(Received for publication, October 23, 1995; and in revised form, January 10, 1996)
From the
Raf is a serine/threonine kinase that binds through its
amino-terminal regulatory domain to the GTP form of Ras and thereby
activates the mitogen-activated protein kinase pathway. In this study,
we have characterized the interaction of the Ras-binding domain of Raf
with Ras using equilibrium binding methods (scintillation proximity
assay and fluorescence anisotropy), rather than with more widely used
nonequilibrium procedures (such as enzyme-linked immunosorbent assay
and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257
or 1-190 of Raf showed that although it was possible to detect
Ras binding using an enzyme-linked immunosorbent assay or affinity
precipitation, it was substoichiometric; under equilibrium conditions
with only a small excess of Raf almost no binding was detected. This
difference was probably due to the presence of a high percentage of
inactive Raf protein. Further studies used protein containing residues
51-131 of Raf, which expressed in Escherichia coli as a
stable glutathione S-transferase fusion. With this protein,
binding with Ras could readily be measured under equilibrium
conditions. The catalytic domain of neurofibromin inhibited binding of
Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin
showing that Raf and neurofibromin cannot be bound simultaneously to
Ras. The affinities of interaction of neurofibromin and Raf with
Harvey-Ras were similar. The rate constant for
dissociation of Raf from Ras was estimated to be >1
min
, suggesting that Ras, Raf, and neurofibromin may
be in rapid equilibrium in the cell. In contrast to previous reports,
under equilibrium conditions there was no evidence for a difference in
affinity between the minimal Ras binding domain of Raf (residues
51-131) and a region containing an additional 16
carboxyl-terminal amino acids, suggesting that residues 132-147
do not form a critical binding determinant.
The proto-oncogene ras encodes a membrane-associated
guanine nucleotide binding protein (M 21,000) that
is activated by mutation in approximately 30% of human tumors (for
reviews, see (1, 2, 3, 4) ). It acts
as a molecular switch in signaling from growth factor receptors at the
cell membrane to nuclear transcription factors via the cytoplasmic
mitogen-activated protein kinase cascade(5, 6) .
Activation of a growth factor receptor by ligand binding results in the
conversion of Ras from its inactive GDP-bound to active GTP-bound
state. This occurs through nucleotide exchange catalyzed by exchange
factors. Signaling is generally thought to be terminated by the binding
of the GTPase-activating proteins, p120GAP and neurofibromin
(NF1-334), which increase the intrinsically low rate of GTP
hydrolysis on Ras(7, 8) . GAPs, (
)like
other putative effectors, bind to the Switch 1 region of
Ras(8) .
The c-raf-1 proto-oncogene encodes a
serine/threonine kinase (Raf) that is a 648-amino acid protein with an
apparent M of 74,000(9) . Comparison of
the sequence of Raf with that of invertebrate homologues identified
three conserved regions, CR1, CR2, and CR3(10) . CR1 (residues
61-192) consists of the Ras-binding domain (11, 12, 13) followed by a cysteine-rich
region (residues 139-184) with a characteristic zinc finger
motif(14) . CR2 (residues 251-266) is rich in serine and
threonine residues, some of which are phosphorylated on
activation(15) , and CR3 (residues 333-625) is the
catalytic kinase domain.
Ras binds directly to the amino-terminal region of Raf in a GTP-dependent manner(11, 12, 13, 16) . Since Raf activates the mitogen-activated protein kinase cascade that is regulated by Ras (17, 18, 19) , and dominant-negative forms of Raf block Ras-dependent activation of mitogen-activated protein kinase(20) , it seems clear that Raf is an important effector of Ras signaling. A minimal region of Raf required for binding to Ras has been defined between residues 51 and 131(11, 21, 22, 23, 24) , but whether other regions of Raf are also involved in binding to Ras is still the subject of debate. In particular, it has been claimed that the addition of residues 132-147 (or 132-149) increases the affinity of Raf-(51-131) for Ras (23, 25, 26) and that the cysteine-rich region (residues 139-184) of Raf constitutes a second Ras-binding site(27) .
