(Received for publication, December 7, 1995; and in revised form, February 22, 1996)
From the
While Ras proteins are activated by stimulated GDP release,
which enables acquisition of the active GTP-bound state, little is
known about how guanine nucleotide exchange factors (GEFs) interact
with Ras to promote this exchange reaction. Here we report that
mutations within the switch 2 domain of Ras (residues 62-69)
inhibit activation of Ras by the mammalian GEFs, Sos1, and
GRF/CDC25. While mutations in the 62-69 region
blocked upstream activation of Ras, they did not disrupt Ras effector
functions, including transcriptional activation and transformation of
NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF
responsiveness of a Ras(69N) mutant was due to a loss of GEF binding,
with no change in intrinsic nucleotide exchange activity. Furthermore,
structural analysis of Ras(69N) using NMR spectroscopy indicated that
mutation of residue 69 had a very localized effect on Ras structure
that was limited to
-helix 2 of the switch 2 domain. Together,
these results suggest that the switch 2 domain of Ras forms a direct
interaction with GEFs.
Ras proteins are guanine nucleotide-binding proteins that
function as molecular switches to mediate downstream signaling from a
variety of cellular receptors that promote cell growth and
differentiation(1) . Switching of Ras proteins between the
active GTP-bound and inactive GDP-bound states in response to cellular
stimulation is tightly regulated in vivo by two classes of
regulatory molecules; guanine nucleotide exchange factors (GEFs; Sos,
GRF/CDC25) (
)that promote the acquisition of
GTP and GTPase-activating proteins (GAPs; p120- and NF1-GAP) that
stimulate its rapid hydrolysis to
GDP(1, 2, 3) . While the Ras-GTP/GDP ratio
can be elevated by inhibition of GAP activity, most receptor-mediated
stimuli promote Ras activation via GEF activation (1) .
Based on the x-ray crystallographic structure of Ras, only two
regions of the molecule undergo significant changes in conformation
upon switching between the GTP- and GDP-bound
states(4, 5) . These regions are generally referred to
as the switch 1 (loop 2 and -sheet 3, amino acids 32-40) and
switch 2 (loop 4 and
-helix 2, amino acids 60-76) domains.
While mutational analyses have revealed that the switch 1 domain and
proximal portion (loop 4) of the switch 2 domain are responsible for
binding and responsiveness of Ras to p120 GAP (reviewed in (6) ), it is less clear how Ras interacts with its positive
regulators, GEFs. Mutation of a number of Ras residues results in a
reduction or loss of its activation by Ras GEFs. These mutations are
located in the switch 1 (7) and switch 2
domains(7, 8, 9, 10, 11, 12, 13) and
between residues 95-110 and
130-140(6, 8, 14) . While these studies
indicate that multiple Ras residues can critically influence its
activation by GEFs, there is little information indicating the
mechanism of this inhibition or how GEFs interact with Ras.
Measurements of K
and K
values indicated that GEF interaction with the poorly responsive
Ras(35A), Ras(62H), and Ras(63H) mutants were not lost(7) .
However, no enzymatic or direct binding assays were performed to
determine if a reduction in GEF responsiveness of other Ras mutants
correlated with decreased GEF association. It is anticipated that
identification of the site(s) of GEF interaction will lead to a better
understanding of how Ras and other regulatory GTPases, e.g. Rab, Rho, and heterotrimeric G protein family
members(15) , are activated and will identify a potential
site(s) for pharmacological intervention of growth factor-induced
mitogenesis.
