From the
We examined c-Ha-Ras harboring an aspartate to asparagine
substitution at position 119 (mutation D119N). The Asp-119 is part of
the conserved NK XD motif shared by members of the regulatory
GTPase family. This asparagine residue has been proposed to participate
in direct bonding to the guanine ring and to determine the
guanine-nucleotide binding specificity. The D119N mutation was found to
alter nucleotide specificity of Ha-Ras from guanine to xanthine, an
observation that directly supports the essential role of hydrogen
bonding between the side chain of the aspartic acid residue and the
guanine ring in nucleotide binding specificity. Besides nucleotide
binding specificity, the D119N mutation has little or no effect on the
interaction of Ha-Ras with SDC25C, SOS1, GAP, or Raf. Neither does it
affect the hydrolysis of nucleotide triphosphate. Like
xanthine-nucleotide-specific EF-Tu, xanthine-nucleotide-specific Ras
and related proteins will be useful tools for elucidating cellular
systems containing multiple regulatory GTPases.
The Ha-Ras is a member of the regulatory GTPase family
(1, 2) . Regulatory GTPases bind guanine-nucleotide and
function as molecular switches in regulating cellular processes. The
activity of a regulatory GTPase is dependent on the phosphorylation
state of the bound guanine-nucleotide. GTP binding activates the
regulatory GTPase, promotes effector interaction, and elicits
subsequent biological effects. This interaction leads to the hydrolysis
of the
Despite functional diversity,
regulatory GTPases share distinct patterns of sequence homologies
(11) . Three most prominent conserved motifs are
G XXXXGK(S/T), D XXG and NK XD. These consensus
sequences lie in the vicinity of the bound guanine-nucleotide and form
direct contacts either with the moieties of guanine-nucleotide or with
the essential cofactor magnesium ion
(12, 13, 14, 15, 16, 17, 18, 19) .
It is likely that the conserved sequences serve as the foundation for
the conserved mechanistic functions of regulatory GTPase, such as
guanine-nucleotide binding, conformational changes, GTP hydrolysis, and
nucleotide exchange
(1, 2) .
Aspartate 119 of Ha-Ras
is part of the conserved motif NK XD, the domain interacting
with guanine base. The x-ray structures of regulatory GTPases have
proposed that the carboxylate side chain of the aspartic acid in the
NK XD domain interacts with the endocyclic N-1 and the
exocyclic 2-amino group through hydrogen bonding, and such interactions
provide the specificity for nucleotide binding
(12, 13, 14, 15, 16, 17, 18, 19) .
This hypothesis was confirmed in EF-Tu by the finding that the
EF-Tu(D138N) mutant preferentially binds and uses xanthine-nucleotide
for its activities including aminoacyl-tRNA binding and peptide
elongation in the ribosomes
(20, 21, 22) . The
observation of the EF-Tu(D138N) mutant activities suggests that the
interaction between the aspartic acid residue and guanine ring can be
fully substituted by the interaction of asparagine and xanthine ring, a
base containing a 2-carbonyl group. The nucleotide substrate
specificity of adenylosuccinate synthetase also appears to be achieved
through the same aspartic acid-guanine ring interaction. Similarly to
EF-Tu, the substitution of an aspartic acid to asparagine at position
333 of adenylosuccinate synthetase converts substrate specificity from
GTP to XTP
(23) . In this study, we examined the nucleotide
specificity of Ha-Ras harboring an Asp-119 to Asn substitution, the
corresponding mutation of EF-Tu(D138N). This mutant was isolated and
partially characterized earlier by Feig et al. (24) as
a GTP-binding deficient mutant.
The intact Ha-Ras(D119N) has proved to be very difficult to
purify by the nondenaturing protocol we routinely used
(27) .
The majority of the expressed Ha-Ras(D119N) is in the insoluble
fraction after cell disruption, and repeated chromatography of the
soluble fraction always resulted in small amounts of protein of
variable purity. This problem was largely eliminated by using
GST-fusion protein; therefore, GST-fusion proteins were used in this
study except when stated otherwise.