Much of the previously reported data comparing
the interactions of different Raf constructs with Ras have been
obtained using nonequilibrium binding procedures involving separation
steps(13, 23, 25, 26, 27, 28) ,
and little attempt has been made to ensure that all Raf proteins are
equally active on a molar basis with respect to Ras binding.
Furthermore, there is little data on the kinetics of dissociation of
the RasRaf complex. In this study, we have concentrated on using
equilibrium binding methods, scintillation proximity and fluorescence
anisotropy assays, to compare the ability of Raf fragments to bind to
Ras and to estimate the dissociation rate. An important aspect of these
comparisons was to establish to what extent reported differences in
apparent affinity might be due to differences in the fraction of active
protein rather than in the intrinsic affinity.
The products were purified using Magic polymerase chain reaction Preps(TM) (Promega), digested with BamHI and EcoRI, repurified, and ligated into digested and dephosphorylated pGEX2T vector. The sequences were confirmed using Taq terminator chemistry on an Applied Biosystems 373A automated DNA sequencer.
Raf-(51-147) in BL21 was purified as a GST fusion and after thrombin cleavage as above, except that the buffer used was 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol. The thrombin-cleaved protein eluted in a sharp peak using this higher ionic strength buffer.
The GST-Raf proteins were concentrated either by ammonium sulfate precipitation (78% saturation) or by ultrafiltration using an Amicon PM10 membrane. Concentration of cleaved proteins was by ultrafiltration (Amicon YM3 membrane for Raf-(51-131) and Raf-(51-147), Amicon PM10 membrane for Raf-(1-190)). The concentrate was dialyzed overnight against 10 mM Tris/HCl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, and quick frozen in aliquots for storage at -80 °C. Precipitation of Raf-(51-131) and Raf-(51-147) (both as GST-fusions and after thrombin cleavage) sometimes occurred on freeze/thawing.
Figure 1: SDS-polyacrylamide gel electrophoresis of purified Raf proteins. Proteins were purified as described under ``Materials and Methods'' and analyzed by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue. Samples are as follows: GST-Raf-(1-257) after affinity chromatography (track 1) and after gel filtration (track 2); GST-Raf-(1-190) after affinity chromatography (track 3) and after gel filtration (track 4); GST-Raf-(51-147) (track 5); Raf-(51-147) (track 6); GST-Raf-(51-131) (track 7); Raf-(51-131) (track 8).
Figure 2:
Raf gives a Ras-dependent signal in an
ELISA when Ras is bound to the plate, but not when Raf is bound to the
plate. Ras was bound to the ELISA plate and then incubated with various
concentrations of either GST-Raf-(1-190) () or
GST-Raf-(1-257) (
). Bound GST fusion was detected with an
anti-GST antibody as described under ``Materials and
Methods.'' Experiments with either GST-Raf-(1-190) (
)
or GST-Raf-(1-257) (
) were also performed in which Ras
was omitted. In a separate set of experiments, GST-Raf-(1-190)
was bound to the plate, which was then incubated with various
concentrations of Ras, and bound Ras was detected by reaction with
anti-Ras antibody (
). In a similar experiment,
GST-Raf-(1-190) was omitted
(
).
Binding of Ras to Raf has frequently been
monitored by using glutathione-agarose to precipitate a GST-Raf/Ras
complex (affinity precipitation). Both GST-Raf-(1-190) and
GST-Raf-(1-257) fusion proteins were able to precipitate
RasGTP. However, the amount of Ras precipitated was markedly
substoichiometric in that using microgram quantities of GST-Raf, the
bound Ras could not be detected by SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining but only by immunodetection
on Western blots (data not shown).
