(Received for publication, November 3, 1995; and in revised form, January 10, 1996)
From the
We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H-Ras. The significance of our findings is discussed in the light of the GTP-induced conformational change model.
Like all other members of the regulatory GTPases, Ras p21 cycles
between two states: the active, GTP-bound state and the inactive,
GDP-bound state(1, 2) . The cycle is controlled by the
balance of guanine nucleotide exchange and GTP hydrolysis, which are in
turn regulated by guanine nucleotide exchange factors and factors that
possess GAP ()activity(3) . The differences between
these two states are characterized by the differences in their ability
to exert biological effects and to be recognized by several protein
factors, such as GAP/NF1, Raf-1, ralGDS, and
others(4, 5, 6, 7, 8, 9) .
GTP induces conformational change in Ras(3) ; it is clear that
the difference in the protein structures between the GDP-bound state
and the GTP-bound state must account for the difference in Ras
activities.
The GTP binding induces conformational change primarily
in two regions of H-Ras termed the switch I (residues 30-38, loop
2, and the effector domain) and the switch II (residues 60-76,
loop 4, and helix 2) domains(10, 11, 12) .
Therefore, any Ras activity that is associated with RasGTP would
be likely to involve these two regions. The requirement of the switch I
region for the action of Ras is well
documented(3, 4, 5, 13) . Numerous
mutations in the switch I region have been shown to disrupt the
biological and biochemical functions of Ras(3, 4) . On
the other hand, although the interaction of H-Ras with exchange factors
appears to require the switch II
domain(14, 15, 16, 17, 18) ,
the role of the switch II region for the activity of Ras is still not
clear. In fact, studies using deletion mutants as well as Ras-Rap
chimeras have concluded that the switch II region is not essential for
the biological activity of H-Ras(19, 20) .
In the
process of conformational change, residues in the loop 4 undergo
significant movements and reorientations to adapt the -phosphate
moiety of GTP(21) . The key feature of the loop 4 is a highly
conserved glycine residue, which is also a part of the consensus
DXXG motif of regulatory GTPase family(22) . Because
glycine exhibits a much broader range of
and
dihedral
angles than other amino acids and will allow sharp bend around the
residue(23) , it is uniquely suitable for serving as a pivot
for conformational change. Although the glycine in the DXXG
motif is not conserved in all regulatory GTPases (24, 25) , its importance to the function of
regulatory GTPases has been demonstrated in heterotrimeric G protein,
EF-Tu, and
H-Ras(26, 27, 28, 29, 30) .
In H-Ras, the substitution of the glycine residue by alanine (G60A
mutation) was found to perturb the GTP-induced conformation change and
to abolish the biological activity(29) . A preliminary NMR
study supports the hypothesis that the region affected by the G60A
mutation is the switch II domain, whereas the switch I domain appears
to be unaltered. (
)This finding is in accord with the
studies of G
and EF-Tu harboring the corresponding
mutation (26, 27, 28) and supports the
crucial functional role of the switch II region for regulatory GTPases.
In this study, we examined the effects of the G60A mutation on the
GTPase activity of H-Ras and on the interaction of H-Ras with four
factors that interact with H-Ras
GTP.
The biochemical property of c-H-Ras(G60A) was first examined by determining the GDP/GTP dissociation constants (Table 1). The equilibrium binding indicates that the c-H-Ras(G60A) appears to have slightly lower GDP/GTP binding affinity than c-H-Ras. This result is opposite to our measurements with v-H-Ras(G60A), where we observed a slight increase in GDP/GTP binding over v-H-Ras(29) . The intrinsic GDP/GTP release also shows that nucleotide dissociates faster from c-H-Ras(G60A) than from c-H-Ras (Fig. 1). Nevertheless, considering the intracellular concentrations of GTP/GDP, these differences are small and are unlikely to have physiological significance. Therefore, we conclude that the G60A mutation does not significantly alter the GDP/GTP binding affinity of either c-H-Ras or v-H-Ras.
Figure 1:
Time course of GTP/GDP release from
c-H-Ras and c-H-Ras(G60A). GDP/GTP dissociation was measured by
labeling p21 with [H]GDP (
and
) or
[
H]GTP (
and
) to equilibrium and
then chased with a 500-fold excess of cold respective guanine
nucleotides. The chase reactions were carried out in the presence of
0.5 mM free Mg
ion at 30 °C and
quantified by membrane filtration method as described (29) . C
represents the amounts of GDP/GTP at time zero,
and C
represents the amounts of GDP/GTP at the
indicated time point.
and
, c-H-Ras;
and
,
c-H-Ras(G60A).