The SOS1-stimulated
nucleotide exchange was also examined Fig. 2). Similarly to
SDC25C, GST-SOS1 readily promotes nucleotide exchange to GST fusion Ras
proteins regardless of the nucleotide (Fig. 2). The nucleotide
exchange is not the outcome of the GST and GST interaction as GST by
itself has no detectable nucleotide exchange stimulating activity on
GST-Ras (data not shown). The time course of the wild type and GTP
exchange is nearly identical with that of the D119N mutant and XTP
exchange (Fig. 2). In fact, the steady state kinetics reveals
little difference between the wild type and the D119N mutant in the
SOS1-stimulated nucleotide exchange (). Again, similarly
to SDC25C, SOS1 does not differentiate between XTP and GTP.
Interestingly, although SOS1 and SDC25C appear to interact with Ras
with similar affinity, the rate of nucleotide exchange stimulated by
SOS1 was significantly lower than that stimulated by SDC25C
().
The x-ray structures of regulatory GTPases suggest that the
specificity of guanine-nucleotide binding is accomplished through the
interaction of the conserved NK XD domain with the guanine
ring, specifically, through the hydrogen bond between the carboxylate
side chain of the aspartic acid residue and the endocyclic N-1 as well
as the exocyclic 2-amino group of the guanine ring
(12, 13, 14, 15) . This hypothesis has
been confirmed in EF-Tu and adenylosuccinate synthetase by using an Asp
to Asn substitution mutation, which converts nucleotide specificity
from GTP to XTP
(20, 21, 22, 23) . In
Ha-Ras, the importance of the aspartic acid-guanine ring interaction to
the nucleotide binding specificity also has been implicated using an
Asp to Ala substitution mutant in which Ras(D119A) was found to greatly
affect the binding of guanine-nucleotide but not inosine-nucleotide
(34) . In this study, we examined the biochemical properties of
the D119N mutant, a mutant expected to utilize xanthine-nucleotide.
We first analyzed the binding of GTP and XTP to the wild type and
the D119N Ras. As expected from the x-ray model of Ha-Ras, the D119N
mutation drastically weakens GTP binding but greatly enhances XTP
binding (). The difference in binding strength is in the
neighborhood of 2 to 3 orders of magnitude, a difference equivalent to
about 3 to 4 kcal/mol. The nonbonding pair is predicated to have one
fewer hydrogen bond than the bonding pair, but the actual difference in
energy is much greater than one would anticipate from a single hydrogen
bond in aqueous solution. We cannot explain this discrepancy; however,
it is possible that the guanine ring binding pocket of Ha-Ras may
provide a hydrogen bonding environment which is very different from
that of aqueous solution. The GST-Ras(WT) appears to bind nucleotide
(either GTP or XTP) with greater strength than the D119N mutant binds
its counterpart. For example, there is an approximately 5-fold
difference between the wild type and the D119N mutant in binding to the
nonbonding nucleotide (). This phenomenon may be due to the
ability of the charged carboxylate group of aspartic acid to form
stronger bonds with the guanine ring than the uncharged carbonyl side
chain of asparagine in the D119N mutant. The nucleotide binding
strength is reflected in the intrinsic nucleotide exchange rates as the
nonbonding pairs, particularly GTP exchange of the D119N mutant, have
faster rates of intrinsic exchange than the bonding pairs
(Fig. 1).