It was possible that the
inability to detect binding of Ras to GST-Raf-(1-190) when the
latter was prebound to the plate and the low stoichiometry seen in the
affinity precipitation assay might have been because both are
nonequilibrium binding assays involving separation steps. Therefore we
attempted to measure Ras/Raf binding using a scintillation proximity
assay. A SPA procedure, employing protein A fluoromicrospheres coated
with anti-GST, has recently been used to measure the binding of
GST-NF1-334 to Ras complexed with [H]GTP
under equilibrium conditions(29) . However, when this assay was
performed using 0.03 µM Ras
[
H]GTP with either
GST-Raf-(1-257) or GST-Raf-(1-190), the signal obtained was
at least 40-fold lower than that with the same molar concentration of
GST-NF1-334 (data not shown).
As observed by others, GST-Raf-(51-131) expressed at high levels in E. coli as a soluble protein, which showed little degradation. The fusion protein was virtually pure after glutathione elution and could also be cleaved with thrombin to produce pure Raf-(51-131) (Fig. 1, tracks 7 and 8). The proteins were demonstrated to be intact by their reactivity with the YL1/2 antibody (which recognizes the Glu-Glu-Phe epitope placed at the carboxyl terminus of the construct) and by electrospray mass spectrometry (data not shown).
GST-Raf-(51-131) was added to
protein A SPA beads, anti-GST, and
Ha-Ras[
H]GTP in a
scintillation proximity assay. In contrast with the longer Raf
constructs described above, a signal similar to that with
GST-NF1-334 was produced. As has been reported with
GST-NF1-334(29) , the signal increased with increasing
concentrations of GST-Raf until a maximum was reached, and it then
decreased (Fig. 3a). These results are consistent with the
hypothesis that with increasing concentration of GST-Raf, the signal
increases as more Ras
GST-Raf complex is formed and then decreases
due to competition between free and Ras-bound GST-Raf for binding to
the limiting amount of anti-GST antibody. The specificity of the signal
was confirmed in that it was dependent upon the presence of both
anti-GST and GST-Raf and was abolished by the neutralizing anti-Ras
antibody, Y13-259 (Fig. 3b).
Figure 3:
Binding of GST-Raf to Ras in a
scintillation proximity assay gives a signal, which is abolished by
Y13-259. Panel a, the indicated concentrations of
GST-Raf-(51-131) were incubated with 0.03 µM Ras[
H]GTP, anti-GST, and protein A SPA
beads as described under ``Methods and Materials'' (
).
Blanks were performed in which anti-GST was omitted (
). Panel
b, the indicated concentrations of Y13-259 antibody were
added to SPAs containing 0.03 µM GST-Raf-(51-131)
and 0.03 µM Ras
[
H]GTP. After
subtraction of blanks (without GST-Raf-(51-131)), the SPA signal
was expressed as percentage inhibition relative to control values
obtained in the absence of Y13-259.
In
order to establish that the rapid dissociation rates observed in the
SPA were not an artifact of the assay procedure, an alternative method
of monitoring the dissociation of the RasRaf complex was used.
The fluorescence anisotropy signal given by
Ha-Ras
mant-GTP has been shown to increase when
binding to p120-GAP or to NF1-334 occurs(34) . A similar
increase in anisotropy was seen after the addition of
GST-Raf-(51-131) to a solution of
K-Ras
mant-GTP (Fig. 4) or
Ha-Ras
mant-GTP (data not shown). Subsequent
addition of an excess of unlabeled Ha-Ras
GTP
reduced the signal to that of Ras
mant-GTP alone within the time
taken to make the addition (<0.5 min) at 25 °C, suggesting a
rate constant for dissociation of >1 min
(Fig. 4). As expected, after further addition of excess
GST-Raf-(51-131), the fluorescence anisotropy signal increased
again to the level before addition of unlabeled Ras (data not shown).
Figure 4:
Measurement of the rate of dissociation of
Raf from Ras using fluoresence anisotropy. The fluorescence anisotropy
of K-Rasmant-GTP (0.3 µM) was
measured every 2 s as described under ``Materials and
Methods.'' At times A, B, C, D, and E, GST-Raf-(51-131) (0.3
µM) was added. At time F, unlabeled
Ha-Ras
(5 µM) was
added.