In contrast to GDP/GTP binding, the GTPase activity of
c-H-Ras was markedly reduced by the G60A mutation. We observed a
30-fold reduction in k for the intrinsic GTPase (Table 2). More strikingly, the GTP hydrolysis stimulated by GAP
was largely abolished by the G60A mutation (Fig. 2). The k
of GAP-stimulated GTPase of the c-H-Ras is
over 4 orders of magnitude greater than that of c-H-Ras(G60A) (Table 2). Nevertheless, c-H-Ras(G60A) still displays a
measurable level of GAP-stimulated GTPase activity, which is
distinguishable from that of v-H-Ras (Fig. 3).
Figure 2:
Time course of intrinsic and
GAP-stimulated GTP hydrolysis. GTP hydrolysis was measured by
monitoring the release of P from
[
-
P]GTP in a 100-µl reaction mixture
containing 50 mM Tris-HCl, pH 7.4, 2 mM MgCl
, 50 mM KCl, 50 µg/ml BSA, 1 mM dithiothreitol, and 100 nM of
[
-
P]GTP-labeled H-Ras (specific activity,
1500 Ci/mmol). GTP hydrolysis was initiated by the addition of 4 nM of full-length GAP. A set of reaction without adding GAP was used
for measuring the intrinsic rate. The experiments were performed at 30
°C, and at the specified times the free phosphate was analyzed by
the isobutanol-benzene extraction method as described under
``Materials and Methods.''
, c-H-Ras intrinsic;
,
c-H-Ras + GAP;
, c-H-Ras(G60A) intrinsic;
,
c-H-Ras(G60A);
, v-H-Ras intrinsic;
, v-H-Ras +
GAP.
Figure 3:
Stimulation of GTP hydrolysis by GST-NF1.
NF1-stimulated GTP hydrolysis was performed in a 100-µl reaction
mixture containing 50 mM Tris-HCl, pH 7.4, 2 mM MgCl, 50 mM KCl, 50 µg/ml BSA, 1 mM dithiothreitol, and 62.5 nM [
-
P]GTP-labeled H-Ras (specific
activity 450 Ci/mmol). The reactions were started by the addition of
the indicated amounts of GST-NF1 and allowed to proceed for 10 min at
30 °C. The release of P
was analyzed similar to the
reaction involving GAP. The results were corrected for the intrinsic
rate before plotting.
, c-H-Ras;
,
c-H-Ras(G60A).
Previous study
showed that the G G226A mutant exhibited a reduction
in GTPase activity when the Mg
concentration in the
reaction mixture was lower than 100 nM; however, the reduction
was restored by supplying millimolar concentrations of
Mg
(27) . Thus, we determined the intrinsic
and the GAP-stimulated GTPase at Mg
concentrations
ranging from 0.1 to 5 mM. For the G60A mutant, we were unable
to detect a significant variation in the rate of GTP hydrolysis due to
Mg
concentration (data not shown). Routinely, we
performed GTP hydrolysis at 2 mM Mg
.
Although the GAP-stimulated GTPase is drastically reduced, the affinity
of H-Ras for GAP appears to be unaffected by the G60A mutation (Table 2). In fact, the apparent K
for the
GAP-stimulated GTP hydrolysis reaction suggests that the affinity of
H-Ras for GAP may be slightly enhanced, a result similar to that of
v-H-Ras(G60A)(29) . This observation is not unique for the G60A
mutant because a neighboring mutation at position 61 has been shown to
enhance GAP binding(36, 39) .
The effect of the
G60A mutation on the NF1-stimulated GTPase was also examined. A GST
fusion protein containing 367 residues of the NF1 catalytic domain was
used in the study(40) . This fusion NF1 fragment was able to
promote GTP hydrolysis, but its activity was lower than that of the
full-length GAP under the assay condition. The k for the NF1-stimulated GTP hydrolysis was estimated to be 10.7
min
, as compared with 432 min
for
the GAP-stimulated reaction (Table 2). On the other hand, NF1
appears to have higher affinity for H-Ras than GAP as indicated by the K
of the reaction. Similar to the GAP-stimulated
reaction, the G60A mutation drastically reduced the NF1-stimulated GTP
hydrolysis (Fig. 4). In fact, we were unable to detect GTP
hydrolysis stimulated by NF1 under the assay condition. In order to
measure the affinity of the G60A mutant for NF1, an indirect
competition assay was performed(29, 41) . In this
assay, either cold Ras
GDP or Ras
GTP was used as the
competing agent for the activity of NF-1. Fig. 4shows that both
v-H-Ras
GTP and v-H-Ras(G60A)
GTP display comparable levels
of inhibitory activity against GST-NF1, whereas v-H-Ras
GDP and
v-H-Ras(G60A)
GDP were much less effective. This observation
indicates that the G60A mutation does not perturb the binding of H-Ras
and NF-1.
