149). In this case (Fig. 2A), the apparent dissociation rate constant was decreased from 4.9·10-3 min-1 to 3·10-3 min-1, which corresponds to a 40% inhibition of the intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras p21 (Fig. 2B). The estimated K'd between Ha-Ras·[gamma -35S]GTPgamma S and GST-Raf (1-149) was ~150 nM, i.e. higher than that calculated for GST-Raf (51-131) and in agreement with the lower affinity of GST-Raf (1-149) for Ras p21 (25-27).


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  		Fig. 1.   Effect of GST-Raf (51-131) concentration on the intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras. In A, the intrinsic dissociation rate constants of p21·[gamma -35S]GTPgamma S complexes (30 nM) were determined from 120-min kinetic experiments in the presence of increasing concentrations of GST-Raf (51-131) (black-triangle); inset, a log concentration scale. In B, the percentages of inhibition of intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras were determined versus the intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras in the absence of GST-Raf (51-131) (open circle ) that was taken as 100% activity.


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  		Fig. 2.   Effect of GST-Raf (1-149) concentration on the intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras. In A, the intrinsic dissociation rate constants of p21·[gamma -35S]GTPgamma S complexes (30 nM) were calculated from 120-min kinetic experiments in the presence of increasing concentrations of GST-Raf (1-149) (black-square). In B, the percentages of inhibition of intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras were determined versus the intrinsic GTPgamma S/GTPgamma S exchange on Ha-Ras in the absence of GST-Raf (1-149) () that was taken as 100% activity. The values represent the mean of three experiments.

The GTPgamma S/GTPgamma S exchange on Ha-Ras can be stimulated by different guanine nucleotide exchange factors, including the catalytic domain of S. cerevisiae Sdc25p (C-Sdc25p), a model GEF that acts equally well on Ha-Ras p21 and Ras2p (8, 28). As shown in Fig. 3, A and B, C-Sdc25p enhances the GTPgamma S/GTPgamma S exchange on Ha-Ras, but this effect can be relieved by the addition of increasing concentrations of GST-Raf (51-131) (Fig. 3A) or GST-Raf (1-149) (Fig. 3B). By repeating these experiments using different concentrations of GST-Raf (51-131) and 20 nM C-Sdc25p, the k'-1 of the Ha-Ras·[gamma -35S]GTPgamma S complex decreased from 3·10-2 min-1 to 0.08·10-2 min-1 in a concentration-dependent manner (Fig. 3C). From these results, one can see that GST-Raf (51-131) has the ability to inhibit the GEF-stimulated GTPgamma S/GTPgamma S exchange on Ha-Ras up to approximately 100% (Fig. 3D). In the case of GST-Raf (1-149), the k'-1 of the Ha-Ras·[gamma -35S]GTPgamma S complex decreased in a concentration-dependent manner from 3·10-2 min-1 to 0.7·10-2 min-1, which corresponds to an 80% inhibition of the GEF-induced GTPgamma S/GTPgamma S exchange on Ha-Ras (Fig. 3D). By comparing the inhibition due to Raf RBD fragments on GTPgamma S/GTPgamma S exchange on Ha-Ras in the presence and absence of GEF, our results show that Raf RBDs inhibit not only the intrinsic exchange on the active form of Ras but also the capacity of GEF to stimulate this exchange. Substitution of GTPgamma S with GTP gave the same results (data not shown).