Raf-(51-147) was expressed as a GST fusion again with
the added C-terminal tripeptide sequence, Glu-Glu-Phe. This produced
soluble protein, which was apparently homogeneous by SDS-polyacrylamide
gel electrophoresis. It reacted with anti-GST, but it showed a very low
reactivity with the YL1/2 antibody compared with the
GST-Raf-(51-131) (data not shown), suggesting degradation at the
carboxyl terminus. Electrospray mass spectrometry revealed several
species; the major one (M 36874) corresponded to
loss of residues 144-147 and would have been produced by
proteolytic cleavage between the paired basic residues Arg-143 and
Lys-144. This degradation was largely prevented by expression in the ompT-deficient E. coli strain BL21(31) .
Electrospray mass spectroscopy showed that a high percentage of the
protein purified from this strain both as a GST-fusion and as a
thrombin-cleaved fragment was now of the calculated M
for the intact protein. GST-Raf-(51-147) gave a significant
signal in the SPA (Fig. 5).
Figure 5:
Effect of the concentration of
Ras[
H]GTP on the SPA signal with
GST-NF1-334, GST-Raf-(51-131), GST-Raf-(51-147),
GSTRaf-(1-257), and GST-Raf-(1-190). The indicated
concentrations of Ras
[
H]GTP were included
in SPAs containing GST-NF1-334 (
), GST-Raf-(51-131)
(
), GST-Raf-(51-147) (
), GST-Raf-(1-257)
(
), and GST-Raf-(1-190) (
), each at a final
concentration of 0.03 µM. The signal obtained from blank
wells containing Ras, but no Raf was subtracted from data before
plotting.
In order to compare the affinity
of Ras for Raf-(51-131) and Raf-(51-147), it was necessary
to ascertain whether both Raf proteins were similarly active. The
titration of Ras[
H]GTP into a mixture of
protein A SPA beads, anti-GST, and a fixed concentration of GST-Raf was
used to estimate the relative proportion of active protein in the
samples. At saturation, the amount of Ras bound should reflect not
differences in affinity but only the amount of protein active in
binding Ras. There was very little difference in the maximum SPA signal
between GST-NF1-334 and GST-Raf-(51-131), but the same
concentration of GST-Raf-(51-147) gave 50% of this signal (Fig. 5). At that concentration of Ras required to saturate
GST-Raf-(51-131) binding, GST-Raf-(1-257) and
GST-Raf-(1-190) gave about 20 and 2% of this signal,
respectively, consistent with the hypothesis suggested above that these
two proteins contain high proportions of inactive protein. The apparent K
for the Ras binding interaction estimated from
titrations with increasing Ras concentrations using the model described
under ``Materials and Methods'' was found to be similar for
GST-Raf-(51-147) and GST-Raf-(51-131) (Table 1). When
this calculation was repeated making the assumption that the
GST-Raf-(51-147) was only 50% active, the apparent K
was only increased by 6%, which is within the
experimental error.
The interactions between Raf fragments and Ras
were further monitored by assessing the ability of Raf or NF1-334
(as thrombin-cleaved proteins) to compete with GST-Raf or
GST-NF1-334 for binding to Ras[
H]GTP
in the SPA ( Fig. 6and Table 2). NF1-334 abolished
the binding of Ras to GST-NF1-334, GST-Raf-(51-131), or
GST-Raf-(51-147). The potency of inhibition was identical in all
three assays (I
= 50 nM) (Table 2).
Similarly, Raf-(51-131) and Raf-(51-147) abolished the
signal from SPAs using Ras binding to GST-Raf-(51-131),
GST-Raf-(51-147), or GST-NF1-334, with similar potencies (Table 2). Thus, again there was no evidence of any significant
difference in Ras binding affinity between the two Raf fragments.
