©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Inhibition by Arachidonic Acid of the Catalytic Activity of Ras GTPase-activating Proteins (*)

(Received for publication, June 6, 1995; and in revised form, August 29, 1995)

Beth A. Sermon John F. Eccleston Richard H. Skinner (1) Peter N. Lowe (1)(§)

From the Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA and the Biology Division, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ras is a guanine nucleotide-binding protein that acts as a molecular switch controlling cell growth. The Ras GTPase-activating proteins (GAPs) p120-GAP and neurofibromin are candidates as Ras effectors. The GTPase-activating activity of both proteins is inhibited by mitogenic lipids, such as arachidonic acid and phosphatidic acid, and differential inhibition of the two GAPs led to the hypothesis that both were effectors in a Ras-controlled mitogenic pathway (Bollag, G., and McCormick, F.(1991) Nature 351, 576-579). We have studied the mechanism of inhibition by arachidonic acid in three ways: first, by measurements of catalytic activity under multiple turnover conditions; second, using p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid as a fluorescent probe for ligands binding to GAPs; and third, by using a scintillation proximity assay to measure direct binding of Ras to neurofibromin. We found no significant differential inhibition between p120-GAP and neurofibromin by arachidonic acid. The inhibition by arachidonic acid included a major component that is competitive with RasbulletGTP. These data suggest that insomuch as the mitogenic effects of lipids are mediated via inhibition of GAPs, GAPs are not Ras effector proteins. Additionally, lipids can exert a non-competitive type effect, consistent with a protein denaturing activity, making difficult extrapolations from in vitro data to the situation within cells, and possibly explaining the variability of literature data on inhibition by lipids.


INTRODUCTION

The ras genes encode guanine nucleotide-binding proteins that act as molecular switches for signal transduction pathways controlling cell growth and differentiation(1, 2, 3, 4, 5) . In the GTP-bound form, Ras is active and interacts with effector proteins to propagate a signal from the outside of the cell to the nucleus or cytoskeleton(6, 7) . A region on Ras has been mapped out through mutagenesis and structural studies as the effector binding region(7) . Ras has a low intrinsic GTPase activity, which is accelerated by the GTPase-activating proteins (GAPs) (^1)p120-GAP and NF1(6, 8) . GAPs can thus act as negative regulators by converting Ras to the inactive GDP form. Activation of Ras to the GTP form occurs by nucleotide exchange, catalyzed by exchange factors(6) .

Several candidate effector proteins have been proposed. Thus, very convincing evidence has emerged for the role of the serine-threonine kinase, c-Raf, as an effector controlling the activation pathway for MAP kinase(9, 10, 11, 12, 13) . Phosphatidylinositol 3-OH-kinase interacts with the effector binding region of Ras and might well be an effector for a Ras signaling pathway controlling phosphoinositide metabolism(14) . Ral-GDP dissociation stimulator also binds to the effector binding region of Ras, but is not known to have a biological activity associated with an effector function(15) . Both p120-GAP and NF1 have many properties expected from a Ras effector, in that they bind preferentially to RasbulletGTP rather than RasbulletGDP and interact with Ras at the effector binding region(6, 7, 8) . Evidence both for and against such a role has been presented. There is much experimental data to support a role of p120-GAP in signaling, other than just to down-regulate Ras, whereas with NF1 most data are consistent purely with a negative regulatory role(8) . However, an effector role for both p120-GAP and NF1 was suggested by Bollag and McCormick (16) based on their data on the differential inhibition of p120-GAP and NF1 activity by lipids.

An early response to mitogenic stimulation is a rapid alteration in levels of various lipids such as diacylglycerol, phosphatidic acid, arachidonic acid, and metabolites of phosphatidylinositol(17, 18, 19, 20, 21) . Phosphatidic acid itself acts as a mitogen in specific cells(22) . Microinjection of a neutralizing anti-Ras antibody blocked the mitogenic activity of phosphatidic acid showing that its activity is completely Ras-dependent(22) . Among other lipids, phosphatidic acid and arachidonic acid inhibit p120-GAP and NF1 in vitro(16, 19, 23, 24, 25) . This suggested the possibility that the mitogenic effects of these lipids might be mediated by inhibition of p120-GAP, leading to an increase in RasbulletGTP, and hence an activation of Ras. Bollag and McCormick (16) reported that phosphatidic acid inhibited NF1 catalytic activity but did not block binding of Ras to NF1. This led to an hypothesis in which NF1 was a Ras effector, modulated by lipid inhibition(16) .