To determine the site(s) of GEF interaction with Ras,
we previously took advantage of the biochemical properties of two Ras
dominant inhibitory mutants. Both Ha-Ras(15A) and Ha-Ras(17N) mutants
have higher affinity for GEFs than Ras(WT) and function biologically by
sequestering these exchange factors, thus preventing GEF activation of
the normal endogenous Ras proteins, resulting in inhibition of cell
growth(3) . By coupling random mutagenesis of the dominant
inhibitory Ha-Ras(17N) mutant with biological selection we recently
identified Ras residues 75, 76, and 78 to be critical for its growth
inhibitory phenotype, presumably through loss of interaction with the
mammalian GEFs, Sos1, and GRF/CDC25(13) . In
contrast, mutational analysis of Ha-Ras(15A) revertants in Saccharomyces cerevisiae identified distinctly different
residues (62, 63, 67, and 69) that impaired Ras interaction with the
yeast CDC25(12) . The 63K mutant has previously been reported
to transform NIH 3T3 cells, suggesting a gain rather than loss of
function(16) . Furthermore, a 76K mutant that was found to
disrupt Ras-GEF interaction without affecting Ras effector function in
NIH 3T3 cells (13) attenuated oncogenic Ras signaling in S.
cerevisiae(12) . These observations indicated that
specific Ras mutants could have divergent effects in these two
biological systems. Whether these contrasting results reflected
differences between the yeast and mammalian GEFs, the different
biological systems, or between the properties of the Ras(15A) and
Ras(17N) dominant inhibitory proteins was unclear.
To determine the
importance of residues 62-69 in Ras interaction with mammalian
GEFs, we characterized the 62K, 63K, 67I, and 69N mutations, previously
identified in yeast, in Ha-Ras and expressed them in NIH 3T3 murine
fibroblasts to examine their effects on Ras function. Our results
establish that, like residues 75-78, residues 62-69 are
essential for Ras stimulation by both Sos1 and GRF/CDC25.
The lack of responsiveness of the 69N mutant was due to a loss of
Ras:GEF interaction, without disrupting Ras effector functions.
Furthermore, in vitro analysis of the Ras(69N) protein by
multidimensional NMR spectroscopy indicated that minor structural
alterations associated with this mutation were localized to
-helix
2 in the switch 2 domain. Thus we conclude that helix 2 defines a
region of Ras that is directly involved in Ras-GEF interaction.
Figure 4:
Ras(63K) is impaired in GAP-stimulated
GTPase activity. Five pmol of (His) Ras(WT) and Ras(63K)
were loaded with [
-
P]GTP as described under
``Materials and Methods'' and incubated ± 0.1 pmol of
p120-GAP, 30 °C. The percentage of GTP hydrolyzed after 2-min
incubation, as determined by nitrocellulose filter binding
assay(24) , is indicated. Results are mean ± S.D. for
triplicate samples.
Figure 6:
Mutation of Ras residue 69 does not
disrupt its intrinsic nucleotide exchange activity or its intrinsic or
GAP-stimulated GTPase activities. A, recombinant Ras(WT) or
Ras(69N)(1-166) was loaded with [8,5`-H]GDP
and the rate of loss of nucleotide measured in the presence of excess
(0.5 mM) cold GTP at room temperature. B, recombinant
Ras(WT) or Ras(69N)(1-166) was loaded with
[
-
P]GTP and incubated in the presence or
absence of 0.2 nM NF1-GRD for the indicated times. GTP
hydrolysis was determined by measuring the accumulation of labeled
inorganic phosphate. All results are mean ± range for duplicate
samples and are representative of at least two
experiments.
Figure 1: Mutations at residues 62, 63, 67, and 69 reverse the growth inhibitory phenotype of Ras(17N). NIH 3T3 cells were transfected with 500 ng of pZIP-ras constructs encoding Ras(WT), Ras(17N), Ras(17N,62K), Ras(17N,63K), Ras(17N,67I), and Ras(17N,69N) and selected on growth medium supplemented with 400 µg/ml G418 for 14 days. G418-resistant colonies were then visualized by staining with 0.5% crystal violet.
Figure 2:
Morphology of NIH 3T3 cells stably
expressing Ras(WT) and Ras(12R) with mutations at residues 62-69.
NIH 3T3 cells were transfected with pZIP constructs encoding the
indicated Ras mutants and stable transfectants selected in
G418-containing growth medium. Pooled populations were obtained by
combining >100 drug-resistant colonies for each Ras mutant and
photographed at 100 magnification to demonstrate
morphology.
