Human cells contain four homologous Ras proteins,
but it is unknown whether these homologues have different biological
functions. As a first step in determining if Ras homologues might
participate in distinct signaling cascades, we assessed whether a given
Ras guanine nucleotide exchange factor could selectively activate a
single Ras homologue in vivo. We found that
Ras-GRF/Cdc25Mm activates Ha-Ras, but does not activate
N-Ras or K-Ras 4B, protein in vivo. Moreover, our results
suggested that residues within the C-terminal hypervariable domains of
Ras proteins may dictate, at least in part, the specificity of
Ras-GRF/CDC25Mm for Ha-Ras protein. Our studies represent
the first biochemical evidence that a Ras GEF can selectively activate
a single Ras homologue in vivo. Selective activation of a
single Ras homologue by Ras-GRF/Cdc25Mm or other Ras
guanine nucleotide exchange factors could potentially enable each of
the Ras homologues to participate in different signal transduction
pathways.
 |
INTRODUCTION |
Ras proteins lie at the heart of signal transduction pathways,
which link cell surface receptors to the nucleus. Ras proteins are
activated by guanine nucleotide exchange factors
(GEFs),1 which catalyze the
exchange of free GTP for bound GDP, and they are inactivated by
GTPase-activating proteins (GAPs), which accelerate the intrinsic rate
that Ras proteins hydrolyze bound GTP to GDP (1). By cycling between
their active, GTP-bound state and inactive, GDP-bound state, Ras
proteins function as molecular switches in signaling pathways that
regulate cell growth and differentiation (2, 3). Although many of the
components involved in Ras-mediated signal transduction have been
elucidated, many critical questions remain. For instance, human cells
contain four homologous Ras proteins (Ha-Ras, N-Ras, K-Ras 4A, and
K-Ras 4B), but despite years of intensive study, it is still not known
whether these four homologues have different biological functions or
participate in distinct signal transduction cascades.
Several different GEFs that activate mammalian Ras proteins have been
identified. These include p140 Ras-GRF (also called Cdc25Mm) (4-6), mSOS-1 and -2 (7, 8), and Vav (9),
although the last is controversial (10, 11). It is currently unknown whether each of these GEFs activates all four Ras proteins or whether a
given GEF selectively activates a single Ras protein in
vivo. The question of GEF specificity is critical, however, because recent studies have shown that a given type of
ligand-stimulated receptor can selectively trigger a specific GEF to
activate Ras proteins. For instance, stimulated tyrosine kinase
receptors or receptors associated with tyrosine kinases (such as
epidermal growth factor, platelet-derived growth factor, insulin, T
cell, and cytokine receptors), induce mSOS to activate Ras proteins (12-22), while some G protein-coupled receptors (such as muscarinic receptors) induce Ras-GRF to activate Ras proteins (23). In addition,
membrane depolarization-induced Ca2+ influx selectively
induces Ras-GRF (24). If, therefore, a given GEF selectively activates
a single Ras homologue, this specificity could potentially enable a
given extracellular ligand to selectively activate a single Ras
homologue and thereby enable each of the Ras homologues to participate
in distinct, parallel signal transduction pathways. Accordingly, the
purpose of the studies reported herein was to determine if a given GEF
could selectively activate a single Ras homologue in
vivo.
