(Received for publication, April 4, 1995; and in revised form, August 25, 1995)
From the
The effector binding domain and the switch II region of c-Ha-Ras
are necessary for p120-stimulated GTP hydrolysis. We
report a third region of c-Ha-Ras located within the
3 helix
(amino acids 101-103) which is also required for efficient
p120
, but not neurofibromin-mediated hydrolysis. This
highly conserved region of the Ras protein was investigated using an
insertion-deletion mutant (Ras-100LIR104) originally characterized by
Willumsen et al. (Willumsen, B. M., Adari, H., Zhang, K.,
Papageorge, A. G., Stone, J. C., McCormick, F., and Lowy, D. R(1989) in The Guanine Nucleotide Binding Proteins; Common Structural and
Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A.,
eds) pp. 165-178, Plenum Press, New York). The 100LIR104
substitution did not alter the intrinsic hydrolytic rate of the
protein. The p120
-stimulated hydrolysis of Ras-100LIR104,
however, was decreased by 2-3-fold compared to wild type Ras.
This decrease in p120
-stimulated hydrolysis was not due
to its inability to physically associate with Ras-100LIR104
GTP
(as determined by competitive binding assays). Surprisingly,
neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation.
Finally, no differences were observed in the ability of either the
p120
catalytic domain or the neurofibromin GRD to
accelerate Ras-100LIR104 GTPase activity, indicating that the
amino-terminal noncatalytic GAP region is critical for
p120
-stimulated GTP hydrolysis. This is the first report
of a Ras mutation which differentiates between p120
and
neurofibromin activity.
Ras proteins, which function in signal transduction pathways
critical for cell growth and differentiation, are guanine nucleotide
binding proteins with a M of approximately 21,000.
The membrane-associated Ras protein cycles between an inactive
GDP-bound and active GTP-bound state. The activity of Ras is negatively
regulated by the hydrolysis of bound GTP, and positively regulated by
specific guanine nucleotide exchange factors which facilitate the
replacement of bound GDP by GTP (Lowy and Willumsen, 1993). Growth
factor stimulation of quiescent cells increases the proportion of
Ras
GTP, resulting in the initiation of a cascade of intracellular
protein kinases (Satoh et al., 1990; Zhang et al.,
1992).
GTPase activating proteins negatively regulate Ras activity
by accelerating the hydrolysis of bound GTP. Two such proteins have
been extensively characterized, p120 and neurofibromin
(Lowy and Willumsen, 1993). The region of p120
which is
responsible for stimulating GTP hydrolysis is located in the
carboxyl-terminal third of the protein and is termed the p120
catalytic domain. The second GTPase activating protein for Ras,
neurofibromin, is the product of the neurofibromatosis type-1 gene and
has a M
of 250,000. Neurofibromin contains a
350-amino acid stretch located in the central portion of the protein
which contains extensive sequence homology to the GAP (
)catalytic domain and is therefore termed the GAP related
domain (GRD; Xu et al., 1990; Martin et al., 1990).
Both the GAP catalytic domain and the neurofibromin GRD are highly
active in accelerating the GTP hydrolysis by Ras.
Mutations in
multiple regions of the Ras protein effect its interactions with
p120 and neurofibromin. Mutations in the Ras effector
binding domain (amino acids 32-40) result in the inability of
p120
to enhance the intrinsic GTPase activity (Adari et al., 1988; Cales et al., 1988). Although not all
mutations in this region abolish GAP activity, the effector region is
thought to be essential for p120
binding. In addition,
analysis of Ras/Rap1A chimeras shows that deletion of residues
61-65 also renders Ras insensitive to p120
stimulation (Zhang et al., 1991; Maruta et al.,
1991). Many Ras mutations that are resistant to GAP stimulation have
also been found to be resistant to stimulation by neurofibromin-GRD.
Thus the effector binding domain (amino acids 32-40) and switch
II region (amino acids 61-65) of Ras are thought to be the sites
required for interaction with GTPase activating proteins. Recently a
third GTPase activating protein has been identified. Its full
characterization has not been reported (Maekawa et al., 1994).
In this paper we identify an additional region within the Ras
protein which is important for p120-stimulated
hydrolysis. This region of the Ras protein, consisting of amino acids
101-103, is highly conserved among a number of small GTP binding
proteins (Santos and Nebreda, 1989). The functional significance of
this highly conserved region of the Ras protein was investigated using
an insertion-deletion mutant originally characterized by Willumsen et al.(1989). This mutation, in which the amino acids KRV were
substituted for LIR at position 101-103 was designated
Ras-100LIR104. The intrinsic hydrolytic activity of Ras-100LIR104 was
unaltered compared to wild type Ras. The p120
-stimulated
hydrolysis by Ras-100LIR104, however, was reduced 2-3-fold, while
neurofibromin hydrolytic activity remained unaffected. No differences
were observed in the ability of either the p120
catalytic
domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase
activity, indicating that regions outside of the catalytic GAP domain
are involved in protein-protein interactions with Ras necessary for
efficient stimulation of GTP hydrolysis.