This effect was further quantitated using a NIH 3T3 focus-formation assay. As seen in Fig. 3, introduction of mutations into Ras residues 62, 67, and 69 greatly impaired (75-100%) the ability of Ras(WT) to induce transforming focus formation, while only partially reducing the activity of the GTPase-defective/constitutively GTP-bound oncogenic Ras(12R). Most significantly, the 69N mutation completely inhibited the focus-forming ability of Ras(WT) while having no effect on the transforming ability of Ras(12R). This suggested that the 69N mutation most severely impairs GEF interaction with Ras without disrupting Ras activation of downstream effector protein(s).
Figure 3: Mutation of residues 62-69 drastically inhibits Ras(WT)-induced, but not Ras(12R)-induced, transformation in NIH 3T3 cells. A, transformed focus formation of Ras(WT) mutants. Cells were transfected with 2 µg, or 0.2 µg for Ras(63K), of the indicated pZIP-ras constructs per 60-mm dish and incubated in growth medium for 14 days prior to quantitation of transformed foci. B, transformed focus formation of oncogenic Ras(12R) mutants. Cells were transfected with 20 ng of the indicated pZIP-ras constructs and treated as in A. Results are shown as mean ± S.D. for triplicate plates and are representative of at least three independent experiments.
In contrast to studies in yeast, but in agreement with the observation of Fasano et al.(16) , expression of the Ras(63K) mutant in NIH 3T3 cells resulted in an apparent gain of function as evidenced by morphological transformation (Fig. 2, upper panels). This observation again indicates that conflicting results can be obtained in the yeast and mammalian biological assay systems. The transforming potential of Ras(63K), as quantitated by focus forming activity (Fig. 3a) was found to be approximately 25-fold greater than by Ras(WT). The potent transforming activity caused by oncogenic mutations at codons 12, 13, and 61 of Ras is due to decreased intrinsic and GAP-stimulated GTPase activity, resulting in the proteins accumulating in the active GTP-bound state(29) . While the GAP-stimulated GTPase activity of a Ras(63Q) mutant was similar to that of Ras(WT)(30) , the mild transforming activity of a Ras(63H) mutant has previously been correlated with a reduced sensitivity of its GTPase activity to GAP stimulation(31) . As shown in Fig. 4, the transforming activity of the 63K mutation also correlated with a significant decrease in intrinsic and p120 GAP-stimulated GTPase activity.
Figure 5:
Mutation of Ras residues 62-69
blocks Sos1- and GRF-stimulated transcriptional activation of
Ras-responsive elements. NIH 3T3 cells were cotransfected with 2 µg
of pZIP or pZIP-ras mutant and either 200 ng of empty pBABE
vector, pBABE-Sos1 (A), or 100 ng of pJ4-GRF (B) along with 1 µg of pB4X-CAT reporter construct.
Chloramphenicol acetyltransferase activity was determined 48 h
post-transfection as described previously(13) . Similar results
were obtained following cotransfection of Ras mutants with the isolated
catalytic domain of Sos1 or a membrane-targeted form of the GRF
catalytic domain (not shown).
The
lack of responsiveness of the Ras 62K, 67I, and 69N mutants to GEF
stimulation and their inability to induce transformation could possibly
be due to reduced intrinsic nucleotide exchange activity, thereby
maintaining Ras in its inactive GDP-bound state. To address this
possibility, we compared the biochemical properties of the Ras(69N)
mutant to those of Ras(WT). As shown in Fig. 6A, the
intrinsic off-rate of GDP from Ras(69N) protein was not significantly
reduced from that of Ras(WT). Furthermore, in contrast to the Ras(63K)
mutant, there was no effect of the 69N mutation on intrinsic or
GAP-stimulated GTPase activity (Fig. 6B). Therefore,
the biological properties of Ras(69N) are consistent with a defect in
GEF stimulation. This hypothesis was confirmed using a GST-CDC25
catalytic domain fusion protein (23) that promoted nucleotide
exchange on Ras(WT) but was completely ineffective on the 62K, 63K,
67I, and 69N mutants in vitro. ()
Figure 7:
Mutation of residues 62, 63, 67, and 69 of
Ras disrupts interaction of Ras with the catalytic domain of the yeast
GEF, CDC25. 100 µg of recombinant Ras(WT), 62K, 63K, 67I, and 69N
proteins were incubated (60 min, 4 °C) with a GST-CDC25 catalytic
domain fusion protein (10 µg) immobilized on glutathione-agarose
beads. Following extensive washing, bead associated Ras was quantitated
by Western blotting with a Ha-Ras-specific monoclonal antibody, as
described under ``Materials and Methods.'' Mutation of each
residue significantly reduced association with CDC25 as compared with
Ras(WT), with no detectable binding of the Ras(63K) or Ras(69N)
mutants. Binding of all four mutant proteins, but not that of Ras(WT),
to CDC25 was lost following more stringent
washing.