 |
EXPERIMENTAL PROCEDURES |
Generation of Molecular Constructs and Cell Lines--
To
distinguish exogenous from endogenous Ras proteins, a Glu-Glu (EE)
epitope tag (EEEEYMPME) (25) was added to the N termini of wild type
human Ha-Ras, N-Ras, and K-Ras 4B cDNA by the polymerase chain
reaction. cDNA clones encoding mutant Ha-Ras protein, containing Ser residues substituted for the two palmitylated Cys residues of
Ha-Ras (Cys-181 and -184), and mutant K-Ras 4B protein, containing 12 neutral Glns substituted for the 6 charged amino acids of the charged
domain (CD) and the 6 basic amino acids of the polylysine domain (KD)
of K-Ras 4B (CD and KD are amino acids 164-169 and 175-180,
respectively; see Fig. 5), were generated from wild type EE-tagged
Ha-Ras and K-Ras 4B cDNA, respectively, by polymerase chain
reaction mutagenesis (26). EE-tagged wild type and mutant Ras cDNAs
were cloned into the pZIP-Neo-SV(x)1 retroviral vector (27). To
immunologically identify Ras-GRF, a Myc epitope tag (EQKLISEEDL) was
added to the N terminus of murine p140 Ras-GRF (28) by cloning Ras-GRF
cDNA in frame into a modified pBabe puro retroviral vector (29),
containing a Kozak and Myc epitope sequence immediately 5
of its
cloning site. Ras constructs (10 µg) were transfected into NIH 3T3
cells, and Ras-GRF constructs (or the empty pBabe puro vector, as
control) (10 µg) were transiently transfected into the viral
packaging cell line, BOSC 23 (30) by calcium phosphate precipitation
(26). Ras-transfected NIH 3T3 cells were selected with G418 (400 µg/ml), selected cells were infected with BOSC cell supernatants
containing recombinant Ras-GRF or control retrovirus, and infected
cells were selected with puromycin B (2.5 µg/ml). Selected cells were
metabolically labeled with [35S]Met/Cys; Ras or Ras-GRF
protein contained in these cells was immunoprecipitated with anti-EE
(25) or anti-Myc 9E10.2 (31) antibody, respectively; and levels of
protein expression were evaluated by SDS-polyacrylamide gel
electrophoresis and fluorography, as described previously (26). Cell
lines expressing comparable amounts of Ras-GRF and/or each of the Ras
homologues were utilized for all studies.
In Vivo GTP/GDP Binding Assays--
Subconfluent (60-75%)
cells expressing comparable amounts of Ha-Ras, N-Ras, or K-Ras 4B
protein and comparable amounts of Ras-GRF (or the pBabe puro vector, as
control) were metabolically labeled with
[32P]orthophosphate (1 mCi/ml) in phosphate-free,
serum-free medium for 16 h and lysed. Ras proteins contained in
cell lysates were immunoprecipitated with anti-EE antibody, and guanine
nucleotides bound to Ras proteins were eluted and fractionated by thin
layer chromotography. The cpm in GTP and GDP were quantitated on an Ambis
scanner, as described previously (32), except the NaCl concentration in the lysis buffer was reduced to 150 mM.
The percentage of total exogenous Ras protein bound to GTP was
calculated according to the following formula: % GTP = cpm in
GTP/(cpm in GTP + cpm in GDP), using cpm normalized for moles of
phosphate.
Cell Proliferation Assays--
Cell lines expressing Ras-GRF
and/or Ras proteins were plated at a density of 1 × 104 cells/60-mm dish in normal growth medium, and 16 h
following plating, cells were changed to and maintained in growth
medium containing 0.5% calf serum. 3, 5, 7, 9, and 11 days following plating, cells were trypsinized and counted using a hemocytometer.
In Vitro Mitogen-activated Protein (MAP) Kinase Assays--
Cell
lines expressing Ras-GRF and/or Ras proteins were transiently
transfected with an ERK2/pSR
plasmid construct (2 µg). 32 h
following transfection, cells were serum-starved for 16 h, and
ERK2 protein was subsequently immunoprecipitated from these cells with
ERK2/C-14 antibody (Santa Cruz). The expression of ERK2 was evaluated
by immunoblot, and the kinase activity of immunoprecipitated ERK2 was
evaluated by assessing its ability to phosphorylate myelin basic
substrate protein (MBP; Sigma) in vitro (33).