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  		Fig. 3.   Specific inhibition by GST-Raf-1 RBDs of the C-Sdc25p-stimulated nucleotide dissociation activity of Ha-Ras. 30 nM p21·[gamma -35S]GTPgamma S were incubated for 10 min at 30 °C with increasing concentrations of GST-Raf (51-131) (A) or GST-Raf (1-149) (B). The dissociation reaction was started by adding a 500-fold excess of cold nucleotide and 20 nM C-Sdc25p. At the given times, 10-µl samples were withdrawn, and the amount of p21-bound [gamma -35S]GTPgamma S was determined by the nitrocellulose filtration procedure. In the equation ln(Ct/Co)=-k'-1·t, Co represents the initial concentration of the p21·nucleotide complex, and Ct represents the concentration at time t. In C, the dissociation rate constants of p21·[gamma -35S]GTPgamma S complexes in the presence of C-Sdc25p and the given concentrations of GST-Raf (51-131) (open circle ) or GST-Raf (1-149) (black-square) were calculated from four independent experiments. In D, the percentages of inhibition of C-Sdc25p activity were determined by taking the C-Sdc25p-dependent p21 exchange activity in the absence of GST-Raf (51-131) or Raf (1-149) proteins as 100% activity. Inhibition values were expressed as a function of different concentrations of GST-Raf (51-131) (bullet ) or Raf (1-149) ().

We also checked whether the Raf effect was dependent on the active state of Ha-Ras, the target of Raf. As expected, because Raf does not interact with the inactive GDP-bound state of Ha-Ras, no effect of Raf RBDs on the GEF-dependent GDP/GDP exchange on p21 could be detected (data not shown).

PI3K RBD Also Inhibits the GEF-dependent GTP/GTP Exchange on Ha-Ras p21-- The ability of Raf to interfere with GEF prompted us to carry out a similar experiment with PI3K to clarify whether the dual effect of Raf to act upstream and downstream on Ras could also be extended to other ligands of Ras, as suggested in the case of PI3K by in vivo results (18, 19). To this purpose, we have used the RBD (amino acid residues 133-314) of PI3K p110alpha (29) and determined its effect on the activity of the exchange factor. Fig. 4A illustrates the action of increasing concentrations of GST-PI3K RBD on the k'-1 of the Ha-Ras·[3H]GTP complex in the presence and absence of C-Sdc25p. At a concentration of 20 nM C-Sdc25p, the k'-1 of Ha-Ras·[3H]GTP decreased from 3·10-2 min-1 to 1.3·10-2 min-1 in a dose-dependent manner. Substitution of GTP with GTPgamma S (Fig. 4B) gave the same results, showing a complete inhibition of the C-Sdc25p-dependent exchange on Ha-Ras at 3.6 µM GST-PI3K RBD. We could estimate that the IC50 of GST-PI3K RBD on C-Sdc25p activity was about 500 nM. In contrast to Raf-1 RBDs, the GST-PI3K RBD does not seem to influence the intrinsic exchange activity of Ha-Ras (Fig. 4, A and B).


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  		Fig. 4.   Effect of PI3K RBD concentration on the GEF-dependent GTP/GTP exchange on Ha-Ras p21. In A, the dissociation rate constants of p21·[3H]GTP complexes (30 nM) in the presence of C-Sdc25p (20 nM) and the given concentrations of PI3K RBD were calculated from 35-min kinetic experiments. In the absence of C-Sdc25p, the dissociation rate constants were calculated from 60-min kinetic experiments. In B, the dissociation rate constants of p21·[gamma -35S]GTPgamma S complexes (30 nM) in the presence of C-Sdc25p (20 nM) and the given concentrations of PI3K RBD (black-triangle) were calculated from 35-min kinetic experiments. In the absence of C-Sdc25p (), the dissociation rate constants were calculated from 60-min kinetic experiments. The inset illustrates the different dissociation rates of the p21·[gamma -35S]GTPgamma S complexes using 20 nM C-Sdc25p in the presence (bullet ) and absence (triangle ) of 3.6 µM PI3K RBD.