By using N, we could remove all NMR signals not attached to the
N nucleus, thereby simplifying the NMR spectrum of this
18.9-kDa protein. Comparison of
H-
N
correlation maps between Ras(WT) and Ras(69N) showed chemical shift
differences in only a few H
resonances, indicating that the
mutation at position 69 produced only minor perturbations in the
protein. We were able to assign all of the H
residues in
the mutant, with the exception of residue 69, by comparing
N-edited three-dimensional NOESY and TOCSY data acquired
on Ras(WT) and Ras(69N). The only residues that showed sizable changes
in both
H and
N chemical shifts were localized
near residue 69 in helix 2 as well as residues 26 and 28 in loop 2.
Although the chemical shift perturbations associated with residues near
the site of the mutation were expected, we were surprised by the
chemical shift alterations observed for residues N26 and F28 in loop 2.
However, we have previously observed that residue 28 is very sensitive
to changes in temperature and environment, which could cause
significant changes in the chemical shift of this residue.
To more
definitively assess structural changes resulting from the 69N mutation,
we examined NOE cross-peaks in Ras(WT) and Ras(69N) three-dimensional N-edited NOESY data sets. Variations in short range (<5
Å) inter-residue proton contacts are identifiable through
analysis of the NOE cross-peak intensities. NOE differences observed
between Ras(WT) and Ras(69N) provide information regarding alterations
in the spatial distribution of protons, which can be related to changes
in tertiary structure. Close inspection of three-dimensional
N-edited NOESY data revealed a partial disruption of
inter-residue connectivities in residues spanning from 64 to 76 of
switch 2 and also between residues 28 and 147. A ribbon diagram of Ras
derived from the Ras(WT)-GDP NMR solution structure is shown in Fig. 8and illustrates the location of residues that exhibit loss
of NOEs associated with the 69N mutation.
Figure 8: Structural analysis of 69N 1-166 (compared with WT). A ribbon diagram derived from the Ras(WT)-GDP NMR solution structure (28) is shown. Residues that demonstrate loss of sequential NOEs in the Ras(69N) mutant are shown in black. NOE changes are localized to residues 64-76 in switch 2 and between residues 28 and 147. Side chains for residues 28, 69, and 147 are illustrated in the ribbon diagram.
Residues that make up an
-helix are expected to show consecutive strong amide-amide (dNN)
cross-peaks with adjacent amino acids. NOE correlations ranging from
weak to medium intensity are also observed for amino acids up to 4
residues away. Although consecutive dNN cross-peaks between residues 70
and 76 were detected in NOE spectra of Ras(69N), which are usually
indicative of an
-helix, we also observed a reduction in the
number of medium and long range cross-peaks, indicative of some
perturbation of the helix.
We also observed a loss in an NOE
connectivity between residue Phe and Lys
.
These results are intriguing as mutation of Phe
has been
shown to drastically increase the nucleotide dissociation rate of Ras,
presumably due to disruption of a critical hydrophobic interaction with
the guanine nucleotide base (33) . Binding of GEFs to Ras
facilitates dissociation of the bound guanine nucleotide substrate, but
the mechanism by which this occurs is unknown. While we did not observe
loss of NOE cross-peaks between Phe
and the H-8 proton of
the guanine base or the C-1`-H proton of the ribose sugar in the 69N
mutant, consistent with its unaltered guanine nucleotide dissociation
rate, it is tempting to speculate that binding of GEFs alters the
interaction between Phe
, Lys
, and possibly
the guanine nucleotide. However, it is difficult to understand how a
mutation in helix 2 could affect the interaction between Phe
and Lys
(on the opposite side of the protein),
since no significant structural alterations in residues outside of
helix 2 were observed that might propagate structural perturbations to
loop 2. Given the sensitivity of residue 28 to slight differences in
solvent and temperature, it is possible that the loss of an NOE between
residues 28 and 147 may result from small variation in sample
conditions between Ras(WT) and Ras(69N), rather than from mutation of
residue 69.