Phosphorylation of MBP was quantitated on an Ambis
scanner.
 |
RESULTS |
Activation of Ras Proteins by Ras-GRF--
To determine whether a
given GEF might selectively activate a specific Ras homologue in
vivo, we expressed exogenous Ras-GRF protein and/or wild type
Ha-Ras, N-Ras, or K-Ras 4B protein in NIH 3T3 cells and determined the
percentage of each exogenous Ras protein bound to GTP
(versus GDP) in the absence or presence of Ras-GRF. As can
be seen in Fig. 1, approximately 2.2-fold
more (p < 0.001; t test) exogenous Ha-Ras
protein was bound to GTP in cells co-expressing Ras-GRF
versus cells expressing Ha-Ras alone (6.0 ± 0.05 versus 2.7 ± 0.4% GTP-bound Ha-Ras, respectively). In
contrast, no significant (p > 0.05) increase in the
percentage of N-Ras (3.3 ± 0.3 versus 3.3 ± 0.4%) or K-Ras 4B (4.0 ± 0.3 versus 3.3 ± 0.1)
protein bound to GTP was detected in cells co-expressing Ras-GRF
versus cells expressing N-Ras or K-Ras 4B protein alone, respectively. To confirm that the observed 2.2-fold increase in the
amount of GTP bound to Ha-Ras in the presence of Ras-GRF was biologically significant, we examined the effect of Ras-GRF on two
downstream indicators of Ras activation. Specifically, we assessed the
effect of exogenous Ras-GRF on MAP kinase activation and cell
proliferation in serum-starved NIH 3T3 cells co-expressing Ha-Ras,
N-Ras, or K-Ras 4B protein. As demonstrated in Fig.
2, the kinase activity of p42 MAP kinase
(ERK2) was 4-fold higher (p < 0.001; t
test) in cells co-expressing Ras-GRF and Ha-Ras versus cells
expressing Ha-Ras protein alone (323,082 ± 67,209 versus 79,675 ± 14,484 cpm of MBP phosphorylation,
respectively). However, there was no significant difference
(p > 0.05) in MAP kinase activity in cells
co-expressing Ras-GRF and N-Ras or K-Ras 4B versus cells
expressing N-Ras or K-Ras 4B protein alone (175,952 ± 49,540 versus 155,972 ± 28,208 and 118,948 ± 17,911 versus 134,231 ± 20,525, respectively). (As indicated
in Fig. 2, the expression of ERK2 was comparable in each of the cell
lines tested.) In addition, as shown in Fig.
3, Ras-GRF increased the growth rate of
serum-starved Ha-Ras-expressing cells approximately 2-fold but had no
effect on the growth rate of N-Ras- or K-Ras 4B-expressing cells.
Importantly, each of the experiments described in Figs. 1, 2, and 3
utilized cells that expressed comparable amounts of Ras-GRF and/or
Ha-Ras, N-Ras, or K-Ras 4B proteins (see Fig.
4). Our combined results indicate,
therefore, that Ras-GRF selectively activates Ha-Ras but does not
activate N-Ras or K-Ras 4B, protein in vivo.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 1.
Ras-GRF increases the amount of GTP bound to
Ha-Ras, but not N-Ras or K-Ras 4B, protein in NIH 3T3 cells.
Subconfluent, serum-starved NIH 3T3 cells expressing comparable amounts
of Ha-Ras, N-Ras, or K-Ras 4B protein and/or Ras-GRF were metabolically
labeled with [32P]orthophosphate; Ras proteins contained
in these cells were immunoprecipitated with anti-EE antibody; and
guanine nucleotides bound to Ras proteins were eluted, fractionated by
thin layer chromotography, and quantitated on an Ambis scanner. The
migration positions of GDP and GTP standards are shown. Data shown are
representative of five independent experiments. The indicated
percentages of Ras bound to GTP represent the mean of the percentage of
Ras bound to GTP from five individual experiments.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Ras-GRF increases the kinase activity of ERK2
in serum-starved NIH 3T3 cells expressing exogenous Ha-Ras, but not
N-Ras or K-Ras 4B, protein. The kinase assay shows the level of ERK2-mediated MBP phosphorylation; and the immunoblot shows the relative amount of ERK2 protein expression. The cell lines described in
the legend to Fig. 1 were transiently transfected with an ERK2/pSR plasmid construct and serum-starved. 48 h following transfection, ERK2 proteins contained in these cells were immunoprecipitated with
ERK2 antibody; the expression of ERK2 was evaluated by immunoblot; and
the kinase activity of immunoprecipitated ERK2 was evaluated by
assessing its ability to phosphorylate myelin basic substrate protein
(MBP) in vitro. Data shown are representative of five independent experiments.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Ras-GRF increases the growth rate of
serum-starved NIH 3T3 cells expressing exogenous Ha-Ras, but not N-Ras
or K-Ras 4B, protein. The cell lines described in the legend to
Fig. 1 were plated at a density of 1 × 104
cells/60-mm dish and serum-starved. At the indicated time points, cells
were trypsinized and counted using a hemocytometer. Data shown
represent the mean ± S.E. of three independent experiments. Where
no error bars are shown, the S.E. is less than 2% of the y
axis coordinate scale.