Figure 1:
Intrinsic rate of GTP hydrolysis by
Ras and Ras-100LIR104. Ras or Ras-100LIR104 were allowed to associate
with [-
P]GTP and then incubated at 30
°C for the indicated lengths of time. The extent of hydrolysis was
calculated after incubation by determining the amount of label
remaining associated with Ras protein in a filter binding assay. The %
hydrolysis was determined as the ratio of binding at zero time to the
reduction in the binding after incubation. In this and each of the
experiments described, care was taken to ensure that the extent of GTP
hydrolysis within a given experiment was between approximately 20 and
80% hydrolysis so that the extent of hydrolysis could be roughly
equated to the amount of GTPase activation.
Figure 2:
Hydrolysis by immunoprecipitated GAP and
neurofibromin (NF). Neurofibromin and p120 were
separately immunoprecipitated from rabbit brain lysate and bound to
Protein A-Sepharose. The indicated volumes of this immobilized
immunoprecipitate were incubated for 40 min at 30 °C with Ras or
Ras-100LIR104 bound to [
-
P]GTP. The GTPase
filter binding assay was then used to determine hydrolysis (see Fig. 1). Data is representative of three experiments: (a) immunoprecipitated neurofibromin; and (b)
immunoprecipitated p120
. The data presented were obtained
in a single experiment, while three separate experiments were analyzed
together statistically as described under ``Experimental
Procedures'' yielding the following results: for
p120
, n = 9, p = 0.0012,
ratio is 1.8-2.6; and for neurofibromin, n = 12, p = 0.5, ratio is
0.9-1.1.
These results were
repeated multiple times and the data collectively analyzed using the
methods described under ``Experimental Procedures.'' The
probability that neurofibromin stimulated the GTPase activity of wild
type Ras and mutant Ras to different extents was not significant (p = 0.5); while the probability that p120 stimulated wild type Ras and mutant Ras differently was highly
significant (p = 0.0012).
In order to confirm the
above results, neurofibromin and p120 proteins were
separated from each other utilizing an entirely different approach.
Neurofibromin was affinity purified by passage of rabbit brain lysate
over a Protein A-Sepharose column bound to neurofibromin-specific
antibodies. The neurofibromin-antibody complex was then released from
the Protein A-Sepharose. The neurofibromin thus purified appeared as a
single band on a silver-stained gel (together with the immunoglobulin,
data not shown). The p120
was prepared from a recombinant
baculovirus expression system (from R. Jove: Park et al.,
1992). Lysates of baculovirus expressing cells were used as a source of
p120
. These lysates had no endogenous GTPase activating
activity in the presence of Ras unless the p120
protein
had been expressed.
With a GTPase filter binding assay, the
affinity-purified neurofibromin protein was found to stimulate
Ras-100LIR104 hydrolysis to the same extent as with wild type Ras (Fig. 3a). In contrast, the ability of baculovirus
p120 to stimulate the intrinsic hydrolysis of
Ras-100LIR104 was reduced by 2-3-fold compared to wild type
protein (Fig. 3b). These conclusions are highly
significant when all results are statistically analyzed (see figure
legend for statistical data). These results were similar to the
differences in activity observed using p120
and
neurofibromin immunoprecipitated from rabbit brain lysate. It is also
important to note that the GTPase filter binding assays discussed above
were also performed at Ras or 100LIR104 Ras concentrations of 12
µM, the concentration of Ras required to bind 50% of
p120
molecules. At this Ras concentration the same
decrease in baculovirus p120
-stimulated hydrolysis of
100LIR104 Ras compared to wild type Ras was observed (data not shown),
indicating that the differences seen are not a result of low Ras
concentrations. In summary, the results obtained using affinity
purified neurofibromin and baculovirus p120
accurately
reflect those observed in the previous determination, and indicate that
only p120
has reduced activity with the mutant Ras
protein.