Figure 4:
Inhibition of GST-NF1 activity by v-H-Ras
and v-H-Ras(G60A). The experiments were performed by measuring the
GST-NF1-stimulated GTP hydrolysis of c-H-Ras
[
-
P]GTP in the presence of the
indicated amounts of competitor H-Ras p21. The experiments were
performed similarly as described in the legend to Fig. 3in a
50-µl reaction mixture containing constant amounts of substrate (25
nM [
-
P]GTP labeled H-Ras; specific
activity, 450 Ci/mmol) and GST-NF1 (6 nM). H-Ras species used
as the competitor were v-H-Ras
GDP (
),
v-H-Ras(G60A)
GDP (
), v-H-Ras
GTP (
), and
v-H-Ras(G60A)
GTP (
).
Figure 5:
The Interaction of H-Ras and Raf-1. H-Ras
and Raf-1 binding was determined by a co-precipitation assay as
described(29) . The reactions were carried out in a 200-µl
reaction mixture containing 50 mM Hepes, pH 7.5, 100 mM KCl, 20 µM ZnCl, 5 mM MgCl
, 1 mg/ml BSA, 62.5 nM H-Ras
[
-
P]GTP (specific activity,
450 Ci/mmol) and indicated amounts of GST-Raf-N275. The binding was
allowed to proceed for 30 min at 4 °C and followed by an additional
30-min incubation interval with glutathione-Sepharose. The bound H-Ras
was then separated from the reaction mixture by passage through glass
fiber filter and quantified. The percent bound was calculated by
dividing the amounts of radioactivities collected on glass fiber filter
by the total amounts of input H-Ras as determined by nitrocellulose
binding assay.
, c-H-Ras;
,
c-H-Ras(G60A).
The effect of the G60A mutation on H-Ras and Raf-1 interaction was
further examined by the two-hybrid system(42) . This assay uses
vectors containing the full-length H-Ras fused to the Gal4 DNA-binding
domain and Raf-N275 to the Gal4 activation domain. In addition, an
intragenic C186S mutation, a mutation that blocked the membrane
association of H-Ras(3) , was incorporated into the H-Ras gene.
This mutation has been shown to enhance the sensitivity
(-galactosidase activity) of the assay employing H-Ras and
Raf-1(43) . The results of the two-hybrid system expressed in
Miller units showed that the G60A mutation reduced the interaction of
v-H-Ras and Raf-N275 in vivo by about 3-fold (Table 4),
a result similar to that of the in vitro binding
assay(29) .
We then examined the effect of the G60A mutation
on Raf-1 binding in c-H-Ras background. The in vitro binding
assay indicated that c-H-Ras bound more tightly to Raf-N275, and it was
comparatively less affected by the G60A mutation than v-H-Ras (Table 3). The dissociation constants were found to be 29 and 64
nM, respectively, for the c-H-RasRaf-1 and
v-H-Ras(G60A)
Raf-1 complexes, a difference of about 2-fold (Table 3). This observation is in agreement with the result of
the two-hybrid system, in which c-H-Ras was also found to interact more
strongly with Raf-1 than v-H-Ras and the effect of the G60A mutation on
Raf-1 binding reduction was smaller (30% versus 3-fold) (Table 4).
Interestingly, in contrast to GAP and Raf-N275 binding, the binding of ralGDS-C127 to H-Ras is drastically reduced by the G60A mutation (Table 5). The reduction is about 30- and 57-fold for c-H-Ras and v-H-Ras, respectively. These levels of reduction were about an order of magnitude greater than that of Raf-N275 binding reduction (Table 3). The interaction of H-Ras and ralGDS-C127 was further examined by the two-hybrid system (Table 6). The experiment was performed similarly to that of H-Ras and Raf-N275 assay, except that the Gal4 activation domain contained the ralGDS-C127 instead of Raf-N275. Table 6shows that the difference in signal output between the wild type and the G60A mutant in vivo paralleled that of in vitro binding. Again, the results show that the G60A mutation more significantly reduces ralGDS-C127 binding than Raf-N275 binding. However, our construct of Gal4 activation domain-ralGDS-C127 fusion vector displayed a relatively low signal when compared with the same vector harboring Raf-N275 fusion. The difference in readout signal is not necessarily reflecting the strength of binding when heterologous vectors are used for the assay because the output signal of the two-hybrid system depends not only on the distance but also on proper juxtaposition of the Gal4 DNA binding and transcription activation domains.
