(Received for publication, June 6, 1995; and in revised form, August 29, 1995)
From the
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 RasGTP. 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.
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) ()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 RasGTP rather than Ras
GDP 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 RasGTP, 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.
The initial
rate of fluorescence change was measured directly from the
photomultiplier output versus time plot in units of
voltss
. To compensate for changes made in the
applied photomultiplier voltage to allow measurements over a wide range
of concentration of Ras
mantGTP, it was necessary to establish the
relationship between volts and molar concentration of Ras
mantGTP.
This was done in parallel experiments in which the Ras
mantGTP
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 Ras
mantGTP and photomultiplier output
to be established. The conversion of Ras
mantGTP to
Ras
mantGDP 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
RasmantGTP hydrolysis, under multiple turnover conditions.
Stopped-flow fluorescence experiments were performed in which 1
µM Ras
mantGTP 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
, 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
Ras
mantGTP solutions (trace b). To convert fluorescence
changes into molar concentrations, in separate experiments 1 µM Ras
mantGTP was mixed with500 µM GDP dissolved
in 20 mM Tris/HCl, pH 7.5, 1 mM MgCl
, 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.
Figure 1:
Inhibition of the GAP-344- and
NF1-334-stimulated Ras GTPase by arachidonic acid. A solution
containing 7 µM Ras[
H]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
, 0.1 mM dithiothreitol at 25 °C. Arachidonic acid (
) was added
to the mixture from a stock solution in ethanol. After 10 min, the
extent of [
H]GTP hydrolysis was measured by HPLC
separation of [
H]GTP from
[
H]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.
Using 1 µM RasmantGTP and 0.1 µM NF1-334, the
initial rate of Ras
mantGTP hydrolysis was calculated from Fig. 2a to be 1.1
µM
s
. 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 Ras
mantGTP concentration being well above K
and the NF1-334 being fully active.
Inclusion of 20 µM arachidonic acid reduced the initial
rate of Ras
mantGTP 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
(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
.
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 RasmantGTPase with I
20
µM. The biphasic effect on inhibition with increasing
ionic strength is likely to be caused by an increase in the K
for Ras
mantGTP(27) , resulting in
more potent inhibition countered by a reduction in the affinity for
arachidonic acid.
To measure values of K and k
, initial rates of hydrolysis were measured
varying both the concentrations of Ras
mantGTP and of arachidonic
acid. The experiment was performed in the presence of 50 mM NaCl such that the K
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
. 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
(or K
if the components are
in rapid equilibrium) was around 1 µM and
K
is
5, where
represents the ratio
of K
to K
().
Figure 3:
Steady-state kinetic analysis of the
inhibition of NF1-334-catalyzed RasmantGTPase by
arachidonic acid. Initial rates (k
) of
NF1-334 catalyzed Ras
mantGTP hydrolysis were measured in
experiments similar to those shown in Fig. 3. Experiments were
performed with 0 µM (
), 12 µM (
),
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
µM
s
, 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 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
/k
(Fig. 3C) or 1/k
(not
shown) against arachidonic acid concentration are non-linear, parabolic
upward.
Figure 4:
Effect of GAP-344, NF1-334 or bovine
serum albumin on the fluorescence of DPH-carboxylic acid. GAP-344
(), NF1-334 (
), or bovine serum albumin (
) 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 () or 2 µM NF1-334
(
). 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-Ras
GTP 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 Ras
GTP. 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
Ras
GTP was binding to NF1-334 or to the probe. Therefore,
Ras
GTP 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 Ras
GTP. These data are
consistent with Ras binding to NF1-334 rather than to
DPH-carboxylic acid. As a further control, normal Ras
GDP 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 Ras
GDP as
compared with Ras
GTP.
Figure 6:
Effects of RasGTP and of arachidonic
acid on the fluorescence of a mixture of DPH-carboxylic acid and
NF1-334. [Leu
]Ras
GTP 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 (
), 5 µM (
), 10 µM (
), 15 µM (
), or 20 µM (
) 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 Ras
GTP
was taken to be 100. In B, the effect of arachidonic acid on
the observed fluorescence in the presence of 8 µM Ras
GTP 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).
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-Ras[
H]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 RasGTP 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
]Ras
[
H]GTP
and 0.09 µM GST-NF1-334 (
) or 0.04 µM GST-[Leu
]Ras
[
H]GTP
(
).
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 RasGTPase,
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 RasGTPase
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
RasGTPase was investigated in more detail by using the
fluorescent analogue of GTP, mantGTP. The fluorescence decrease that
occurs between Ras
mantGTP and Ras
mantGDP 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
Ras
mantGTP and arachidonic acid, the effect of arachidonic acid
on K
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 RasGTP 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
Ras
GTP, since the fluorescence of bound DPH-carboxylic acid was
reduced on the binding of Ras
GTP (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]Ras
GTP 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 RasGTP 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
RasGTPase ( Fig. 1and 3) show a sigmoid or non-hyperbolic
dose-response curve. In addition, the effect of arachidonic acid on the
binding of Ras
GTP 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 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 without affecting K
,
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 Ras
GTP 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 RasGTP 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.