Figure 3:
Hydrolysis by separately purified GTPase
activating proteins. a, neurofibromin (NF1) was
affinity purified from rabbit brain lysate and the indicated volumes of
purified protein were incubated with RasGTP or
Ras-100LIR104
GTP for 30 min at 30 °C. b, the
indicated volumes of baculovirus p120
cell lysate were
incubated with Ras
GTP or Ras-100LIR104
GTP for 30 min at 30
°C. Reaction mixtures were then analyzed using a GTPase filter
binding assay. (Statistical analysis of data from two separate
experiments yielded for baculovirus GAP, n = 14, p = 0.0001, ratio 3.2-4.6; and for purified
neurofibromin, n = 9, p = 0.1, ratio
0.8-1.0.)
A final experiment was performed to confirm that
p120 (and not neurofibromin) has reduced ability to
stimulate the GTPase activity of Ras-100LIR104. The detergent n-dodecylmaltoside has been shown to specifically inactivate
neurofibromin in brain lysates (Bollag and McCormick, 1991). When a rat
brain lysate was treated with maltoside to neutralize the neurofibromin
activity, a 2-3-fold decrease in its ability to stimulate GTPase
was evident with the Ras-100LIR104 mutant compared to wild type Ras,
similar to results obtained above. In untreated brain lysates on the
other hand, where a preponderance of the GTPase activating activity is
due to neurofibromin, the decrease in GTPase stimulation of
Ras-100LIR104 was not observed (data not shown; see
``Discussion'' for details).
Because of the difficulty in
obtaining purified neurofibromin or p120, together with
the loss of activity of these two reagents experienced during
purification, we elected to rapidly separate the two activities from
each other using three entirely separate approaches. With each of these
approaches almost identical results were obtained indicating that the
101-103 region is required for efficient p120
stimulated hydrolytic activity of Ras; but not for
neurofibromin-stimulated hydrolysis. While the means of separating
p120
from neurofibromin differed in each experimental
approach, the similarity in results obtained with each, together with
the high degree of reproducibility of the data, strongly argue in favor
of the above conclusion.
Figure 4:
GTPase activation by catalytic domains. a, the indicated volumes of purified neurofibromin GRD were
incubated with RasGTP or Ras-100LIR104
GTP for 30 min at 30
°C. b, the indicated volumes of purified p120
catalytic domain were incubated with Ras
GTP or
Ras-100LIR104
GTP for 30 min at 30 °C. Reaction mixtures were
then analyzed using a GTPase filter binding assay. (Statistical
analysis of data from three separate experiments yielded for GAP
catalytic domain, n = 13, p = 0.50,
ratio 1.0-1.1; while for the GRD of neurofibromin, n = 15, p = 0.5, ratio
0.8-1.0.)
Figure 5:
Determination of binding affinities.
Binding affinities were determined by a competitive inhibition
approach. Ras protein was allowed to bind
[-
P]GTP and the unbound label removed by
gel filtration. Then, 6 nM of the labeled Ras was incubated 4
min with enough (a) affinity purified neurofibromin or (b) baculovirus p120
to induce approximately 50%
hydrolysis. In these determinations the extent of hydrolysis was
determined by a charcoal binding assay (see ``Experimental
Procedures''). This hydrolysis was then assayed in the presence of
the indicated increasing concentrations of either Ras or Ras-100LIR104
which had been allowed to bind a nonhydrolyzable analogue of GTP. The
concentration of unlabeled competitor which results in a 50% decrease
in the extent of hydrolysis is the IC
. Data is
representative of three experiments.
These experiments rule out the possibility that the decrease
in p120 activity in the presence of Ras-100LIR104 results
from a decreased binding affinity. The decrease in GTPase stimulation
of Ras-100LIR104, therefore, is due to the inability of full-length
p120
to efficiently stimulate the hydrolysis of the
mutant protein once it has bound. Clearly, amino acids in the region of
101-103 of Ras are important for the interaction between
p120
and Ras proteins, an interaction which apparently is
important for full activity of p120
. It is interesting,
however, that this region is apparently not involved in the interaction
between Ras and neurofibromin. It is possible that the amino-terminal
regions of p120
(which are necessary to achieve efficient
GTPase activity, and which are totally distinct from any sequence in
neurofibromin) physically interact with the carboxyl terminus of the
3 region of Ras or another region of Ras altered by the
101-103 mutation.
Lastly, In order to address the biological
significance of the Ras-100LIR104 mutation, wild type Ras or the
Ras-100LIR104 mutant were microinjected into NIH3T3 cells. The cells
were subsequently labeled with [H]thymidine. The
results indicated that there was no difference in the ability of the
cell microinjected with Ras or Ras-100LIR104 to enter S phase (data not
shown). In the future, experiments designed to transfect different cell
types with Ras-100LIR104 may provide further insight regarding the
biological role of this mutant.