|
|

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 4.
Expression levels of Ras and/or Ras-GRF
proteins in NIH 3T3 cells. The cell lines described in the legend
to Fig. 1 were metabolically labeled with [35S]Met/Cys;
Ras or Ras-GRF protein contained in these cells was immunoprecipitated
with anti-EE or anti-Myc 9E10.2 antibody, respectively; and levels of
protein expression were evaluated by SDS-polyacrylamide gel
electrophoresis and fluorography.
|
|
Effect of Hypervariable Domain Ras Residues on Ras-GRF
Specificity--
The only area where the highly homologous Ha-Ras,
N-Ras, and K-Ras 4B proteins substantially differ from each other is in their 25 C-terminal amino acids, termed the hypervariable domain (see
Fig. 5). We wondered, therefore, whether
residues within the hypervariable domain of Ha-Ras might be required
for Ras-GRF activation or whether residues within the hypervariable
domain of N-Ras or K-Ras 4B might prevent Ras-GRF activation.
Accordingly, as a first step in determining what factors might account
for Ras-GRF specificity for Ha-Ras, we determined if Ras-GRF could activate a mutant Ha-Ras protein (2S) containing 2 nonpalmitylated Ser
residues substituted for its 2 normally palmitylated Cys residues or a
mutant K-Ras 4B protein (12Q) containing 12 neutral Gln residues substituted for the 12 charged amino acids of its CD and KD. We also
determined if Ras-GRF could activate a nonposttranslationally modified
mutant Ha-Ras protein (SAAX) containing a Ser substituted for the Cys
of its CAAX motif (where C is a Cys, A is an aliphatic amino
acid, and X is a Ser or Met) (see Fig. 5). As demonstrated in Fig.
6A, there was a significant
(p < 0.001; t test) increase in the
percentage of nonpalmitylated Ha-Ras protein bound to GTP in cells
co-expressing Ras-GRF versus cells expressing 2S alone (4.7 ± 0.4 versus 2.4 ± 0.2%, respectively),
although the magnitude of increase of Ras-GRF-induced 2S GTP binding
was slightly lower than that observed with wild type Ha-Ras protein
(6.6 ± 0.6 versus 2.1 ± 0.1). In addition, there
was a small, but significant (p < 0.05; t
test) increase in the percentage of nonposttranslationally modified
Ha-Ras protein bound to GTP in cells co-expressing Ras-GRF versus cells expressing SAAX alone (3.65 ± 0.3 versus 2.48 ± 0.18%, respectively). Surprisingly, as
shown in Fig. 6B, unlike its wild type counterpart, the
neutralized K-Ras 4B mutant, 12Q, was efficiently (p < 0.05; t test) activated by Ras-GRF (13.7 ± 1.2 versus 7.9 ± 0.7% GTP binding in the presence or
absence of Ras-GRF, respectively). Indeed, the -fold increase in the
amount of GTP bound to 12Q in the presence of Ras-GRF was comparable to
that seen with Ras-GRF and wild type Ha-Ras. Moreover, in addition to
being activated by Ras-GRF, 12Q was also constitutively active.