Both SDC25C and SOS1 promote XTP exchange to the
D119N mutant to about the same extent that they stimulate GTP exchange
to the wild type Ras (Figs. 1 and 2); therefore, we conclude that the
D119N mutation does not affect the interaction of Ha-Ras with either
SDC25C or SOS1. This conclusion is in agreement with the findings of
the steady state kinetics determinations (). The D119N
mutant appears to have a higher SDC25C-stimulated exchange rate than
the wild type; however, a difference of about 25% may not be
significant. Another interesting finding of our study is that the
nucleotide specificity is strictly the property of Ha-Ras, and neither
SDC25C nor SOS1 distinguishes between XTP and GTP. Despite the fact
that GST-SOS1 has a similar affinity as SDC25C for Ras
The D119N mutant appears to have higher
affinity for binding SDC25C than the wild type Ras under equilibrium
conditions (Fig. 2); however, this phenomenon is likely to be due
to the reduced guanine-nucleotide binding affinity associated with the
D119N mutation. Nucleotide and SDC25C compete in binding to Ha-Ras; the
reduction in binding affinity for one would appear as an enhancement of
affinity for the other. Therefore, the ratio of nucleotide required for
complex dissociation () to the nucleotide dissociation
constant () is a better assessment of SDC25C binding
affinity. The XTP ratio for the D119N mutant is similar to the GTP
ratio for the wild type Ras. Thus, we conclude that the interaction
with SDC25C is not significantly altered by the D119N mutation, a
conclusion in agreement with the steady-state kinetics study.
D119N
mutant is capable of hydrolyzing XTP. In fact, the intrinsic XTP
hydrolysis rate of D119N is similar to the intrinsic GTP hydrolysis
rate of wild type Ras ( Fig. 4and ). GAP greatly
enhances the rate of GTP hydrolysis in the wild type Ras as well as
that of XTP hydrolysis in the D119N mutant, and the ratios of GAP
stimulation appear to be similar ( Fig. 3and ).
These observations suggest that the mechanism of GTP hydrolysis is not
affected by the D119N mutation. Nevertheless, we found that the GAP
stimulation ratio was a few fold lower than that reported by others
(35) . Furthermore, GAP-stimulated XTP (in D119N mutant) and GTP
hydrolysis (in the wild type) reactions have similar apparent
K
The D119N mutation also does not perturb the ability of Ras to
interact with Raf; however, the interaction is XTP-dependent in the
D119N mutant while it is GTP-dependent in the wild type (Fig. 5).
This observation, together with the finding of GAP interaction,
suggests that nucleotide triphosphate-induced conformational changes
are not affected by the D119N mutation. Similarly to SDC25C, SOS1, and
GAP, Raf does not differentiate between XTP and GTP.
Overall, our
results suggest that the primary role of the Asp-119 residue in Ha-Ras
is to determine nucleotide binding specificity. They also indicate that
Asp-119 is not directly involved in binding SDC25C, SOS1, GAP, or Raf.
Asp-119 also is not involved in nucleotide triphosphate hydrolysis or
nucleotide triphosphate-induced activities of Ha-Ras. Therefore,
Ras(D119N) is predicated to behave like the wild type Ras if sufficient
guanine-nucleotide or xanthine-nucleotide is provided. The reduction in
the guanine-nucleotide affinity by D119N mutation is in the
neighborhood of three orders of magnitude, a reduction that should be
readily overcome by the intracellular guanine-nucleotide. This explains
why Ha-Ras(D119N) still possesses transforming activity similar to that
of the wild type
(24) .
The xanthine-nucleotide binding
mutant of EF-Tu, D138N, has been used to probe the involvement of EF-Tu
in the elongation cycle of protein synthesis
(21, 22) .
The advantage of using such a mutant is that the action of EF-Tu in the
peptide elongation cycle can be monitored in the presence of EF-G (the
peptide translocation factor), a necessary factor for protein
biosynthesis, which also utilizes and hydrolyzes GTP
(36) . This
approach led to the intriguing finding that not one but two molecules
GTP are hydrolyzed by EF-Tu in each step of peptide elongation
(37) . Since the homologous mutations in EF-Tu
(20) ,
adenylosuccinate synthetase
(23) , and Ha-Ras produce similar
XTP binding properties, the conversion to XTP binding preference is
likely to be a general phenomenon for regulatory GTPases. There are
many cellular processes involving multiple regulatory GTPases. One
example is the vesicular transport system
(38) . We believe that
mutation to the XTP-specific mutant will be an invaluable tool for
elucidating the role of specific regulatory GTPases in these complex
systems.
The dissociation constants were
determined by the equilibrium binding method in the presence of EDTA as
described under ``Materials and Methods.''