It is now known that in addition to activation by point mutation, Ras is responsible for mediating cellular transformation induced by deregulated upstream GEFs and by tyrosine kinase oncogenes that act via GEF stimulation(22, 34, 35, 36) . While it has also been established that activation of GEFs, rather than inhibition of GAPs, is the major physiological mechanism for elevating the GTP-bound state of Ras(1) , much less is known about Ras interaction with GEFs than with GAPs(6) . To better understand the GEF-mediated GDP/GTP exchange reaction, we set out to identify sites of Ras interaction with GEF molecules. Our initial studies identified a domain (residues 75-78) as being critical for GEF-mediated activation of Ras, while yeast studies pinpointed a juxtaposed region (residues 62-69)(12, 13) . Since these two independent screens did not identify overlapping regions, we have examined the role of Ras residues 62-69 in mammalian GEF stimulation of Ras and addressed the role of this region in mediating GEF interaction.
Analysis of the consequences of 62K,
67I, and 69N mutations in NIH 3T3 cell transformation and
transcriptional activation assays revealed that they abolished the
dominant negative Ras(17N) phenotype, inhibited focus formation by WT
but not oncogenic Ras, and were poorly or unresponsive to mammalian GEF
(Sos1, GRF) stimulation. These data all support the involvement of this
region of Ras in GEF-mediated activation. Sos1 and GRF were similarly
affected by different Ras mutants (the 62 mutant was slightly more
sensitive to GRF than Sos1 stimulation). Thus although the catalytic
domains of Sos1 and GRF are only 30% identical, they presumably
function similarly to promote nucleotide exchange. A slight stimulation
of transcriptional activation was observed with the 67IK mutant in the
presence of both GEFs and with the 62K mutant in the presence of GRF.
This is consistent with the weak interaction of these, but not the 63K
or 69N mutants, with CDC25 in vitro (Fig. 7).
In contrast to the 62, 67, and 69 mutants, but in agreement with a previous observation(16) , Ras(63K) was found to be transforming and induced transactivation of Ras-responsive promoter elements. We show here that this enhanced biological activity can be attributed to a defect in GAP-stimulated GTPase activity, similar to that of oncogenic Ras mutants with mutations at residues 12 or 61(29) . The stimulatory phenotype was not observed in yeast, presumably due to the relative differences in GAP activity between cell types. Since mammalian Ras(WT) is insensitive to the yeast GAPs, IRA1 and IRA2, and so accumulates in the active GTP-bound state(29) , Mosteller et al. (12) used a strain of yeast that expressed the catalytic domain of the mammalian p120 GAP from a strong promoter. High expression of this protein presumably offset the decreased sensitivity of Ras(63K) to GAP stimulation. Since Ras(63K) lacks responsiveness to Sos1 and GRF in NIH 3T3 cell transcriptional activation assays, this mutant also appears to be defective in GEF regulation as found in yeast. Loop 4 of Ras contains residues critical for both GEF and GAP regulation(6, 31) . Since Ras(63K) is transforming in mammalian cells, it would appear that the GTPase defect is dominant over the exchange defect as was also observed for the 12R double mutants (Fig. 2).
A number of studies have identified regions
of Ras whose integrity are required for GEF-stimulated activation.