Finally, the effect of the G60A mutation on the interaction between H-Ras and ralGDS-C127 was examined in a competition assay. The experiment was performed by measuring the amounts of v-H-Ras precipitated by GST-Raf-N275 in the presence of increasing concentrations of MBP fusion protein of ralGDS-C127. This MBP-ralGDS-C127 construct contains the same ralGDS-C127 fragment as the GST-ralGDS-C127 fusion and binds H-Ras with an affinity that was about one-third that of the GST-fusion counterpart (Table 5). Similar to GST fusion ralGDS-C127, the G60A mutation also strongly reduced the binding of H-Ras to MBP-ralGDS-C127 (Table 5). Unfortunately, the binding of v-H-Ras(G60A) to MBP-ralGDS-C127 was too low to be measured reliably by the co-precipitation assay (Table 5). Fig. 6shows that the binding of v-H-Ras to GST-Raf-N275 gradually diminished with increasing concentrations of MBP-ralGDS-C127; this indicates that the binding of Raf-N275 and ralGDS-C127 to H-Ras is mutually exclusive. Nevertheless, under the same condition, MBP-ralGDS-C127 was unable to displace GST-Raf-N275 for binding to v-H-Ras(G60A). This result further confirms that the binding of v-H-Ras to ralGDS-C127 is significantly impaired by the G60A mutation.
Figure 6:
Competition of ralGDS and Raf-1 for
binding to H-Ras. The assay were performed in a 200-µl reaction
mixture containing 62.5 nM H-Ras[
-
P]GTP, 167 nM GST-Raf-N275 (precipitating agent), and various amounts
(0.3-4.8 µM) of MBP-ralGDS-C127 (competing agent)
similarly as described in the legend to Fig. 5. The percent of
H-Ras bound to Raf-1 was calculated by using the amounts of
H-Ras
[
-
P]GTP precipitated without
MBP-ralGDS-C127 as 100%.
, c-H-Ras;
,
c-H-Ras(G60A).
The glycine residue of the conserved DXXG motif plays a pivotal role in GTP-induced conformational change. Substitution of this residue by alanine has been shown to disrupt the normal functions of regulatory GTPases(26, 27, 28, 29, 30) . Specifically, the G60A mutation was found to hinder GTP-induced conformational change and to eliminate the biological activity of v-H-Ras. Among the regions that respond to GTP, the switch II domain appears to be the region affected by the G60A mutation. In this study, we examined the effects of the G60A mutation on the GTPase activity and the ability of H-Ras to interact with four cellular factors, GAP, NF1, Raf-1, and ralGDS. All of these factors interact with the GTP-bound form of H-Ras.
The G60A mutation did not significantly alter the
GTP/GDP binding affinity of H-Ras. This result was somewhat surprising,
considering that the Gly-60 residue has to reorient in order to accept
the -phosphate of GTP, and the Gly to Ala substitution should
curtail this process(23) . Nevertheless, the G60A mutant is not
unique in its position because the corresponding mutants of other
regulatory GTPases also exhibit GTP/GDP binding affinity that is not
significantly different from those of the wild
types(26, 27, 28, 30) .
In
contrast to GTP/GDP binding, the GTPase activity of H-Ras was greatly
attenuated by the G60A mutation. The reduction in GTP hydrolysis was
most dramatic when the rates of GAP- and NF1-stimulated GTP hydrolysis
were compared ( Table 2and Fig. 2-3). NF1 had no
measurable activity toward the G60A mutant under our assay condition.
Although GAP was still able to stimulate GTP hydrolysis on the G60A
mutant, the reduction was so great that it was equivalent to a total
elimination of GAP activity. These results suggest that proper
positioning of the switch II region is required for the GTP hydrolysis,
particularly the factor-stimulated GTP hydrolysis. The reduction of
GTPase activity by the G60A mutation was in sharp contrast to that of
the corresponding mutation in G or EF-Tu. In
G
(G226A), the intrinsic GTPase was unchanged as long
as enough Mg
was provided to support a stable binding
of GTP to the protein(27) . In EF-Tu, the G83A mutation
actually enhanced the intrinsic GTPase by about 10-fold(30) .
These differences in GTPase activities indicate that different members
of regulatory GTPase employ different mechanisms for hydrolyzing GTP.
Another example of the possible nonconserved mechanism of GTP
hydrolysis is that Rad (Ras-related protein associated with diabetes)
has similar intrinsic GTPase activity regardless of whether the residue
in question is a Gly or a Glu(44) .