The parameters were determined for the bonding pairs only. The
concentrations of exchange factor used were 20 nM SDC25C or
100 nM GST-SOS1. The concentration of NTP used in the assay
was 5 µM.
All experiments were performed
using 0.5 pmol of labeled intact Ras and 5 pmol of SDC25C. ND, not
determined.
The concentration of GAP used
in the GAP-stimulated assays was 0.2 nM.
We thank Drs. Frank McCormick and Gideon Bollag of
Onyx Pharmaceuticals for providing Ras GAP, Andrea Parmeggiani for
supplying SDC25C, Ulf R. Rapp for the human Raf clone, and David
Bowtell for the mouse SOS1 DNA. We also thank Drs. Carl Dobkin and
David Miller for critical reading of this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-phosphate and returns the regulatory GTPase to the
inactive GDP-bound state. The inactive regulatory GTPase can then be
reactivated by exchanging the bound GDP with an exogenous GTP
(1, 2) . Thus, the activity of regulatory GTPase is
controlled through the balance of GTP hydrolysis and guanine-nucleotide
exchange
(1, 2) . Factors that interact with and
regulate the activity of Ras p21 include a group of CDC25-related
proteins (such as SDC25C and SOS), which stimulate guanine-nucleotide
exchange reaction; GAP,
(
)
which promotes GTP
hydrolysis; and the putative effector Raf protein
(3, 4, 5, 6, 7, 8, 9) .
Recently, phosphatidylinositol 3-kinase has also been suggested as a
possible target of Ras
(10) .
Mutant Construction and Protein Purification
The
Ha-Ras(D119N) was constructed by oligonucleotide site-directed
mutagenesis as described by Kunkel and co-workers
(25) .
Single-stranded DNA from M13 phage clone containing the entire c-Ha-Ras
was used as the template for mutagenesis. The following
oligonucleotide, GGCCAGGTTACACTTGTT, was used for mutant construction.
The Ha-Ras(D119N) was subsequently constructed as a GST-fusion protein
using a modified pGEX3 vector
(26) . The Ha-Ras(D119N) was also
cloned into an Escherichia coli expression vector containing
the T7 RNA polymerase promoter for in vitro labeling as
described
(27) . The procedures for preparing GST fusion
proteins Ras and Raf were described elsewhere
(26) . SDC25C was
expressed from the vector pTTQ-SDC25
(28) and purified as
described by Créchet et al. (29) . Preparation of
[-
P]XTP-[
-
P]XTP
was synthesized according to a modified procedure of Glynn and Chappell
(30) . Briefly, reaction in 1 ml of mixture containing 50
mM Tris-HCl, pH 8.0, 3 mM MgCl
, 5
mM dithiothreitol, 0.5 mM XTP, 0.5 mM NAD,
0.5 mM 1,3-diphosphoglycerate, 50 mg/ml
glyceraldehyde-3-phosphate dehydrogenase, 5 mg/ml phosphoglycerate
kinase, and 500 µCi/ml phosphorus-32 (specific activity 285 Ci/mg
of phosphorus) was allowed to proceed at room temperature for 15 h. The
incorporation of
P into XTP was monitored by a charcoal
adsorption assay and is usually equal to 60-65% of total input
phosphorus-32. [
-
P]XTP was partially
purified from the reaction mixture using ion exchange resin
Q-Sepharose. The elution of phosphate and XTP was performed using 50
mM NaCl and 300 mM NaCl, respectively.
Preparation of GST-mSOS Fusion Protein
The
GST-fusion protein containing residues 539 to 1046 of mouse SOS1
catalytic domain was constructed similarly as described by Liu et
al. (31) . The mouse SOS1 catalytic domain DNA was
generated from the full-length cDNA clone by polymerase chain reaction
using the following oligonucleotides, CCTTTATCGATGTCAGCTGAAGAGAAAAACAAC
and GGAACTCGAGTCATGGATTTGATGGACGAACCC, respectively, as the 5` and the
3` primer. The 5` primer contains a ClaI site, and the 3`
primer contains a XhoI site. In addition, a stop codon
in-frame with the SOS1 coding sequence was included in the 3` primer.