Competitive Binding of GEF and Raf RBD to the Active Form of Ha-Ras as Monitored by Scintillation Proximity Assay-- The inhibition of GEF activity was very likely the result of a reversible competition between Raf proteins and the exchange factor for binding to the active form of Ha-Ras, because increasing the C-Sdc25p concentration reversed the inhibitory effect of Raf (data not shown). To determine whether the interaction of Raf RBD with the active form of Ras directly affected the binding of GEF to Ras, we performed experiments in which Ras was displaced from the Ras·Raf complex, and the amount of the remaining complex was determined by SPA. This technology can be used to detect the binding or inhibition of binding without requiring a separation step, i.e. under equilibrium conditions of binding. A SPA procedure using protein A fluoromicrospheres coated with anti-GST was used to measure the binding of GST-Raf (51-131) to Ras complexed with [3H]GTP (21, 22). This procedure has allowed us to monitor the effect of GEF on the interaction between GST-Raf and Ha-Ras·[3H]GTP. As shown in Fig. 5A, increasing concentrations of C-Sdc25p result in a decrease in the signal generated by the GST-Raf binding to Ha-Ras·[3H]GTP. These results are consistent with the hypothesis that there is a competition between C-Sdc25p and GST-Raf (51-131) for binding to Ha-Ras·[3H]GTP. The concentration of C-Sdc25p inhibiting 50% of Raf/Ras binding was about 1.2 µM.


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  		Fig. 5.   Raf binding to Ha-Ras·[3H]GTP is inhibited by C-Sdc25p and CDC25Mm. The indicated concentrations of C-Sdc25p (A), CDC25Mm (B), or C-CDC25Mm-285 (C) were added to SPAs with 30 nM Ras·[3H]GTP and 50 nM GST-Raf (51-131). After subtraction of the blanks (without GST-Raf), the SPA signal was expressed as the percentage of inhibition relative to the control value obtained in the absence of GEF proteins.

A useful control to check the validity of our results was the omission of GST-Raf (51-131) from the assay. In this condition, we have not observed any increase in counts. Because recent studies (30) indicate that CDC25Mm selectively activates Ha-Ras in vivo, we have monitored the interactions between Raf and Ras in a homologous mammalian system by assessing the ability of full-length CDC25Mm to compete with GST-Raf (51-131) for binding to Ha-Ras·[3H]GTP. Also, in this system, increasing concentrations of full-length CDC25Mm reduced the signal given by the Raf binding to Ras (Fig. 5B). However, because the isolation of purified CDC25Mm in concentrations higher than the micromolar range is still problematic due to aggregation phenomena leading to the inactivation of the protein (23), we could only use a final concentration of mammalian GEF (430 nM) that induced a 20% inhibition of Ras/Raf binding in the assay. This inhibition corresponds to that obtained with the same concentration of C-Sdc25p. By using higher concentrations (up to micromolar concentrations) of its catalytic domain C-CDC25Mm-285 (10), we could inhibit more than 50% of the Ras/Raf binding (Fig. 5C).

This is the first demonstration that an effector can block the physical interaction between the active form of Ras and GEF, showing that these two proteins cannot be bound simultaneously to the active form of Ras.

Presence of Raf Enhances the Regeneration of the Ha-Ras Active Form By GEF-- To evaluate the implication of the inhibitory effect of Raf RBDs on GEF in the regeneration of the active form of Ha-Ras and, consequently, to examine the possibility of correlating these observations in vitro with the physiological conditions in the cell, we tested the influence of Raf RBD in a system in which all the molecules of p21 were initially in the inactive GDP-bound form, and the molar ratio of the free nucleotides GDP and GTP or GTPgamma S corresponded to the value estimated to occur in the cell. Fig. 6A illustrates the action of full-length CDC25Mm on Ha-Ras p21·[3H]GDP in the presence and absence of 600 nM GST-Raf (51-131) and at a [3H]GDP and GTPgamma S molar ratio of 1:10. At a concentration of 200 nM CDC25Mm, the presence of GST-Raf (51-131) enhances the CDC25Mm-dependent stimulation of the GDP/GTPgamma S exchange on Ha-Ras, as determined by following the dissociation of [3H]GDP from the Ha-Ras p21·[3H]GDP complex. Because the enhancement induced under these experimental conditions by GST-Raf (51-131) on the GEF-dependent stimulation on Ha-Ras, although significant, was not so drastic, we have verified this phenomenon using a complementary system in which Ha-Ras p21 was prebound with unlabeled GDP, and the free GTPgamma S was substituted with [3H]GTP or [gamma -35S]GTPgamma S. In this system, we monitored the exchange reaction as association of labeled free [3H]GTP (Fig. 6B) or [gamma -35S]GTPgamma S (Fig. 6C) stimulated by CDC25Mm in the presence and absence of GST-Raf (51-131). In both cases, the regeneration of the active form of Ras by GEF proteins was increased by the presence of Raf, confirming the results presented in Fig. 6A. These data show that the Raf inhibition of the GEF activity by sequestration of the active form of Ha-Ras favors the interaction between the exchange factor and Ha-Ras·GDP, its physiological substrate.