Large differences in potency and specificity of inhibition of GAPs by lipids have been observed by the various researchers in this field. For example, Bollag and McCormick (16) reported that arachidonic acid inhibits p120-GAP with an I of 200 µM, and the catalytic domain of NF1 (NF1-GRD) with an I of 30 µM, whereas Golubic et al.(23) reported that the catalytic domains of NF1 and of p120-GAP were both inhibited by arachidonic acid with I values between 8 and 16 µM. With phosphatidic acid, Bollag and McCormick (16) found no inhibition of p120-GAP, but an I of 10 µM with NF1-GRD. In contrast, Golubic et al.(23) found little or no inhibition by phosphatidic acid of the catalytic domains of either NF1 or p120-GAP. This diversity appears to be a reflection of different experimental procedures, conditions, and components utilized. Thus, there are reports showing that the inhibitory potency differs dependent upon whether full-length or catalytic domains are expressed(23, 24, 26) . The assay conditions and the procedure by which the lipids are solubilized also appear to be important factors. Lipids have been introduced either as pure micelles or as mixed micelles with apparently differing results(19, 24) . Furthermore, some assays have been performed in the presence of detergents(16, 24) , whereas others have not(16, 23) . In this study, we decided to keep the experimental system as simple as possible by performing experiments with arachidonic acid in the absence of detergents.

We report here an investigation into the mechanism of arachidonic acid inhibition of both p120-GAP and NF1 to see if it was different from that reported for phosphatidic acid. We show that there is a strong competitive element in the mechanism of arachidonic acid inhibition, such that arachidonic acid can block binding of NF1 to Ras. This has important implications for any hypothesis in which arachidonic acid, through interaction with GAPs, acts as a modulator of Ras signaling.


EXPERIMENTAL PROCEDURES

Proteins

NF1-334 was purified as described by Eccleston et al.(27) and GST-NF1-334 and [Leu]Harvey-Ras (residues 1-166) as described by Skinner et al.(28) . Normal Harvey-Ras (residues 1-166) was expressed in Escherichia coli and purified by procedures similar to those in (28) . GAP-344 was purified as described by Skinner et al.(29) .

Arachidonic Acid

Arachidonic acid (Sigma) was dissolved in ethanol (Spectrosol grade, BDH) to form a stock solution at 40 mM. Appropriate dilutions were made in ethanol, and aliquots of these added to the experiments such that the final concentration of ethanol was not more than 2%, and in general was chosen to be 1%.

Nucleotide Complexes

RasbulletGTP or Rasbullet[^3H]GTP complexes were prepared by nucleotide exchange in the presence of a GTP regenerating system (28) . RasbulletmantGTP complexes were prepared as described by Eccleston et al.(27) .

CMC Determination

CMC was determined by two independent procedures, one based on light scattering and the other based on fluorescence changes of the probe DPH. In both cases the experiments were performed in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol. In the former procedure, fatty acids were dissolved in ethanol and titrated into buffer such that a maximum of 2% ethanol was present. Light scattering was monitored in a fluorimeter at 500 nm. The CMC was taken to be the concentration of lipid at which a sharp discontinuity occurred in the light scattering versus concentration graph. Alternatively, CMC was determined by titrating the lipid into a solution of DPH and was taken to be the concentration of lipid at which a discontinuity occurred in the graph of fluorescence intensity versus concentration of lipid added(24) .

Steady-state Fluorescence

Measurements were performed on a Perkin-Elmer LS-50B spectrofluorimeter thermostatted at 30 °C. Experiments were performed in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol.

Scintillation Proximity Assay

This was performed basically as described by Skinner et al.(28) , except that the buffer was 20 mM Tris/HCl, pH 7.5. The assay was performed by first adding 80 µl of a solution of 0.2 µM NF1-GST in 20 mM-Tris/HCl, pH 7.5, 2 mM dithiothreitol to each well, followed by 120 µl of a mixture of 0.07 µM Rasbullet[^3H]GTP, 0.031 mg/ml of anti-glutathione S-transferase antibody and 4.2 mg/ml of Protein A polyvinyltoluene scintillation proximity assay beads (Amersham) suspended in 20 mM Tris/HCl, pH 7.5, 2 mM dithiothreitol, 2 mM MgCl(2). Lipids were either added to 80 µl of the solution of NF1-GST, followed by addition of 120 µl of a mixture containing scintillation proximity assay beads, anti-GST, and radiolabeled Ras, or were added directly to the complete mixture of 200 µl of these two components.