Figure 6:
Ras/GST-Raf binding is abolished by
NF1-334, Ras/GST-NF1-334 binding is abolished by Raf, and
no difference is observed between Raf-(51-131) and
Raf-(51-147). The indicated concentrations of cleaved
NF1-334 (), Raf-(51-131) (
) or
Raf-(51-147) (
) were added to SPAs with 0.03 µM Ras
[
H]GTP and GST-NF1-334 (panel a), GST-Raf-(51-131) (panel b), or
GST-Raf-(51-147) (panel c). Percentage inhibition was
calculated as described in the legend to Fig. 3b.
Our initial studies used proteins containing residues 1-257 and 1-190 of Raf, both of which contain the minimal Ras-binding region (residues 51-131). Using ELISA and affinity precipitation techniques, these longer fragments appear to be unable to bind Ras stoichiometrically. Both techniques involve separation steps and therefore do not measure equilibrium binding but the amount of binding detected in a scintillation proximity assay under equilibrium conditions was also much lower than expected for the amount of Raf proteins added. Significantly, the amount of binding detected by ELISA was much higher when excess Raf was allowed to bind to Ras attached to the plate than when excess Ras was allowed to bind to Raf attached to the plate. These data suggested that the low stoichiometry was not due to the nonequilibrium nature of the binding procedures and that the Raf-(1-257) and Raf-(1-190) proteins were far from being fully active with respect to binding Ras. This was confirmed using the Raf-(51-131) protein, which gave the same amount of Ras binding as an equivalent molar concentration of NF1-334 when measured under the SPA equilibrium conditions, compared with the 5- and 50-fold lower maximum values obtained using GST-Raf-(1-257) and GST-Raf-(1-190), respectively. It was notable that the longer Raf fragments were extensively degraded in E. coli, whereas the Raf-(51-131) was stable. Probably the longer fragments were incorrectly folded and hence both sensitive to proteolytic degradation and also much less active with respect to Ras binding.
We have
examined for the first time the kinetics of dissociation of Ras (either
Leu-61 or Val-12 mutants) from Raf. Experiments were performed in which
Ras was displaced from the RasRaf complex and the amount of
remaining complex determined using both SPA and fluorescence anisotropy
procedures. The rate of dissociation was very fast, and the
dissociation rate constant was greater than 1 min
.
In view of these high dissociation rates, nonequilibrium binding
procedures, such as have been frequently used in studies of the
interaction of Ras with Raf, will not give true measures of affinity.
Given such a high dissociation rate, it is surprising that Ras binding
could be observed by either affinity precipitation or ELISA procedures,
both of which involve extensive washing. The explanation for this is
probably a combination of two factors. First an avidity effect of the
local concentration of protein bound to either the beads or the plate (cf. (35) ) and second the detection procedures
employed (either radioactivity or antibody), which are so sensitive
that grossly substoichiometric binding can be detected. Indeed where it
is possible to calculate data from the literature, it would appear that
the binding was substoichiometric(12, 36) .