Specifically, as indicated in Fig. 6B, the amount of GTP
bound to 12Q, in the absence of Ras-GRF or serum (7.9 ± 0.7), was
more than 2-fold higher than that bound to wild type K-Ras 4B (3.3 ± 0.1) under similar conditions (p < 0.01;
t test). Furthermore, as illustrated in Fig.
7, the growth rate of serum-starved NIH
3T3 cells expressing 12Q alone was significantly higher than that of
cells expressing wild type K-Ras 4B alone.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
25 C-terminal amino acids of Ha-Ras, N-Ras,
and K-Ras 4B proteins. The palmitylation sites of Ha-Ras and
N-Ras, the CD and KD of K-Ras 4B, and the CAAX motif of each
of the Ras proteins are indicated.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of C-terminal Ras residues on Ras-GRF
specificity/Ras activation. A, palmitylation contributes to,
but is not absolutely required for, Ras-GRF activation of Ha-Ras
protein. B, the two charged domains of K-Ras 4B inhibit both
constitutive and Ras-GRF-induced K-Ras 4B activation. 2S is the mutant
Ha-Ras protein containing 2 Ser residues substituted for its 2 palmitylated Cys residues. 12Q is the mutant K-Ras 4B protein
containing 12 neutral Gln residues substituted for the 12 charged amino
acids of the CD and KD. NIH 3T3 cells co-expressing either the 2S
Ha-Ras mutant or the 12Q K-Ras 4B mutant and/or Ras-GRF were generated, and the amount of GTP bound to mutant versus wild type
Ha-Ras or K-Ras 4B protein was determined according to the methods
outlined in the legend to Fig. 1. Data shown are representative of five independent experiments. The indicated percentages of Ras bound to GTP
represent the mean of the percentage of Ras bound to GTP from five
individual experiments.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Serum-starved NIH 3T3 cells expressing mutant
12Q K-Ras 4B protein grow at a significantly faster rate than cells
expressing wild type K-Ras 4B protein. The cell lines described in
the legend to Fig. 6B were plated, maintained, and evaluated
for growth rates, according to the methods outlined in Fig. 3. Data
shown represent the mean ± S.E. of three independent experiments.
Where no error bars are shown, the S.E. is less than 2% of the
y axis coordinate scale.
|
|
 |
DISCUSSION |
Our combined results indicate that Ras-GRF selectively activates
Ha-Ras, but does not activate N-Ras or K-Ras 4B, protein in
vivo. As far as we know, our studies represent the first
biochemical evidence that a Ras GEF can selectively activate a single
Ras homologue in vivo. Our results in intact cells
distinctly differ from previously reported in vitro studies,
which showed that purified Ras-GRF protein can stimulate the GTP/GDP
exchange of purified Ha-Ras (4, 34), N-Ras (4), and K-Ras 4B (34)
proteins. Indeed, one of these studies (34) demonstrated that Ras-GRF was more effective on K-Ras 4B than Ha-Ras protein. It should be noted,
however, that these in vitro studies utilized the C-terminal catalytic domain of Ras-GRF rather than full-length Ras-GRF. Moreover, the catalytic domain was fused to glutathione S-transferase
or oligohistidine. It is possible, therefore, that these in
vitro studies failed to detect Ha-Ras specificity because
residues/domains outside of the catalytic domain of Ras-GRF dictate
Ha-Ras specificity, and/or fusion to glutathione
S-transferase or oligohistidine ablates Ras-GRF specificity.
In addition, it is also possible that spatial determinants (such as
contiguous membrane co-localization, scaffolding proteins, etc.) and/or
other cellular factors mediate Ras-GRF specificity in intact cells. Our
studies underscore the importance, therefore, of utilizing intact cells
and full-length proteins in studies aimed at determining GEF
specificity. Although numerous investigators have added EE, Myc, or
other epitope tags to the N termini of Ras and Ras-GRF proteins without
any known adverse effects, we cannot definitively exclude the
possibility that the EE or Myc epitope tags might have affected the
intrinsic biochemical properties of these proteins. Since, however, one
would not expect an epitope tag-induced effect to be Ras
homologue-specific, it is highly unlikely that EE or Myc epitope tags
account for the disparate ability of Ras-GRF to activate Ha-Ras, but
not N-Ras or K-Ras 4B, protein.