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  		Fig. 6.   Effect of Raf-1 RBDs on the CDC25Mm-induced Ha-Ras GDP/GTP exchange in the presence of an exogenous GDP:GTP molar ratio of 1:10. The reactions were carried out in the presence of 200 nM full-length CDC25Mm, 600 nM GST-Raf (51-131), and 500 nM p21·[3H]GDP (A) or 500 nM p21·GDP (B and C). The concentrations of the exogenous [3H]GDP and GTPgamma S (A), GDP and [3H]GTP (B), or GDP and [gamma -35S]GTPgamma S (C) were set at 500 nM and 5 µM, respectively. At the indicated times, the amount of radioactive guanine nucleotide bound to p21 was determined. In A, the amount of p21·[3H]GDP at time 0 was taken as 100% concentration. B and C show the formation of p21·[3H]GTP and p21·[gamma -35S]GTPgamma S complexes, respectively, expressed as the percentage of the total p21·GDP concentration. The standard error of the single points in A and B was calculated from four different experiments.


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the GDP/GTP cycle of Ras proteins, the reversibility of all partial steps of the nucleotide·p21 interaction, except for the hydrolysis of GTP, could allow the occurrence of futile reactions, such as GTP/GTP exchange. Even if rasGEFs show a slightly higher binding affinity for the GDP-bound state of Ha-Ras, this differential property is comparatively small and is not sufficient by itself to support a marked preference for the physiologically productive GDP/GTP exchange on Ras. Therefore, it is likely that additional mechanisms exist favoring the interaction between the exchange factor and Ha-Ras·GDP, its biological substrate.

In our previous work (17), we have shown that stimulation by GEF of the Ha-Ras GTPgamma S/GTPgamma S exchange was specifically inhibited by p120-GAP in vitro. This inhibition by GAP of the GEF activity via sequestration of the active form of Ha-Ras was found to favor the GEF-induced Ha-Ras GDP/GTPgamma S exchange, revealing an additional function of GAP that could contribute to regulate the Ha-Ras activity in the cell. Consistent with this is the observation that under specific conditions in vivo, the GTPase- activating effect of p120-GAP may be inhibited (12, 31-34), suggesting that the protective association of p120-GAP with the active form of p21 is correlated with its proposed function as transmitter of downstream signals of Ha-Ras (35, 36).

Because the active form of Ha-Ras has been found to interact with an increasing number of ligands (1-5), other effectors may be involved in a mechanism of protection of Ha-Ras bound to GTP that is similar to that described for p120-GAP. This would constitute a simple and efficient system to coordinate the multiple interactions of Ha-Ras and would help favor the physiological course of the GEF-dependent GDP/GTP exchange reaction in the cell. However, one should take into account that the competitive phenomena at the level of Ras are very likely further modulated in the cell. Raf-1 activity appears to be regulated by phosphorylation/dephosphorylation events, ligand proteins, and lipids (37). It has been reported that Raf can be phosphorylated by protein kinase A, and that phosphorylation of Raf-1 reduces its affinity for Ras p21 (38). As a consequence, Raf-1 phosphorylation was suggested to be one of the mechanisms by which Ha-Ras distinguishes between its ligands. In turn, phosphorylation, calmodulin, and calpain have been found to be involved in controlling the activity of neuronal rasGEF CDC25Mm, although the nature of these effects and how they are coordinated remain in large part to be clarified (23, 39, 40).