The resulting 1550-base pair polymerase chain reaction product was
digested with ClaI and XhoI and cloned into the
unique ClaI- XhoI site of the modified pGEX3 vector
(26) to yield the GST fusion clone. The procedures for
expression and purification of GST-SOS1 were as described
(26) .
Nucleotide Binding and Exchange Assay
The GTP and
XTP dissociation constants were determined by equilibrium binding and
calculated from the Scatchard plot as described
(32) . The
equilibrium binding was performed in a reaction mixture containing 50
mM Tris-HCl, pH 8.0, 2 mM MgCl, 50
mM KCl, 1 mM dithiothreitol, 1 µM (100
pmol) GST-Ras protein, 4 mM EDTA, and varying concentrations
of [
H]GTP (specific activity 1500 cpm/pmol) or
[
-
P]XTP (specific activity 1400 cpm/pmol)
for 10 min at 30 °C. The concentrations of nucleotide triphosphate
used were in the range of 10
to 10
M for the bonding pairs and 10
to
10
M for the nonbonding pairs. The time
course SDC25C- and SOS1-stimulated guanine-nucleotide exchange reaction
was performed using 1 µM (200 pmol) GST-Ras protein, 60
nM SDC25C or 300 nM GST-SOS1, and 5 µM
[
H]GTP (specific activity 1500 cpm/pmol) or
[
P]XTP (specific activity 1400 cpm/pmol) as
described
(27) . The kinetic parameters of SDC25C- and
SOS1-stimulated reactions were determined in 100 µl of reaction
mixture using 0.25, 05, 1, and 2 µM substrate and either
20 nM SDC25C or 100 nM GST-SOS1 as described
previously
(27) .
In Vitro Ha-Ras p21 Labeling and SDC25C Binding
Assay
The intact wild type and D119N Ha-Ras were labeled in
vitro with [S]methionine as described
(27) . The Ha-Ras
DC25C complex formation and dissociation
were performed in a Pharmacia Superdex 75 HR 10/30 column attached to a
high performance liquid chromatography column as described
(27) .
Measurement of Nucleotide Triphosphate
Hydrolysis
Nucleotide triphosphate hydrolysis was measured by
monitoring the release of the -phosphate group from
[
-
P]GTP or
[
-
P]XTP as described below. GST-Ras(WT) and
GST-Ras(D119N) were labeled in the presence of EDTA to equilibrium with
[
-
P]GTP and
[
-
P]XTP, respectively. The
[Mg
] in the reaction mixture was brought up
to 10 mM, and then the free nucleotide in the reaction mixture
was removed by passing through a G-25 column. The column was eluted
with a buffer containing 20 mM Na-HEPES, pH 7.5, 2 mM
MgCl
. GTPase reaction was performed in 200 µl of
reaction mixture containing 20 mM Na-HEPES, pH 7.5, 2
mM MgCl
, 0.125 µM labeled GST-Ras.
The reaction was initiated by the addition of the full-length human GAP
(final concentration 0.4 nM) and incubated at 30 °C. At
the indicated time interval, an aliquot of 25 µl was withdrawn, and
the amounts of free phosphate were measured as described
(33) and used to calculate nucleotide hydrolysis. The apparent
K
and k
were
determined in two sets of reactions which contained four different
substrate concentrations (0.125, 025, 0.4, and 0.5 µM
concentrations of either
GST-Ras
[
-
P]GTP or
GST-Ras(D119N)
[
-
P]XTP) in 200 µl
of HEPES-Mg
buffer. One set contained only substrate
and the other contained substrate and GAP. The reactions were initiated
by adding GAP (final concentration 0.2 nM) and allowed to
proceed for 5 min at 30 °C. The extent of GTP hydrolysis was
determined as described above and used for calculating kinetic
parameters. The intrinsic k
of nucleotide
triphosphate hydrolysis was determined similarly as described above
using 0.25 µM GST-fusion protein and 2 to 20
µM nucleotide triphosphate.