Despite the reduction in GAP- and NF1-stimulated GTP hydrolysis, the actual physical interaction between H-Ras and GAP (or NF1) is not affected by the G60A mutation ( Table 2and Fig. 4). In fact, the affinity for GAP and NF1 was slightly elevated by the G60A mutation. This situation is not uncommon because many mutations in the loop 4 region are known to heighten the affinity for binding GAP(36, 39) . Furthermore, the interaction of the G60A mutant with GAP and NF1 still displays the GTP-dependent characteristic. This result indicates that not all aspects of GTP-induced conformational change is abolished by the G60A mutation. Because the G60A mutation perturbs only the switch II region, it is also indicates that the binding of H-Ras to GAP or NF1 may not involve switch II region. However, without the involvement of the switch II region, GAP- or NF1-stimulated GTPase cannot proceed. This conclusion is further supported by the findings of another switch II domain mutation Y64W, which binds but has no GAP-stimulated GTPase(45, 46) . Intriguingly, H-Ras harboring a Gly substitution at the same Tyr-64 residue produces a protein that fails to associate NF1(47) . At present, we do not have an explanation for this discrepancy.
The G60A mutant was found to
moderately reduce the interaction of H-Ras and the Raf-N275 fragment (Table 3-4). Similar results were obtained with Raf-1 fragment
containing the first 150 residues (data not shown). This result is
expected because the interaction between H-Ras and the Raf-1 has been
shown to involve the switch I domain of H-Ras and Raf-1 RBD, a fragment
containing Raf-1 residues
50-130(43, 48, 49, 50, 51, 52, 53, 54) .
Similarly, the switch I domain of H-Ras also appears to be essential
for binding the ralGDS RBD(6, 7) . However, unlike the
Raf-1 RBD, the interaction of the ralGDS RBD with H-Ras is severely
reduced by the G60A mutation (Table 5-6). This finding supports
the importance of the switch II domain in the interaction with ralGDS
RBD. Therefore, it appears that tight binding of H-Ras to the ralGDS
RBD requires the cooperation of both switch I and II domains, whereas
the interaction of H-Ras with the Raf-1 RBD may need only the switch I
domain. These results indicate that although GAP, NF1, Raf-1, and
ralGDS all interact with H-RasGTP and they are able to compete
against each other for binding to H-Ras, the binding sites of these
factors on H-Ras are not identical. A similar conclusion has also been
reached by a recent study using switch II domain mutations at positions
64, 65, and 71(47) . Because the role of the ralGDS in the Ras
signaling pathway has not been established, it is not known whether the
reduction in ralGDS binding by the G60A mutation is the basis for its
lack of transforming activity associated with the G60A mutant. The
ability to differentiate different downstream effector is not a
property solely associated with the switch II domains, the switch I
domain mutation Glu-37 to Gly substitution is also able to
differentiate different downstream effectors, such as Raf-1 and Byr2, a
structural homolog to mammalian MEK kinase(55, 56) .
Several lines of evidence suggest that Raf-1 is the cellular
effector of Ras p21(5, 13) . The function of Ras is to
recruit Raf-1 to the cell membrane, and such process by itself appears
to be sufficient for the activation of Raf-1 and subsequent
events(57, 58) . However, the properties of the G60A
mutant are not consistent with that view; the G60A mutant is capable of
binding Raf-1 to a significant level, but it lacks transforming
activity(29) . These observations imply that the contribution
of H-Ras to the activation of Raf-1 may be more than membrane
recruitment. Recently, a second Raf-1 RBD (residues 130-196),
which is capable of independent binding to H-Ras, was
identified(59) . The binding affinity for the second Raf-1 RBD
is weaker than that of the first RBD (residues 50-128) and can be
masked by the first Raf-1 RBD when combined. In contrast to the first
Raf-1 RBD, the binding of the second Raf-1 RBD to H-Ras was abolished
by the G60A and Y64W mutations(60) . This finding offers an
interesting possibility that the activation of Raf-1 by Ras may require
at least two distinct steps: an initial interaction between switch I
and the first Raf-1 RBD and a subsequent interaction between switch II
and the second Raf-1 RBD. The reason for the failure of the G60A mutant
to exhibit biological activities may be due to its inability to
complete the second step. The situation may be similar to when the G60A
mutation uncouples the GAP and NF1 catalytic activity from their
binding to H-Ras. Consistent with the two-step hypothesis of Raf-1
activation, we have found that the G60A mutant can sequester and
inhibit Raf-1 activity. ()With its ability to differentiate
the switch I and II domains of H-Ras and possibly to block the progress
of target activation, the G60A mutant will provide a valuable tool for
elucidating the interaction and activation of H-Ras downstream
effectors.