HPLC Analysis

[^3H]GTP and [^3H]GDP were quantified by HPLC separation using ion-pair chromatography on a Lichrosorb RP18 (5-µm particle size; 250 times 4 mm) eluting isocratically at 1 mlbulletmin with 12% acetonitrile, 88% tetrabutylammonium hydroxide (2.25 mM) dissolved in 56 mM potassium phosphate buffer, pH 5.9, essentially as described by Pingoud et al.(30) . Radionucleotides were detected and quantified by in-line mixing with scintillant, using a Berthold radiochemical detector.

Catalytic Activity Measurements

RasbulletGTP Hydrolysis

The rates of GAP-344- or NF1-334-stimulated RasbulletGTPase were measured by incubating a stoichiometric normal Rasbullet[^3H]GTP complex with the appropriate GAP, in the presence and absence of lipid. Incubations were performed in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol at 25 °C for 10 min. The concentration of Ras was about 7 µM. Final concentrations of GAP-344 and NF1-334 were 0.040 µM and 0.014 µM, respectively. The concentrations of GAPs were chosen so that in the absence of inhibitor the maximum extent of RasbulletGTP hydrolysis was 80% and generally was less and so gave a reasonable estimate of the true initial rate. The extent of hydrolysis in the absence of GAP-344 or NF1-334 was negligible as compared to that in its presence. At the end of the incubation, samples were transferred to ice and immediately quenched by addition of an equal volume of HPLC running buffer. The samples were stored at -70 °C prior to HPLC analysis to determine the extent of conversion of Rasbullet[^3H]GTP to Rasbullet[^3H]GDP. Fatty acids were added from ethanolic stocks such that the final concentration of ethanol was less than 2%. Control experiments showed that 2% ethanol had no effect on the GTP hydrolysis reaction.

RasbulletmantGTP Hydrolysis

Hydrolysis was measured under multiple turnover conditions in which catalytic amounts of NF1-334 (leq10% the molar concentration of Ras) were mixed with RasbulletmantGTP. Experiments were performed either on the home-built stopped flow fluorimeter (27) or on a Hi-Tech SF-61 instrument with the excitation monochromator set at 365 nm and emission monitored through a Wratten 47B filter. In these experiments, stock solutions of RasbulletmantGTP were diluted in buffer to which arachidonic acid was also added when required. This Ras solution was mixed in the instrument with an equal volume of a solution of NF1-334 in buffer, again containing arachidonic acid if required. All concentrations subsequently quoted are after mixing.

The initial rate of fluorescence change was measured directly from the photomultiplier output versus time plot in units of voltsbullets. To compensate for changes made in the applied photomultiplier voltage to allow measurements over a wide range of concentration of RasbulletmantGTP, it was necessary to establish the relationship between volts and molar concentration of RasbulletmantGTP. This was done in parallel experiments in which the RasbulletmantGTP solution of known concentration was mixed in the stopped flow fluorimeter with a solution consisting of 500 µM GDP, 40 mM EDTA, and 400 mM ammonium sulfate. This caused complete displacement of bound mantGTP from the Ras within 20 s (cf.Fig. 2, trace c), and the amplitude of the resultant fluorescence decrease was measured. This allowed the relationship between total RasbulletmantGTP and photomultiplier output to be established. The conversion of RasbulletmantGTP to RasbulletmantGDP results in about a 10% decrease in fluorescence(31) . Although this was not precisely determined in our experiments, we used a figure of 10% to allow us to to calculate rates in units of molar concentration.


Figure 2: Effect of arachidonic acid on the rate of NF1-334 catalyzed RasbulletmantGTP hydrolysis, under multiple turnover conditions. Stopped-flow fluorescence experiments were performed in which 1 µM RasbulletmantGTP was mixed with 0.1 µM NF1-334 at 30 °C. Both proteins were in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol. Fluorescence recordings are shown for experiments in the absence of arachidonic acid (trace a) and with 20 µM arachidonic acid added to both the NF1-334 and RasbulletmantGTP solutions (trace b). To convert fluorescence changes into molar concentrations, in separate experiments 1 µM RasbulletmantGTP was mixed with500 µM GDP dissolved in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol, containing additionally 40 mM EDTA and 400 mM ammonium sulfate to promote rapid nucleotide exchange (trace c) (see ``Experimental Procedures''). The inset shows the initial phases of traces a and b on a larger scale.