Raf and Ras Binding
S-labeled Ha-Ras
p21 was charged with nucleotide triphosphate (GTP or XTP) or
diphosphate (GDP or XDP) at 30 °C to equilibrium in 35 µl of
binding solution containing 25 mM Tris, pH 7.5, 2 mM
MgCl
, 100 mM NH
Cl, 0.2 mg/ml bovine
serum albumin, 20 µM nucleotide, and 5 mM EDTA
for 5 min. The reaction was terminated by the addition of 20
mM MgCl
. Complex formation between Ras and Raf was
determined by mixing the GTP- or XTP-equilibrated
S-labeled Ras (5.6 pmol) with 2.8 nmol of GST-Raf fusion
protein in a 200 µl of Raf binding buffer composed of 50
mM Na-HEPES, pH 7.5, 100 mM KCl, 20 µM
ZnCl
, 5 mM MgCl
, 1 mg/ml bovine serum
albumin. After incubation on ice for 1 h, 20 µl of
glutathione-Sepharose beads (12.5% gel slurry) were added to the
reaction mixture, and the incubation was continued with gentle shaking
for another hour at 4 °C. The free Ras and GST-Raf were removed by
washing the beads 5 times with 1 ml of ice-cold washing buffer (50
mM Na-HEPES, pH 7.5, 100 mM KCl, 5 mM
MgCl
, 20 µM ZnCl
, and 0.1% Triton
X-100). Ras
GST-Raf complexes were eluted from the washed beads by
resuspension in 30 µl of polyacrylamide gel electrophoresis loading
buffer. The coprecipitated
S-labeled Ras protein was
identified by autoradiography following electrophoresis on a 10%
polyacrylamide gel. The amounts of coprecipitated Ras was determined in
a liquid scintillation counter and used to calculate the ratio of Ras
coprecipitated by Raf. The number of coprecipitated Ras represented the
average of at least two trials.
GTP and XTP Binding
The dissociation constants of the
GST-fusion Ras proteins for GTP and XTP were determined by the
equilibrium binding method (). The
Kfor GST-Ras(WT) was
calculated to be 1.6
10
M. This
value is similar to that of the intact protein
(24, 32) , a result indicating that the introduction of
the GST moiety does not perturb nucleotide binding. As expected, the
affinity for GTP is drastically reduced by the D119N mutation. The
K
is 1.9
10
M for the D119N mutant, which is about 3
orders of magnitude weaker than that of the wild type. This difference
in the GTP affinity reported here is about 10-fold greater than that
observed by others
(24) . On the other hand, the protein's
affinity for XTP was significantly enhanced by the D119N mutation. The
K
is 4.1
10
M for the wild type Ras and 3.9
10
M for the D119N mutant, an enhancement
in XTP affinity of about 2 orders of magnitude.
Kinetics of Nucleotide Exchange
The intrinsic and
SDC25C-stimulated nucleotide exchange was measured. As shown in
Fig. 1
, regardless of the nucleotide and Ras species, SDC25C
readily stimulates the nucleotide exchange to reach equilibrium. This
observation shows that SDC25C interaction is not disrupted by the D119N
mutation and SDC25C does not distinguish XTP from GTP. However, the
level of nucleotide binding at equilibrium for the bonding pairs (GTP
with the wild type and XTP with D119N mutant) is much higher than that
of the nonbonding pairs. Under the assay condition (5 µM
nucleotide), about 8% GST-Ras(D119N) binds GTP and about 30%
GST-Ras(WT) binds XTP at equilibrium while the binding was nearly
completed for both the bonding pairs. The level of nucleotide binding
reflects protein's affinity for nucleotide, since the
concentration of nucleotide in the assay is not sufficient to support
the saturation binding for the nonbonding pairs. The higher
dissociation rate with nonbonding nucleotide also results in a higher
intrinsic exchange rate, particularly, GTP exchange of the D119N mutant
(Fig. 1, inset).