One of
the main aims of this work was to compare the affinity of Ras binding
of the two different length Raf fragments, residues 51-131 and
51-147. In view of the high dissociation rates and the
substoichiometric binding observed with the longer fragments, it was
considered particularly important to use an equilibrium binding method
(the scintillation proximity assay) and to establish that the proteins
being compared were intact, pure, and had comparable biological
activities. Initially both proteins were expressed in E. coli RR1M15. However, although both purified proteins appeared
homogeneous by SDS electrophoresis, the 51-147 protein was
proteolytically degraded such that residues 144-147 and the added
Glu-Glu-Phe epitope were missing. Full-length protein was obtained
using the ompT protease-deficient E. coli strain
BL21. The sensitivity to proteases suggests that the region around
residue 144 is not highly structured, whereas Raf-(51-131) has a
well-defined structure(24, 37) . The 51-147
protein gave a maximal signal in the SPA about half that of the
51-131 protein, suggesting that not all of the protein was
functionally active. The affinity of interaction between Ras and Raf
was measured both directly through a titration in which the Ras
concentration was varied and indirectly by measuring the ability of
NF1-334 or Raf to prevent binding of Ras to GST-Raf. The affinity
of interaction between Ras and NF1-334 was nearly identical to
that between Ras and Raf-(51-131), and the latter was similar to
that previously
reported(13, 21, 22, 23) . We
detected no significant differences in affinity for Ras between
Raf-(51-131) or Raf-(51-147) using any of these procedures,
even when corrections were made for the activity of the GST-fusion
proteins assessed by the maximal signal obtained in the SPA. Hence, in
contradiction of the conclusions of Ghosh et al.(25, 26) and Chuang et al.(23) ,
we find no evidence that residues 132-147 form a critical
determinant in the Ras/Raf interaction. We do not know definitively why
there is a discrepancy in our conclusions, but the following factors
would be sufficient to explain it. First, nonequilibrium binding
procedures involving separation steps were used to estimate affinities,
and the apparent affinities would therefore reflect dissociation rates
rather than absolute affinities. Second, the integrity of the proteins
was not established. Third, the stoichiometry of interaction was not
reported, and hence the proteins may not have been fully or equally
active, resulting in measurement of apparent rather than true
affinities. In our opinion, the latter is likely to be particularly
significant. In support of this, longer GST-Raf constructs gave a weak
maximal SPA signal (Fig. 5), and Raf-(51-147), when
expressed as a biotinylated transcarboxylase (Pinpoint) fusion, gave no
SPA signal. (
)
Using the SPA, we showed that the thrombin-cleaved Raf fragments abolished the signal given by GST-NF1-334 binding to Ras (Fig. 6a) and that NF1-334 abolishes the signal given by GST-Raf binding to Ras (Fig. 6, b and c). Other groups have shown that Raf inhibits the stimulation of Ras GTP hydrolysis by p120GAP and neurofibromin(12, 13, 22, 23) , but as this inhibition appeared to be less than 100%, it was unclear whether the proteins were fully competitive. Thus, this is the first demonstration that Raf and NF1-334 block the physical interaction between Ras and NF1-334 and Ras and Raf, respectively, and we conclude that Raf and NF1-334 cannot be bound simultaneously to Ras.
There are conflicting inferences in the literature about the
dissociation rate for the Ras/Raf interaction. Since Ras/Raf binding is
readily observed using nonequilibrium techniques such as affinity
precipitation or ELISA, there has been a tacit assumption that the
dissociation rate must be relatively slow so that a stable complex is
formed. In contrast, Zhang et al.(13) suggest that
their inability to co-immunoprecipitate Ras and full-length Raf from
cell lysates may be due to the transient nature of the interaction. Our
own experiments, which are the first to directly measure the
dissociation kinetics, clearly demonstrate that the Ras binding domain
of Raf dissociates relatively rapidly from the nucleotide binding
domain of RasGTP (Fig. 4). Similar results were obtained
both with Val-12 and Leu-61 Ras variants, consistent with the report
that the affinities of interaction of Raf with Val-12 and Leu-61 Ras,
and also normal Ras, are similar(21) . Based on the assumption
that the dissociation rate of Raf from Ras is very slow, it has been
postulated that the Ras/Raf signal is terminated by the intrinsic GTP
hydrolysis rate(21, 24) . Although the signal might
indeed be, at least in part, terminated by the intrinsic GTPase, as
discussed in (3) and (30) , the data presented here
suggest a rapid equilibration between Ras and Raf (and also between Ras
and neurofibromin and Ras and p120-GAP) in the cell, which would allow
GTPase activating proteins to down-regulate signaling through Raf.
Further work on the kinetics of dissociation of full-length Raf from
processed Ras
GTP attached to the plasma membrane might help to
obtain a better understanding of the system in the cell.