The amino acids KRV located in the 3 helix of Ras at
position 101-103 are highly conserved in Ras and several other
small GTP binding proteins (Santos and Nebreda, 1989). This conserved
region of the Ras protein was investigated using an insertion-deletion
mutant Ras-100LIR104. The 100LIR104 substitution does not alter the
intrinsic GTP binding or hydrolysis of the protein but does diminish
p120
-stimulated hydrolysis by 2-3-fold.
Neurofibromin hydrolytic activity, however, was not altered in
Ras-100LIR104 when compared to wild type Ras. This result was obtained
regardless of the means by which p120
and neurofibromin
were separated from each other. A biochemical purification of these two
GTPase activating proteins was not attempted due to the extensive
manipulations required and the consequent loss and potential alteration
of their activities. Three methods were therefore utilized to rapidly
separate the two proteins and assess their abilities to stimulate the
GTPase activity of mutant and wild type proteins. First, the two
proteins were separately immunoprecipitated and assayed directly.
Second, the neurofibromin was affinity purified on an antibody column
and the neurofibromin-immunoglobulin complex released and utilized in
comparison to a lysate of baculovirus expressed p120
.
Finally, crude brain lysate was assayed after the neurofibromin
activity had been specifically inhibited by a detergent.
In the
first instance the two proteins produced in mammalian cells were
isolated as immunoprecipitates. In the second approach the p120 was produced in an insect cell where no endogenous GTPase
activity has been observed (data not shown). In this case the protein
would be free of other mammalian proteins. In the third procedure the
p120
was assayed in the presence of other soluble
mammalian proteins, but with inactivated neurofibromin. The fact that
essentially identical results were obtained in repeated analyses of
each type clearly indicate that there is a distinction in the
interaction of these two GTPase activating proteins with the mutant
Ras. On the other hand, the catalytic domain of p120
and
the GRD of neurofibromin were found to stimulate the rate of GTP
hydrolysis of Ras and Ras-100LIR104 efficiently, and to the same
extent. It is therefore concluded that an interaction between the
amino-terminal, noncatalytic region of p120
and the
101-103 position of Ras must be essential for efficient GTPase
stimulation, while no such interactions are required for the activity
of neurofibromin. Alternatively, alterations in the structure of
distant regions of Ras resulting from the mutation might also play a
role.
In an attempt to address the biological significance of the
Ras-100LIR104 mutation, this protein was microinjected into NIH3T3
cells. We found, however, no difference in the ability of the cells
microinjected with Ras or Ras-100LIR104 to enter S phase (data not
shown). These results are, however, not unexpected when considering our
data obtained using rat brain lysate. When rat brain lysate was used as
a source of GAP (p120 and neurofibromin) activity, no
difference was observed between Ras and the Ras-100LIR104 mutant. It
was only the separation of p120
from neurofibromin which
resulted in the identification of a decrease in p120
activity in the presence of Ras-100LIR104. It is therefore
conceivable that microinjection of the Ras-100LIR104 mutant into cells
containing both p120
and neurofibromin, would not produce
different effects. Additional experiments in which the Ras-100LIR104
mutant is transfected into different cell types, however, may further
our understanding regarding the biological significance of this
mutation.
It has been previously reported that the Ras-100LIR104
mutation exhibits normal sensitivity to p120. However,
the source of GAP in these studies was an MCF-7 cell extract which
would be expected to contain both p120
and neurofibromin
(Adari et al., 1988). Those experiments were performed at a
Ras concentration of 0.8 µM. Since the binding constant of
Ras for neurofibromin and p120
is 30 nM and 12
µM, respectively, a Ras concentration of 0.8 µM would result predominantly in neurofibromin-mediated Ras
GTP
hydrolysis. Thus, it is understandable that no decrease in GTP
hydrolysis was detected when Ras-100LIR104 was incubated with MCF-7
cell lysate. For the same reason we also did not observe differences in
GTP hydrolysis between Ras and Ras-100LIR104 when rabbit brain lysate
(which contains both neurofibromin and p120
) was used as
a source of GAP (data not shown). Only when steps were taken to
separate the p120
and neurofibromin activity were the
differences seen.