These include the residues 130-140 (8) and 95-110
regions(8, 14) , switch 1 (in particular residues 35
and 38(7) ), and multiple residues in the switch 2 domain:
residues 66, 75(11) , 62, 63, 78, 81(7) ,
73/74(10) , 75/77(9) , 62, 63, 67, and 69 (12) and residues 75, 76, and 78(13) . Although these
mutations of Ras can disrupt GEF-mediated activation, there has been
little insight into how GEFs interact with Ras to promote the exchange
reaction. Only two studies previously addressed whether reduced
responsiveness to GEFs was a result of decreased Ras-GEF binding. While
measurement of K and K
suggested that 35A, 62H, and 63H mutants could still interact
with Ras (7) , the two-hybrid analysis of Mosteller et
al.(12) suggested that 62K, 63K, 67I and 69N mutants were
greatly impaired in their GEF binding ability. This was confirmed by
direct in vitro binding in this study. Although the yeast
CDC25 was used in these binding experiments, we anticipate that similar
results would have been observed using its mammalian counterparts.
Indeed, a recent report by Moodie et al.(37) demonstrated the inability of additional switch 2
mutants to bind to mammalian GEFs. The Y13-259 monoclonal antibody that
specifically interacts with residues 63-73 in the switch 2 of Ras (38) competed for binding of Ras to both the yeast CDC25 and
mammalian SOS1(12) , further indicating the functional
similarity of these GEFs and supporting the involvement of the switch 2
domain of Ras in exchange factor interaction. However, none of these
data could exclude the possibility that 62-69 mutations had not
disrupted the tertiary structure of Ras, so perturbing GEF interaction
with a more remote domain. For example, Stouten et al.(39) have proposed that upon Ras switching between its GDP- and
GTP-bound states, movement of switch 2 is relayed to
-helix 3,
located within another domain of Ras previously implicated in GEF
sensitivity (14) .
Limited structural analysis has been
performed on Ras mutants, with most work focusing on the effects of the
oncogenic mutations that attenuate GTPase
activity(40, 41) . It is evident upon examination of
NMR data that mutation of residue 69 from Asp to Asn, that completely
blocked GEF binding and stimulation of Ras, produced only minor
structural alterations in Ras that appear to be primarily localized to
helix 2 within the switch 2 domain. This is consistent with the fact
that Asp lies on the exterior face of helix 2 in the
GDP-bound form of Ras and that residues in helix 2 form few tertiary
contacts with other secondary elements in the NMR structure of
Ras(WT)(1-166)(28) . Furthermore, NMR analysis did not
reveal structural changes in Ras(69N) at other sites previously
reported to alter GEF sensitivity (i.e. residues 95-110,
130-140, or the switch 1 domain). The loss of a NOE cross-peak
between residues 28 and 147 is an intriguing observation, but further
study is required to ascertain whether it was lost as a result of
slight changes in solvent conditions as opposed to mutation of residue
69.
While the switch 2 domain undergoes significant structural orientation upon Ras exchanging GTP for GDP, both nucleotide-bound states are equally responsive to GEF stimulation(42) . Neither the rate of GDP release from Ras nor its downstream effector functions were disrupted by introduction of the 69N mutation, indicating that conformational switching was not impaired. These observations, taken together with the lack of structural alterations outside of helix 2 of Ras(69N), indicate that the inability of Ras(69N) to interact with GEFs is most likely due to localized disruption of a GEF binding site in the switch 2 domain. Consistent with this conclusion, it has been reported that a swap of positive for negative charge on residue 1374 (Arg to Glu) in the catalytic domain of yeast CDC25 rescued the GEF sensitivity of the Glu to Lys (negative for positive) mutation at Ras residue 63, suggesting that these two residues form a functional ion pair interaction(43) . The ability of the Y13-259 anti-Ras antibody, which binds to residus in helix 2(38) , to disrupt binding of Ras to CDC25 and Sos1 further support a role for this region of Ras in GEF interaction. From biochemical studies with the dominant inhibitory Ras(15A) it is clear that GEFs have a much higher affinity for, and act to stabilize, the nucleotide-free state of Ras(44) . Therefore, it is possible that interaction of GEFs with the switch 2 region helps stabilize the nucleotide-free intermediate state of Ras. Future studies will be required to determine if other regions on the surface of Ras interact with GEFs and how GEF interaction with Ras catalyzes the nucleotide exchange reaction.