RESULTS

Inhibition of GAP- and NF1-activated RasbulletGTPase by Arachidonic Acid

Arachidonic acid was added to standard GAP activity assays from an ethanolic stock. Under these conditions, arachidonic acid inhibited GAP-344 and NF1-334 activities with I values of 10 and 5 µM, respectively (Fig. 1). The inhibitory potency was much higher than that reported by Bollag and McCormick(16) . We noted one experimental difference, which was that these workers did not use pure lipid but included the detergent Nonidet P40 in all assays so that presumably mixed micelles were formed. We assayed the inhibitory potency of arachidonic acid on p120-GAP in the presence of 0.1% Nonidet P40, and found only 18% inhibition at 30 µM, whereas in the absence of Nonidet nearly complete inhibition was obtained at that concentration. This suggested that partitioning of arachidonic acid into detergent micelles reduced its inhibitory potency and so all subsequent experiments were performed with lipids in the absence of detergent.


Figure 1: Inhibition of the GAP-344- and NF1-334-stimulated Ras GTPase by arachidonic acid. A solution containing 7 µM Rasbullet[^3H]GTP was incubated with either 0.040 µM GAP-344 (panel A) or 0.014 µM NF1-334 (panel B) in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl(2), 0.1 mM dithiothreitol at 25 °C. Arachidonic acid (bullet) was added to the mixture from a stock solution in ethanol. After 10 min, the extent of [^3H]GTP hydrolysis was measured by HPLC separation of [^3H]GTP from [^3H]GDP. Inhibition of GTPase activity was calculated as a percentage relative to incubations without arachidonic acid.



With GAP-344, the relationship between inhibitor concentration and inhibition achieved was not strictly hyperbolic, but rather was sigmoid, but with NF1-334 the data were more variable. More pronounced sigmoid curves had been observed with GAP by Serth et al.(24) . In those experiments the steep phase occurred at 40-50 µM, which was the same as their experimentally determined CMC value. We also found that CMC, as determined either by light scattering of arachidonic acid itself or by the use of DPH fluorescence enhancement, was around 40 µM. However, this concentration was clearly much higher than the I values seen under our experimental conditions, showing that micelle formation is not required for inhibition.

Steady-state Kinetic Analysis of the Inhibition of NF1-activated RasbulletmantGTPase by Arachidonic Acid

The mechanism of inhibition was investigated further by analyzing the kinetics under multiple turnover conditions. A continuous record of the hydrolysis process was obtained by replacing GTP by its close analogue mantGTP, as RasbulletmantGTP hydrolysis is accompanied by a 10% decrease in fluorescence(31) . Experiments were performed under multiple turnover (steady-state) conditions, i.e. with the concentration of NF1-334 well below the concentrations of RasbulletmantGTP (Fig. 2).

Using 1 µM RasbulletmantGTP and 0.1 µM NF1-334, the initial rate of RasbulletmantGTP hydrolysis was calculated from Fig. 2a to be 1.1 µMbullets. This corresponds to a turnover rate of 11 s, similar to that seen under single turnover conditions ( (27) and data not shown) and consistent with the RasbulletmantGTP concentration being well above K(m) and the NF1-334 being fully active. Inclusion of 20 µM arachidonic acid reduced the initial rate of RasbulletmantGTP hydrolysis by 45% (Fig. 2, trace b). The shape of the reaction progress curves in the absence of arachidonic acid (trace a) was typical for the substrate concentration being higher than K(m) (linear phase followed by a sharp decrease in rate), whereas with arachidonic acid the progress curve (trace b) fitted a single exponential, consistent with arachidonic acid increasing the value of the K(m).

The inhibitory potency of arachidonic acid was influenced by ionic strength. With increasing concentrations of NaCl up to 300 mM NaCl, an increase in inhibition by arachidonic acid was seen, but further increases in NaCl concentration resulted in a decrease in the level of inhibition (data not shown). Thus, 20 µM arachidonic acid inhibited by 45% with no added NaCl, 80% with 300 mM added NaCl and 41% with 1000 mM NaCl. At 150 mM NaCl, arachidonic acid inhibited NF1-stimulated RasbulletmantGTPase with I 20 µM. The biphasic effect on inhibition with increasing ionic strength is likely to be caused by an increase in the K(m) for RasbulletmantGTP(27) , resulting in more potent inhibition countered by a reduction in the affinity for arachidonic acid.