The 3 helix of Ras has been implicated in
other studies as a site for GAP interaction (Wood et al.,
1994). The yeast RAS2-E99K mutation which exhibited intrinsic
hydrolytic rates similar to wild type Ras, showed reduced sensitivity
to three separate GTPase proteins: IRA2 GAP, E. coli expressed
mammalian GAP, and neurofibromin. Furthermore, Wood et
al.(1994) also showed by a competitive binding assay that the
affinity of RAS2-E99K for neurofibromin was dramatically reduced. These
results differ from ours, where neither the neurofibromin activity nor
binding affinity for Ras-100LIR104 was diminished. The yeast RAS2-E99K
mutation would be located at the beginning of the
3 helix of the
protein, while the 100LIR104 mutation is located at the terminus of
this helix. While the individual characteristics of the two types of
mutants differ, both result in clear alterations in interactions with
GTPase activating proteins supporting the conclusion that this region
of Ras, in addition to the effector binding domain and residues
61-65 of the switch II region, is an important site for
interaction with GTPase activating proteins. It is also interesting to
note that the effector domain, the switch II region, and a region of
the
3 helix, all reside on the same surface of the Ras protein.
These three regions are in close proximity to one another and could
potentially provide a binding site for GAP proteins (Wood et
al., 1994). Finally, the Ras-D38E mutant is not activated by
p120
but is able to bind to p120
with an
affinity similar to wild type. Interestingly, the D38E mutation causes
a chain shift in the 101-103 region of Ras. Thus the D38E
mutation may have low GTPase activity in part due to the chain shift in
the 101-103 region of Ras (Krengel et al., 1990).
Alternatively, both this mutation and the Ras-100LIR104 mutation might
induce conformational changes at other sites of the protein which are
important in modulating GTPase activation.
Other studies have also
examined Ras-100LIR104 in the presence of GAP purified from placenta.
Placental GAP, designated p100, is generated from an
alternative splicing product and encodes a protein product with a
predicted molecular weight of 100,400 (Trahey et al., 1988).
p100
lacks the hydrophobic amino terminus of the
p120
species, but retains catalytic activity. No
difference in hydrolysis was observed when Ras and Ras-100LIR104 were
assayed for p100
activity (Downward et al.,
1990). This observation is consistent with our data where decreased
hydrolysis was observed with Ras-100LIR104 only in the presence of
full-length p120
. Therefore, the hydrophobic, extreme
amino terminus of p120
appears to be responsible for the
differences in GTPase activation observed between Ras-100LIR104 and
wild type Ras. While it is not clear exactly how Ras protein is
positioned with respect to the plasma membrane, it is likely that the
protein is oriented with the lipid-modified carboxyl terminus oriented
toward the membrane. If so, it is further likely that the terminal
region of the
3 helix of Ras containing amino acids 101-103
is located near the plasma membrane (de Vos et al., 1988; Pai et al., 1989). If, as postulated above, the amino terminus of
p120
interacts with the terminus of the
3 region of
Ras, this hydrophobic region of p120
would be expected to
be positioned near, or perhaps even at, the plasma membrane. It is
additionally clear from these studies that this region of p120
plays a critical role in modulating GTPase activation.
Recently, a novel mammalian GTPase activating protein for Ras,
Gap1, has been identified (Maekawa et al., 1994).
Because Gap1
is expressed in brain, it is possible (in
those experiments in which rabbit brain lysate was used as a source of
p120
) that Gap1
function is also impaired in
the presence of the Ras-100LIR104 mutant. Further investigation,
however, is needed in order to determine the interaction of
Gap1
with 100LIR104 Ras.
It is also possible that
associated protein(s) are in a complex with p120. This
possibility, however, does not diminish the importance of the
observation that the conserved 101-103 amino acids in the
3
helix of Ras are specifically involved in p120
-mediated
hydrolysis but not neurofibromin-mediated hydrolysis. The likelihood
that associated proteins are bound to p120
is lessened by
the fact that p120
is overexpressed in insect cells,
therefore, the ratio of an associated protein to overexpressed
p120
would differ significantly to that observed in rat
brain lysate in which p120
is not overexpressed. Thus, if
an associated protein were involved, one would expect different results
from p120
overexpressed in baculovirus lysates compared
to endogenous p120
in rat brain lysates. Since identical
results were obtained in both cases, it is unlikely that an associated
protein contributes significantly to the decrease in GTPase activity.
Another interesting possibility is that the decrease in p120 activity is not due to the deletion of the conserved KRV amino
acids but the substitution of amino acids LIR. Future experiments, in
which single substitutions or a deletion of the one or all three of the
conserved amino acids may help us better answer this question or
perhaps even lead to the identification of additional mutants of this
type.
Lastly, the Ras-100LIR104 mutant may serve as an important
tool to better understand the function of the GAP proteins. For
example, such a mutant may differentiate between different GAPs or
between neurofibromin and p120. The construction of cell
lines transfected with the Ras-100LIR104 mutation could thus provide a
useful in vivo approach to address these types of questions.