To shed more light on the possible molecular mechanisms accounting for a transient activation of Ras as a result of coupling or uncoupling with its regulators and effectors, we have studied the influence of c-Raf-1 on the GEF-induced nucleotide exchange of Ha-Ras in this work, using purified components. As a main result, the Ras binding domains of Raf have been found to be able to inhibit not only the intrinsic GDP/GTP exchange on Ras, as reported by Herrmann et al. using another method (15), but also the GEF-stimulated GTPgamma S/GTPgamma S exchange. The specific nature of this effect is supported by its strict dependence on the active state of Ha-Ras, the target of Raf; no inhibition by Raf-1 RBDs of the GEF-dependent GDP/GDP exchange on p21 was detected. The observation that Raf RBDs behave as nucleotide dissociation inhibitors, trapping GTPgamma S or GTP in the Ras·Raf complex (15), raised the question of whether this effect is the result of a competition between the exchange factor and Raf for binding to the active form of Ras or is only a consequence of the Raf-dependent inhibition of the intrinsic GDP/GTP exchange on p21. To analyze this aspect, Ha-Ras·GTP was displaced by GEF proteins from the Ras·Raf complex, and the amount of residual complex was assessed by using the scintillation proximity assay. With this system, we could demonstrate that the inhibition of the GEF activity was due to a reversible competition between Raf and GEF for binding to the active form of Ha-Ras. Moreover, it has been shown for the first time that the binding of Raf-1 to the active form of Ras blocks the interaction of the latter with its nucleotide exchange factor, showing that the binding of Raf and GEF to Ha-Ras is mutually exclusive.

Extensive analysis by site-directed mutagenesis has pointed to the switch 1 region of Ras as a crucial area for interaction with the various effectors (3). Moreover, it has been shown that residues outside this region, such as those of the switch 2 region, are responsible for molecular recognition and the specificity of the interaction (1, 41-43). With regard to the interaction between Ras and GEF, a direct role has been assigned to helix alpha 2, which is part of the switch 2 region (11, 44-50), and helix alpha 3 (17). During the submission of our manuscript, the three-dimensional model of Ha-Ras in complex with the GEF region of the son of sevenless protein has been reported (51). This model emphasizes a direct interaction of not only the switch 2 region but also of the switch 1 region with GEF, a contact that could not be deduced from site-directed mutagenesis (28). Therefore, the present knowledge of the interaction sites of Ras with Raf and GEF indicates that the switch 1 and 2 regions represent structural elements that are directly involved in the binding of both GEF and effectors. The interferences between these ligands reported in our work thus depend on the overlap of their binding sites on p21, inducing exclusion phenomena.

Recently, it has been demonstrated in intact cells and also in experiments in vitro that protein kinase A regulates the selectivity of Ha-Ras p21 binding to either RalGDS or Raf-1, and that the binding of RalGDS to the GTP-bound active form of Ras p21 inhibits the interaction of Ras p21 with Raf-1 and GAP (16, 38). The human protein Rin has also been identified as a protein with a pronounced affinity for the GTP-bound state of Ras in vivo, competing with Raf-1 for binding to Ha-Ras in vitro (52). This further supports the results of this work indicating the existence of coordinated mechanisms controlling the interactions of Ras with its multiple ligands. These mechanisms are based on mutual competitions for binding to p21 that are very likely regulated in the cell by the level of the active state of the various ligands of Ras.

The complexity of the interactions in the network of Ras can be further illustrated by the relationship between p21 and PI3K (14, 18, 19, 29). In the intact cell, evidence has been obtained that Ras can stimulate PI3K activity; the ability of Ras to contribute to PI3K activation seems to represent an important portion of its downstream signaling (14, 29). Nonetheless, an elevated level of GTP-bound Ras was found in response to constitutively active PI3K expressed in Xenopus oocytes (19), suggesting that PI3K would not only act downstream of p21 but also act upstream to Ha-Ras on the activation mechanism leading to the GDP/GTP exchange. In this context, to shed more light on the issue of the upstream versus downstream role of PI3K, we have attempted to correlate this aspect with our results on Raf by studying the influence of PI3K RBD on the GEF-stimulated exchange on the active form of Ha-Ras. Similar to Raf, we have found that the presence of PI3K RBD reduced the GTP/GTP exchange on Ras stimulated by GEF; in contrast with Raf and other effector molecules of Ras such as RalGDS, no apparent inhibition by PI3K RBD on the intrinsic exchange on Ras was detected.