Our observed approximately 2.2-fold increase in the amount of GTP bound
to exogenous Ha-Ras in Ras-GRF-expressing NIH 3T3 cells is similar in
magnitude to the increase previously observed in 293T cells transiently
co-expressing exogenous Ha-Ras and Ras-GRF (24). However, the
percentage of GTP bound to exogenous Ha-Ras in our control and
Ras-GRF-stimulated NIH 3T3 cells (2.7 and 6%, respectively) was
significantly lower than that reported in 293T cells (19 and 32%,
respectively) (24). To confirm, therefore, that our observed increase
in Ha-Ras GTP binding was biologically significant, we determined
whether this increase was associated with increases in downstream
indicators of Ras activation. In direct correlation with their 2.2-fold
increase in Ha-Ras GTP-binding, cells co-expressing Ras-GRF and Ha-Ras
proteins demonstrated a ~4- and ~2-fold increase in MAP kinase
activation and growth rates, respectively. Likewise, in direct
correlation with their lack of increase in N-Ras or K-Ras 4B GTP
binding, cells co-expressing Ras-GRF and N-Ras or K-Ras 4B proteins
demonstrated no increase in MAP kinase activation or growth rates. Our
results indicate, therefore, that although Ras-GRF only increased
Ha-Ras GTP binding from 2.7 to 6.0% in our cells, this increase was
sufficient to activate downstream Ras signaling pathways and stimulate
cell proliferation. We did not observe a significant difference in the
percentage of Ha-Ras protein bound to GTP when we utilized Y13-259,
rather than anti-EE, antibody (data not shown). We do not believe,
therefore, that our low percentage of GTP binding was due to the
inability of anti-EE antibody to prevent GAP activity. Although we do
not have a definitive explanation for the low percentage of GTP binding
to Ras proteins in our cells, one possibility is that it is simply a
consequence of the high levels of expression of exogenous Ras proteins
in our cells. Thus, although the relative percentage of Ha-Ras protein
bound to GTP (versus total Ha-Ras protein expressed) may be
low, the absolute number of moles of Ha-Ras protein bound to GTP in our
cells could nonetheless be substantial.
Several regions within the Ha-Ras protein that appear to be important
for binding to and/or activation by Ras-GRF have been identified in
studies that utilized the catalytic domain of Ras-GRF or its yeast
homologue, Cdc25. These regions include amino acids 62-78 (35-40) and
amino acids 102-105 (38). In addition, amino acids 32-38 of Ras have
been shown to be important for functional interaction of Ras with
another yeast GEF, Sdc25 (41, 42). Since Ha-Ras, N-Ras, and K-Ras 4B
proteins are completely identical in all three of these regions, it is
unlikely that these regions account for our observed specificity of
Ras-GRF for Ha-Ras. The only area where the highly homologous Ha-Ras,
N-Ras, and K-Ras 4B proteins substantially differ from each other is in
their 25 C-terminal amino acids, termed the hypervariable domain (see
Fig. 5). This domain contains a CAAX motif, which signals
the Ras proteins to undergo a series of posttranslational
modifications. Each of the Ras proteins becomes farnesylated,
truncated, and carboxylmethylated at its CAAX residues
(43-47). However, in addition to these modifications, the Ha-Ras and
N-Ras proteins also become palmitylated on 2 or 1, respectively,
non-CAAX Cys residues within their hypervariable domain (43,
44). K-Ras 4B lacks these Cys residues and does not become
palmitylated. Instead, K-Ras 4B contains two charged domains,
consisting of 6 contiguous basic Lys (KD) and 6 contiguous charged
amino acids (CD; 5 basic and 1 acidic amino acids) within its
hypervariable domain. Given these C-terminal differences between Ha-Ras, N-Ras, and K-Ras 4B proteins, we wondered whether the 2 palmitylated Cys residues of Ha-Ras might be required for activation by
Ras-GRF or the 2 charged domains of K-Ras 4B (or undefined residues
within the C terminus of N-Ras) might prevent activation by Ras-GRF.