To measure values of K(m) and k, initial rates of hydrolysis were measured varying both the concentrations of RasbulletmantGTP and of arachidonic acid. The experiment was performed in the presence of 50 mM NaCl such that the K(m) was around 1 µM. The data obtained are shown in Fig. 3. The pattern of inhibition displayed in the Lineweaver-Burk double-reciprocal plot (Fig. 3B) is typical of mixed inhibition, in which the inhibitor both decreases k and increases K(m). If the data are interpreted as fitting a mixed inhibition model(32) , then it can be calculated from the intersection point of the lines that K(m) (or K(S) if the components are in rapid equilibrium) was around 1 µM and alphaK(m) is 5, where alpha represents the ratio of K to K ().


Figure 3: Steady-state kinetic analysis of the inhibition of NF1-334-catalyzed RasbulletmantGTPase by arachidonic acid. Initial rates (k) of NF1-334 catalyzed RasbulletmantGTP hydrolysis were measured in experiments similar to those shown in Fig. 3. Experiments were performed with 0 µM (bullet), 12 µM (circle), 18 µM (), or 24 µM () arachidonic acid. The data at each concentration of arachidonic acid were fitted by non-linear regression to the Michaelis-Menten equation to obtain values of K and k. In A and B, the solid lines are based on these constants. K at 0, 12, 18, and 24 µM arachidonic acid were 1.1, 1.3, 1.8, and 3.8 µM, respectively. k at 0, 12, 18, and 24 µM arachidonic acid were 2.6, 2.3, 1.7, and 1.1 µMbullets, respectively. In C, the ratio of K to k obtained from this curve-fitting has been plotted against the concentration of arachidonic acid used.



This value of alpha suggests that the mixed inhibition is predominantly of a competitive character, but the uncompetitive component is still significant. However, the data clearly do not fit this simple model in that the relationship between inhibition and the concentration of arachidonic acid at any fixed concentration of Ras is distinctly sigmoid and plots of either K(m)/k (Fig. 3C) or 1/k (not shown) against arachidonic acid concentration are non-linear, parabolic upward.

Characterization of the Interaction of Arachidonic Acid with NF1 and GAP Using the Fluorescent Probe DPH-carboxylic Acid

As the kinetic mechanism of inhibition of GAP activity by arachidonic acid is complex, we examined other methods to show whether arachidonic acid binds at the Ras-binding site, or at some other site, on GAPs. We postulated that p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid (DPH-carboxylic acid) might be a sufficiently close analogue of arachidonic acid that it would act as a fluorescent probe of the lipid-binding site. An increase in fluorescence of DPH-carboxylic acid (1 µM) occurred when it was mixed with NF1-334, GAP-344 or bovine serum albumin (Fig. 4). At 10 µM protein, the increase was 18-, 1.6-, and 32-fold, respectively. Arachidonic acid (10 µM) abolished the enhancement of fluorescence of DPH-carboxylic acid caused by mixture with albumin, as expected if arachidonic acid competed with DPH-carboxylic acid for binding to albumin (Fig. 5). However, at concentrations of arachidonic acid up to 40 µM, there was no significant reduction in the fluorescence of DPH-carboxylic acid mixed with either NF1 (Fig. 5) or GAP. Indeed with GAP, an increase was seen. This suggested that with the latter two proteins the probe was binding at a site distinct from the arachidonic acid-binding site. However, we were still able to use DPH-carboxylic acid as a probe for ligands interacting with GAPs since the fluorescence of the protein-bound probe was sensitive to addition of ligands.


Figure 4: Effect of GAP-344, NF1-334 or bovine serum albumin on the fluorescence of DPH-carboxylic acid. GAP-344 (), NF1-334 (circle), or bovine serum albumin (bullet) was titrated into a solution of 1 µM DPH-carboxylic acid dissolved in 20 mM Tris/HCl, pH 7.5, such that the concentration of DPH-carboxylic acid was not reduced significantly. Fluorescence was recorded with excitation at 357 nm and with emission at 435 nm. Data have been normalized on the initial level of fluorescence so that the fluorescence increases on addition of proteins are directly comparable. The solid lines show the best fit to binding isotherms for simple association of two molecules, with K = 2.6, 0.55, and 62 µM, with NF1-334, albumin, and GAP-344, respectively. Data at concentrations of GAP-344 up to 120 µM were obtained but are not shown on this scale for clarity.