Figure 1:
The intrinsic and SDC25C-stimulated
nucleotide exchange. A, GTP exchange; B, XTP
exchange. The experiments were performed using 1 µM
GST-Ras protein, 60 nM SDC25C, and 5 µM
[H]GTP or [
P]XTP as
described (27). The inset in A is an expansion of the
region corresponding to lower y axis value of A.
Symbols used in the plots are GST-Ras(WT) alone (
),
GST-Ras(WT) with SDC25C (
), GST-Ras(D119N) alone (
), and
GST-Ras(D119N) with SDC25C (
).
The kinetic parameters for the
SDC25C-stimulated reaction were subsequently determined for the bonding
pairs (). The apparent Kof
the SDC25C-stimulated reaction was 1.8 µM for the wild
type and 1.6 µM for the D119N mutant (). This
result supports the conclusion that the interaction of Ras with SDC25C
was not altered by the D119N mutation. However, the D119N mutant
displays a SDC25C-stimulated exchange rate which is about 25% faster
than that of the wild type ().
Figure 2:
The time course of SOS1-stimulated
nucleotide exchange. The experiments were performed on the bonding
pairs (GTP exchange for the wild type and XTP exchange for the D119N
mutant) using 1 µM GST-Ras protein, 300 nM
GST-SOS1, and a 5 µM concentration of either
[H]GTP or [
P]XTP as
described (27).
, GST-Ras(WT) alone;
, GST-Ras(WT) with
SOS1;
, GST-Ras(D119N) alone; and (
), GST-Ras(D119N) with
SOS1.
SDC25C Binding
The SDC25C-stimulated XTP exchange
suggests that the D119N mutant is able to form a complex with SDC25C
similar to that of the wild type
(27) . Thus, we examined the
ability of the intact Ras(D119N) to interact with SDC25C and
nucleotide. As with the wild type Ras, the addition of SDC25C to
Ras(D119N) converts Ras(D119N) into the Ras(D119N)SDC25C complex
which can be distinguished from the uncomplexed Ras in a gel filtration
column (Fig. 3). However, Ras(D119N) appears to bind SDC25C more
efficiently than the wild type Ras because a 10-fold molar excess of
SDC25C causes quantitative conversion of Ras(D119N) to the complex form
but not Ras(WT) (Fig. 3). The lower yield of complex formation
with wild type Ras has been attributed to the presence of
guanine-nucleotide in the translation mixture as well as to the bound
guanine-nucleotide; therefore, a reduction in guanine-nucleotide
affinity such as the D119N mutation, will improve the efficiency of
Ras
SDC25C complex formation
(27) . We subsequently measured
the nucleotide concentration which promotes complete complex
dissociation (I). The concentration of nucleotide for
dissociating the Ras(WT)
SDC25C complex was 0.6 µM for
GDP, 0.2 µM for GTP, or 10 µM for XDP. In
contrast, it requires 25 µM GDP, 0.5 µM XTP,
or 0.8 µM XDP to dissociate the Ras(D119N)
SDC25C
complex. These results show that the amounts of nucleotide required for
complex dissociation parallels the protein's affinity for each
nucleotide.
Figure 3:
The
interaction of the wild type Ras and D119N with SDC25C and nucleotide.
The complex formation and dissociation was performed using
S-labeled intact Ras in a gel filtration column as
described previously (26). A, the wild type with SDC25C and
GTP; B, D119N mutant with SDC25C and XTP. Symbols used for both plots are:
, 0.5 pmol of labeled Ras alone;
, labeled Ras with 5 pmol of SDC25C;
, labeled Ras with 5
pmol of SDC25C in the presence of 0.5 µM GTP ( A)
or XTP ( B).
Nucleotide Triphosphate Hydrolysis and GAP
Interaction
In the normal cycle of its function, Ras utilizes
and hydrolyzes GTP; therefore, we analyzed XTP hydrolysis of the D119N
mutant. GST-fusion Ras proteins were used in this study. GST-Ras(WT)
exhibits a slow intrinsic rate of GTP hydrolysis which can be greatly
enhanced by the addition of GAP (Fig. 4 A). Apparently,
GTP hydrolysis is not affected by the presence of the GST moiety.