Accordingly, as a first step in determining what factors might account
for Ras-GRF specificity for Ha-Ras, we assessed the ability of Ras-GRF
to activate a nonpalmitylated Ha-Ras mutant or a K-Ras 4B mutant whose
two charged domains had been neutralized. As detailed under
"Results," the nonpalmitylated Ha-Ras mutant, 2S, was activated by
Ras-GRF, although the degree of activation was slightly lower than that
observed with wild type Ha-Ras protein. Thus, although Ras-GRF may
activate palmitylated Ha-Ras protein more efficiently than
nonpalmitylated Ha-Ras, palmitylation is clearly not absolutely
required for activation by Ras-GRF, nor is it likely to contribute to
Ras-GRF specificity. Surprisingly, unlike wild type K-Ras 4B, the
neutralized K-Ras 4B mutant, 12Q, was efficiently activated by Ras-GRF.
Our studies suggest, therefore, that the CD and KD of K-Ras 4B may
prevent K-Ras 4B activation by Ras-GRF.
Recent studies from our laboratory have shown that the 12Q mutant,
despite normal levels of posttranslational modifications, is ~50%
membrane-bound and ~50%
cytosolic.2 It could be
argued, therefore, that the cytosolic location of 12Q facilitates its
activation by Ras-GRF. This premise is unlikely for several reasons.
First, Ras-GRF is predominantly membrane-bound (48). Second, as
indicated under "Results," a completely cytosolic, nonposttranslationally modified mutant Ha-Ras protein, SAAX (containing a Ser substituted for the Cys of its CAAX motif), is
approximately 2-fold less responsive to Ras-GRF than wild type Ha-Ras
protein.
It is not yet clear why 12Q is constitutively more active than wild
type K-Ras 4B protein. Since the NIH 3T3 cells employed in our studies
do not express endogenous Ras-GRF, one possibility is that CD and KD
prevent endogenous GEFs, as well as exogenous Ras-GRF, from activating
K-Ras 4B. Therefore, in the absence of CD and KD, these GEFs may
promiscuously activate 12Q. Alternatively, the intrinsic and/or
GAP-stimulated GTPase activity of the 12Q mutant could be decreased in
comparison with wild type K-Ras 4B. This possibility is less likely,
however, because decreased GTPase activity would not readily explain
the ability of Ras-GRF to activate 12Q. Finally, the intrinsic GTP/GDP
exchange rate of the 12Q mutant could be increased in comparison with
wild type K-Ras 4B. Again, however, an increased intrinsic GTP/GDP
exchange rate would not necessarily affect Ras-GRF-induced GTP/GDP
exchange. Additional studies, utilizing purified 12Q protein, will be
required to definitively assess what, if any, role decreased GTPase
activity and/or increased exchange rates may play in constitutive
and/or Ras-GRF-induced activation of 12Q.
In summary, our results demonstrate that Ras-GRF activates Ha-Ras, but
does not activate N-Ras or K-Ras 4B, protein in vivo. Our
studies represent the first biochemical evidence that a Ras GEF can
selectively activate a single Ras homologue in vivo. It will
be important, in future studies, to determine if other Ras GEFs or
extracellular ligands selectively activate a single Ras homologue
in vivo. In addition, it will be important to determine whether the four Ras homologues selectively activate distinct effectors
in vivo. These studies are currently under way and may ultimately enable us to determine whether the four Ras homologues participate in different signaling cascades and have different biological functions.
We thank D. Lowy for the p140 Ras-GRF
construct, M. Karin for the ERK2 construct, J. Morgenstern for the
pBabe puro vector, W. Pear for the BOSC 23 cells, G. Walter for
hybridoma cells expressing anti-EE antibody, and M. Cochrane for typing
the manuscript.