Figure 5: Effect of arachidonic acid on the fluorescence of a mixture of DPH-carboxylic acid with NF1-334 or serum albumin. Arachidonic acid, from a stock solution in ethanol, was added to a solution of 1 µM DPH-carboxylic acid in 20 mM Tris/HCl, pH 7.5, containing either 0.5 µM bovine serum albumin (bullet) or 2 µM NF1-334 (circle). The final concentration of ethanol was <1%. Fluorescence was recorded with excitation at 357 nm and emission at 435 nm. Data were corrected for light scattering caused by proteins and arachidonic acid, and for the fluorescence of 1 µM DPH-carboxylic acid in the absence of added protein. The maximum correction was 3.7% and 11.6% of the maximum fluorescence with albumin and NF1-334, respectively. The corrected fluorescence in the absence of arachidonic acid was taken to be 100. With albumin this represented 2.9-fold more fluorescence than with NF1-334.



The fluorescence of a solution containing 1 µM DPH-carboxylic acid and 2 µM NF1-334 was dramatically reduced by addition of [Leu]Harvey-RasbulletGTP protein (Fig. 6). The reduction in fluorescence was nearly proportional to the concentration of added Ras, with half-maximal effect occurring at about 1 µM RasbulletGTP. This is consistent with a relatively high affinity of interaction of Ras with NF1-334, as expected for the binding of NF1-334 to the [Leu]Ras mutant. Since several species were present in this experiment, it was necessary to determine whether RasbulletGTP was binding to NF1-334 or to the probe. Therefore, RasbulletGTP was titrated into a mixture of 2 µM DPH-carboxylic acid and 1 µM NF1-334. In this experiment, the curve was shifted to the left with half-maximal reduction occurring at 0.5 µM RasbulletGTP. These data are consistent with Ras binding to NF1-334 rather than to DPH-carboxylic acid. As a further control, normal RasbulletGDP was titrated into the mixture of DPH-carboxylic acid and NF1-334, but only 20% reduction of fluorescence occurred at 4 µM Ras, consistent with the known weaker affinity of NF1 for RasbulletGDP as compared with RasbulletGTP.


Figure 6: Effects of RasbulletGTP and of arachidonic acid on the fluorescence of a mixture of DPH-carboxylic acid and NF1-334. [Leu]RasbulletGTP was added to a solution of 1 µM DPH-carboxylic acid in 20 mM Tris/HCl, pH 7.5, and 2 µM NF1-334 containing 0 µM (bullet), 5 µM (circle), 10 µM (), 15 µM (), or 20 µM (box) arachidonic acid. Fluorescence was recorded with excitation at 357 nm and emission at 435 nm. Data were corrected for light scattering caused by proteins and arachidonic acid, and for the fluorescence of 1 µM DPH-carboxylic acid in the absence of added protein. The maximum correction was 12.8% of the maximum fluorescence. The corrected fluorescence in the absence of RasbulletGTP was taken to be 100. In B, the effect of arachidonic acid on the observed fluorescence in the presence of 8 µM RasbulletGTP is shown.



These data suggested that the probe was able to monitor the interaction between Ras and NF1. Therefore, the effect of arachidonic acid on the fluorescence changes induced by Ras was examined (Fig. 6). The decrease in fluorescence caused by addition of Ras to the mixture of DPH-carboxylic acid and NF1-334 was largely abolished by 20 µM arachidonic acid, strongly suggesting competition between Ras and the lipid for binding to NF1. However, the dependence on concentration of arachidonic acid was not hyperbolic (Fig. 6B).

NF1/Ras Binding Scintillation Proximity Assay

The data from the kinetic characterization and use of the fluorescent probe suggested that arachidonic acid might, at least in part, be competing with Ras for binding to NF1 or GAP. We therefore tested whether arachidonic acid might block binding of Ras to NF1 by a more direct method. The recently described scintillation proximity assay procedure (28) was used to monitor the binding of RasbulletGTP to NF1-334 and to test the effects of arachidonic acid on this interaction. In this assay, GST-NF1-334 fusion protein bound via an anti-GST antibody to protein A coated fluoromicrosphere beads interacts with a Rasbullet[^3H]GTP complex. When the Ras is in close proximity to the beads, i.e. when bound to NF1-334, scintillation occurs, whereas Ras in free solution does not cause any light emission. A key feature of the system is that it allows direct measurements of binding at equilibrium.