Taken together, our results show that not only Raf-1 but also PI3K contributes to prevent GEF-dependent exchange on the active form of Ha-Ras p21. The mutual exclusion between GEF and Raf or PI3K, as the one previously reported for p120-GAP and GEF (17), could represent a regulatory process under more physiological conditions, as suggested by the observation that by mimicking the cellular GDP:GTP molar ratio, the presence of Raf enhances the GEF-stimulated GDP/GTP or GTPgamma S exchange on Ha-Ras. This "feedback influence" of interactions of p21 and effectors on the level of the active state emphasizes the dual role of several protein ligands of Ras, such as Raf and PI3K in addition to p120-GAP, and reveals the existence of coordination mechanisms controlling the level of p21·GTP.

To derive a model consistent with our results, one should take into account the following aspects. In the absence of external stimulation, Ras proteins are normally found in the inactive GDP bound state. When an external signal is transmitted by tyrosine kinase- or G protein-coupled receptors, rapid activation of Ras·GDP is achieved by the binding of GEF proteins and subsequent GDP release. After the dissociation of GDP, the nucleotide-free Ras protein, despite its comparable affinity for GDP and GTP, is likely to bind GTP, with the excess of GTP over GDP (10:1) in the cell assuring a preferential GTP binding, at least at the beginning. This leads to the formation of a mixed pool of Ras proteins composed by Ras·GTP and Ras·GDP complexes. Because the role of GEF is to activate as many molecules of Ras as possible, the interaction with already activated Ras proteins would be physiologically useless, delaying a productive GDP/GTP cycle. Futile interactions of Ras could be prevented, or at least greatly reduced, by binding to effectors that sequestrate the active form of Ras. This would favor the interaction of GEF with its biologically relevant substrate, the Ras·GDP complex. The model suggests a general mechanism modulating the activity of RAS through a competitive binding between effectors and GEF regulators that may determine the physiological course of the GDP/GTP cycle of Ras and thus the activity of the Ras pathway in the cell.

    ACKNOWLEDGEMENTS

We are indebted to Drs. M. S. Marshall, P. N. Lowe, and J. Downward for providing the vectors encoding c-Raf-1 (1-149), c-Raf-1 (51-131), and PI3K RBD, respectively. We are grateful to Dr. P. N. Lowe for sending SPA beads and advice on this method. We thank our laboratory colleagues Drs. J. B. Créchet, E. Jacquet, and I. Krab for discussion and advice and Drs. S. Baouz and E. Jacquet for providing purified CDC25Mm and C-CDC25Mm-285.

    FOOTNOTES

* This work was supported by the European Community contract BIOTECH BIO4-CT96-1110, the Ligue Nationale Française Contre le Cancer, the Association pour la Recherche sur le Cancer (Grant 6377), and the Fédération Nationale des Centres de Lutte Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed:Tel.: 33-1-6933-4180; Fax: 33-1-6933-4840; E-mail: andrea{at}poly.polytechnique.fr.

The abbreviations used are: GAP, GTPase-activating protein; p120-GAP, human rasGAP; GEF, guanine nucleotide exchange factor; C-Sdc25p, catalytic domain of Saccharomyces cerevisiae of 550 amino acid residues; CDC25Mm, mouse rasGEF (p140-GRF); C-CDC25Mm-285, catalytic domain of CDC25Mm comprising 285 amino acid residues; RBD, Ras-binding domain; PI3K, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; GTPgamma S, guanosine 5'-O-(thio-triphosphate); SPA, scintillation proximity assay; ME, 2-mercaptoethanol.
    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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