Similarly, GST-Ras(D119N) also exhibits a low rate of XTP hydrolysis
which can be accelerated by the addition of GAP
(Fig. 4 B). The kvalues of
GAP-stimulated nucleotide triphosphate hydrolysis at 0.2 nM
GAP were estimated to be 6.45 and 5.08 s
for
GST-Ras(WT) and GST-Ras(D119N), respectively (). This
represents an approximately 5
10
-fold stimulation
ratio for both GST-Ras(WT) and GST-Ras(D119N). The
K
values of GAP-stimulated hydrolysis are
also similar for both wild type and D119N mutant (). These
observations support the conclusion that the D119N mutation dose not
affect the mechanism of nucleotide triphosphate hydrolysis of Ha-Ras as
well as its interaction with GAP.
Figure 4:
The intrinsic and GAP-stimulated
hydrolysis of nucleotide triphosphate. The experiments were performed
using GST-fusion Ras and the full length human GAP as described under
``Materials and Methods.'' A, GTP hydrolysis of
GST-Ras(WT); B, XTP hydrolysis of GST-Ras(D119N). ,
GST-Ras(WT) without GAP;
, GST-Ras(WT) with GAP;
,
GST-Ras(D119N) without GAP; and
, GST-Ras(D119N) with
GAP.
Raf Binding
Ras was found to interact with Raf in
a GTP-dependent manner
(4, 5, 6, 7, 8, 9) .
Subsequently, we examined XTP-dependent Raf binding activity of D119N
mutant. The intact forms of wild type and D119N mutant were labeled
in vitro with [S]methionine, charged
with nucleotide, reacted with GST-Raf, and then precipitated by
glutathione-Sepharose resin as described under ``Materials and
Methods.'' As expected, GTP induces the interaction of Ras(WT)
with GST-Raf (Fig. 5). Similar to the effect of GTP on Ras(WT),
XTP promotes the interaction of Ras(D119N) with GST-Raf. The amounts of
coprecipitated Ras(D119N) (9.6%) were close to that of Ras(WT) (10.7%),
an observation suggesting that the interaction of Ras with Raf is not
altered by D119N mutation. Compared to nucleotide triphosphates,
nucleotide diphosphates were much less effective for promoting the Ras
and Raf interaction (Fig. 5). The nonbonding nucleotide
triphosphate is also able to promote substantial Raf binding if
sufficient amounts of that are provided. For example, XTP at 0.5
mM produces 8.5% coprecipitated Ras(WT); while GTP at 0.5
mM yields 7.2% coprecipitated Ras(D119N).
Figure 5:
Nucleotide triphosphate-dependent Raf
interaction. The Raf interaction was analyzed using a coprecipitation
assay consisting of S-labeled intact Ras and GST-fusion
Raf N-terminal domain as described (26). The nucleotide concentration
used in the assay was 20 µM. Lane 1, wild type
Ras precharged with GDP; lane 2, wild type Ras precharged with
GTP; lane 3, D119N mutant precharged with XDP; lane
4, D119N mutant precharged with XTP.
NTP, it
exhibits much lower activity in stimulating nucleotide exchange than
SDC25C (). This lower stimulating activity is likely to be
the inherent property of GST-SOS1 since it also has a lower activity
than SDC25C in stimulating guanine-nucleotide exchange onto the intact
Ha-Ras (data not shown).
values, a result indicating that the
interaction of Ha-Ras and GAP is not altered by the D119N mutation. As
with SDC25C and SOS1, we found that GAP does not differentiate XTP from
GTP.
Table:
The apparent GTP/XTP dissociation
constant of GST-fusion Ras
Table:
The apparent Kand
k
of SDC25C- and SOS1-stimulated nucleotide exchange
Table:
Nucleotide concentration for promoting
RasSDC25C complex dissociation
Table:
Kinetic parameters of intrinsic and
GAP-stimulated nucleotide hydrolysis
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.