Arachidonic acid completely abolished the signal produced in this assay, with 50% inhibition occurring at 5-10 µM arachidonic acid (Fig. 7). As a control that the reduction in signal truly represented disruption of binding between Ras and NF1, arachidonic acid was added to a mixture of scintillation proximity assay beads, anti-GST and a GST-Rasbullet[^3H]GTP complex (Fig. 7). In this case, at concentrations of arachidonic acid up to 50 µM, negligible inhibition was observed. This control demonstrated that arachidonic acid was not affecting binding of nucleotide to Ras, or binding between Protein A and antibody or between antibody and GST. In most experiments there was a non-hyperbolic dependence of inhibition on concentration of arachidonic acid, with a distinct lag at low inhibitor concentrations.


Figure 7: Inhibition of binding of GST-NF1-334 to RasbulletGTP by arachidonic acid as monitored by scintillation proximity assay. Arachidonic acid was added at the indicated concentrations to scintillation proximity assays containing protein A scintillation beads, anti-GST and either 0.04 µM [Leu]Rasbullet[^3H]GTP and 0.09 µM GST-NF1-334 (bullet) or 0.04 µM GST-[Leu]Rasbullet[^3H]GTP (circle).




DISCUSSION

There are conflicting literature reports on the potency of arachidonic acid and phosphatidic acid, on their ability to differentially inhibit NF1 and p120-GAP activation of RasbulletGTPase, and the mechanism by which this is achieved. We therefore investigated the effect of arachidonic acid on the interaction of Ras and GAPs using three different techniques in the simplest system possible.

Kinetic methods showed that in the absence of detergent, arachidonic acid inhibited the NF1- and p120-GAP activated activity of RasbulletGTPase to similar extents with I values of 5-15 µM (Fig. 1). These potencies are similar to those observed by Golubic et al.(23) . In the presence of Nonidet detergent, the inhibitory potency of arachidonic acid on GAP activity was markedly reduced to levels reported by Bollag and McCormick (16) when they also used this detergent. We therefore performed all subsequent experiments in the absence of detergent. Under these conditions, we found phosphatidic acid to be only a weak reversible inhibitor of either GAP-334 or NF1-334 catalytic activity (data not shown), as Golubic et al.(23) had concluded, and in contrast to the more potent inhibition seen by other groups. These data suggested to us that some of the reported differential inhibition effects of NF1 and p120-GAP by lipids might be artifactual.

The mechanism of the inhibition of the NF1-activated RasbulletGTPase was investigated in more detail by using the fluorescent analogue of GTP, mantGTP. The fluorescence decrease that occurs between RasbulletmantGTP and RasbulletmantGDP allowed the hydrolysis to be monitored continuously (Fig. 2). This has many advantages over the use of radiolabeled GTP, which requires analysis of many single time points. By varying the concentrations of both RasbulletmantGTP and arachidonic acid, the effect of arachidonic acid on K(m) and k was established (Fig. 3). The inhibition was of a mixed character but predominantly competitive.

The second method to study the effect of arachidonic acid was to use the fluorescent probe DPH-carboxylic acid in equilibrium measurements of the interaction of RasbulletGTP with NF1. Although DPH-carboxylic acid did not compete for the proposed arachidonic acid-binding site on NF1 (Fig. 5), as had been hoped, it still provided a probe on the interaction of NF1 with RasbulletGTP, since the fluorescence of bound DPH-carboxylic acid was reduced on the binding of RasbulletGTP (Fig. 6). Arachidonic acid largely abolished this effect (Fig. 6), strongly suggesting competition between the Ras and lipid for binding to NF1.

The third method to study the effect of arachidonic acid was to use a scintillation proximity assay (Fig. 7). Arachidonic acid completely abolished the interaction between [Leu]RasbulletGTP and NF1, with 50% inhibition occurring at 5-10 µM.

All of the data above support the argument that arachidonic acid competitively inhibits the interaction of RasbulletGTP with NF1. Furthermore, the inhibitory effects occur well below the CMC for arachidonic acid under the conditions used, showing that the effect is not caused by micelle formation, as had been suggested by Serth et al.(24) .

Despite this evidence for competitive inhibition, our results suggest that arachidonic acid can exert other effects on the interaction. For example, the inhibition of the GAP-activated RasbulletGTPase ( Fig. 1and 3) show a sigmoid or non-hyperbolic dose-response curve. In addition, the effect of arachidonic acid on the binding of RasbulletGTP to NF1 as monitored by DPH-carboxylic acid (Fig. 6) or by the scintillation proximity assay (Fig. 7) is not hyperbolic. We do not know the explanation for this phenomenon, but similar behavior has been seen previously with arachidonic acid(23, 24) .It is possible that self-association of arachidonic acid to structures smaller than full micelles is required for maximal inhibitory effects.

A further deviation from a simple competitive pattern was seen from the kinetic analysis (Fig. 3), which showed that arachidonic acid not only raised K(m) but also reduced k. The effect on k is consistent with an irreversible inhibitory component for which we have some additional evidence (data not shown). In the scintillation proximity assay, we noted that when certain lipids (arachidic acid and phosphatidic acid, but not noticeably arachidonic acid) were added to NF1 before addition of Ras they were more potent inhibitors than when added after Ras, suggesting that the inhibitory mechanism was not always rapidly reversible on the time-scale of these experiments. Also, arachidonic acid caused slow time-dependent effects on the fluorescence of DPH-COOH bound to GAP, consistent with denaturation, which were significantly prevented by the inclusion of Ras.

Although these non-competitive effects occur, the predominantly competitive nature of the interaction makes us conclude that arachidonic acid does not act in the way that phosphatidic acid was reported to do by Bollag and McCormick(16) . They reported two mains lines of evidence that phosphatidic acid does not block binding of Ras to NF1(16) . First, the inhibitor displayed non-competitive kinetics, in which the inhibitor reduced V(max) without affecting K(m), typical of depletion of both substrate-bound and free enzyme forms by inhibitor. However, such an inhibitory pattern is also seen with an irreversible or slowly reversible type of action. Second, phosphatidic acid did not block binding of RasbulletGTP to NF1 immobilized on beads(16) . No detailed quantitation (e.g. yield of bound proteins) or controls were given for this assay other than that SDS blocked binding. Experiments from our own laboratory (27, 28) showed that the rate of dissociation of NF1 from Ras is fast, such that no significant binding of Ras to NF1 should have been observed under the experimental conditions of Bollag and McCormick(16) . Thus, the binding observed might not have been true reversible binding of NF1 to Ras. We found that dodecylmaltoside blocks binding of Ras to NF1(28) , again supporting the hypothesis that a primary mode of inhibition of amphipathic molecules such as lipids and detergents might be by preventing Ras binding to GAPs.

On the basis that certain lipids differentially inhibited p120-GAP and NF1 and on the assumption that one could extrapolate from data with phosphatidic acid that lipids in general do not block binding of Ras to GAPs, Bollag and McCormick (16) suggested a hypothesis with these key features. (a) p120-GAP and NF1 are differentially regulated by lipids. (b) Lipids activate Ras signaling by inhibiting NF1 GTPase stimulating activity. (c) Lipid-inhibited NF1 binds to RasbulletGTP propagating a signal through NF1. (d) NF1 and p120-GAP are alternative effectors for Ras. (e) The mitogenic activity of certain lipids is through their modification of GAP activity in the cell. However, the data presented in this paper are not in accordance with this hypothesis, as (a) no significant differential inhibition of activity was observed, and (b) lipid-inhibited GAPs do not bind to Ras. We would suggest two alternative hypotheses consistent with our data. If GAPs are indeed Ras effectors for a mitogenic pathway, the mitogenic activity of arachidonic acid cannot be accounted for through inhibition of GAP catalytic activities, since arachidonic acid also blocks binding between Ras and these putative effectors. Alternatively, the mitogenic activity of arachidonic acid may be accounted for by inhibition of GAP catalytic activity, but in this case GAPs are unlikely to be effectors for a mitogenic pathway.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-181-639-6370; Fax: 44-181-639-6877.

(^1)
The abbreviations used are: GAP, GTPase-activating protein; CMC, critical micellar concentration; mantGTP, 2`(3`)-O-N-methylanthraniloyl-GTP; mantGDP, 2`(3`)-O-N-methylanthraniloyl-GDP; phosphatidic acid, beta-stearoyl--arachidonyl-L-alpha-phosphatidic acid; NF1, neurofibromin; NF1-334 and GAP-344, the catalytic domains of NF1 and p120-GAP, respectively; DPH-carboxylic acid, p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid; DPH, 1,6-diphenyl-1,3,5-hexatriene; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; Ras, protein product of the Harvey-ras gene (in all experiments, this refers to Ras truncated at residue 166, except where otherwise specified).


ACKNOWLEDGEMENTS

We acknowledge the help of Geoff Brownbridge and Martin Webb in obtaining initial data using fluorescence anisotropy suggesting that arachidonic acid blocks Ras/GAP binding. We thank James Rowedder and Al Brown for obtaining some preliminary data on inhibition of GAP activity.


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