Ras-GRF Activates Ha-Ras, but Not N-Ras or K-Ras 4B, Protein in Vivo*

Michael K. Jones and Janis H. JacksonDagger

From The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta  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/pSRalpha 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 beta  scanner.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.


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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 beta  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.


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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/pSRalpha 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.


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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.


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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.


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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.


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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.


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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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, Public Health Service Grant CA-54298 (to J. H. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. The Scripps Research Institute, 10550 N. Torrey Pines Rd., Box IMM 12, La Jolla, CA 92037. Tel.: 619-784-8748; Fax: 619-784-8150.

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; CD, charged domain; KD, polylysine domain; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MBP, myelin basic substrate protein; GAP, GTPase-activating protein.

2 J. H. Jackson and S. Wong, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Downward, J. (1992) Nature 358, 282-283[Medline] [Order article via Infotrieve]
  2. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-828[CrossRef][Medline] [Order article via Infotrieve]
  3. Bourne, H. A., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve]
  4. Shou, C., Farnsworth, C. L., Neel, B. G., Feig, L. A. (1992) Nature 358, 351-354[CrossRef][Medline] [Order article via Infotrieve]
  5. Wei, W., Mosteller, R. D., Sanyal, P., Gonzales, E., McKinney, D., Dasgupta, C., Li, P., Ben-Xian, L., and Broek, D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7100-7104[Abstract]
  6. Martegani, E., Vanoni, M., Zippel, R., Coccetti, P., Brambilla, R., Ferrari, C., Sturani, E., and Alberghina, L. (1992) EMBO. J. 11, 2151-2157[Abstract]
  7. Chardin, P., Camonis, J. H., Gale, N. W., Van Aelst, L., Schlessinger, J., Wigler, M. H., Bar-Sagi, D. (1993) Science 260, 1338-1343[Medline] [Order article via Infotrieve]
  8. Bowtell, D., Fu, P., Simon, M., and Senior, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6511-6515[Abstract]
  9. Gulbins, E., Coggeshall, K. M., Baier, G., Katzav, S., Burn, P., and Altman, A. (1993) Science 260, 822-825[Medline] [Order article via Infotrieve]
  10. Bustelo, X. R., Suen, K.-L., Leftheris, K., Meyers, C. A., Barbacid, M. (1994) Oncogene 9, 2405-2413[Medline] [Order article via Infotrieve]
  11. Khosravi-Far, R., Chrzanowska-Wodnicka, M., Solski, P. A., Eva, A., Burridge, K., Der, C. J. (1994) Mol. Cell. Biol. 14, 6848-6857[Abstract]
  12. McCormick, F. (1992) Nature 363, 15-16
  13. Skolnik, E. Y., Batzer, A., Li, N., Lee, C. -H., Lowenstein, E., Mohammadi, M., Margolis, B., Schlessinger, J. (1993) Science 260, 1953-1955[Medline] [Order article via Infotrieve]
  14. Winitz, S., Russell, M., Qian, N.-X., Gardner, A., Dwyer, L., and Johnson, G. L. (1993) J. Biol. Chem. 268, 19196-19199[Abstract/Free Full Text]
  15. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J. H., Cooper, J. A., Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509-517[Abstract]
  16. Bennett, A. M., Tang, T. L., Sugimoto, S., Walsh, C. T., Neel, B. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7335-7339[Abstract]
  17. Cutler, R. L., Liu, L., Damen, J. E., Krystal, G. (1993) J. Biol. Chem. 268, 21463-21465[Abstract/Free Full Text]
  18. Alblas, J., van Corven, E. J., Hordijk, P. L., Milligan, G., Moolenaar, W. H. (1993) J. Biol. Chem. 268, 22235-22238[Abstract/Free Full Text]
  19. Cook, S. J., Rubinfeld, B., Albert, I., and McCormick, F. (1993) EMBO J. 12, 3475-3485[Abstract]
  20. LaMorte, V. J., Kennedy, E. D., Collins, L. R., Goldstein, D., Harootunian, A. T., Brown, J. H., Feramisco, J. R. (1993) J. Biol. Chem. 268, 19411-19415[Abstract/Free Full Text]
  21. Ravichandran, K. S., Lee, K. K., Songyang, Z., Cantley, L. C., Burn, P., Burakoff, S. J. (1993) Science 262, 902-905[Medline] [Order article via Infotrieve]
  22. van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., Moolenaar, W. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1257-1261[Abstract]
  23. Mattingly, R. R., and Macara, I. G. (1996) Nature 382, 268-272[CrossRef][Medline] [Order article via Infotrieve]
  24. Farnsworth, C. L., Freshney, N. W., Rosen, L. B., Ghosh, A., Greenberg, M. E., Feig, L. A. (1995) Nature 376, 524-527[CrossRef][Medline] [Order article via Infotrieve]
  25. Grussenmeyer, T., Scheidtmann, K. H., Hutchinson, M. A., Eckhart, W., Walter, G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7952-7954[Abstract]
  26. Jackson, J. H., Li, J. W., Buss, J. E., Der, C. J., Cochrane, C. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12730-12734[Abstract/Free Full Text]
  27. Cepko, C. L., Roberts, B. E., and Mulligan, R. C. (1984) Cell 37, 1053-1062[Medline] [Order article via Infotrieve]
  28. Cen, H., Papageorge, A. G., Vass, W. C., Zhang, K., Lowy, D. R. (1993) Mol. Cell. Biol. 13, 7718-7724[Abstract]
  29. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract]
  30. Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 90, 8392-8396[Abstract/Free Full Text]
  31. Kramer, S. M., Aggarwal, B. B., Eessalu, T. E., McCabe, S. M., Ferraiolo, B. L., Figari, I. S., Palladino, M. A., Jr. (1988) Cancer Res. 48, 920-925[Abstract]
  32. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., Cantrell, D. A. (1990) Nature 346, 719-723[CrossRef][Medline] [Order article via Infotrieve]
  33. Han, J., Lee, J.-D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886-2891[Abstract/Free Full Text]
  34. Orita, S., Kaibuchi, K., Kuroda, S., Shimizu, K., Nakanishi, H., and Takai, Y. (1993) J. Biol. Chem. 268, 25542-25546[Abstract/Free Full Text]
  35. Verrotti, A. C., Crechet, J. B., Di Blasi, F., Seidita, G., Mirisola, M. G., Kavounis, C., Nastopoulos, V., Burderi, E., De Vendittis, E., Parmeggiani, A., Fasano, O. (1992) EMBO J. 11, 2855-2862[Abstract]
  36. Quilliam, L. A., Kato, K., Rabun, K. M., Hisaka, M. M., Huff, S. Y., Campbell-Burk, S., Der, C. J. (1994) Mol. Cell. Biol. 14, 1113-1121[Abstract]
  37. Mosteller, R. D., Han, J., and Broek, D. (1994) Mol. Cell. Biol. 14, 1104-1112[Abstract]
  38. Segal, M., Marbach, I., Willumsen, B. M., Levitzki, A. (1995) Eur. J. Biochem. 228, 96-101[Abstract]
  39. Howe, L. R., and Marshall, C. J. (1993) Oncogene 8, 2583-2590[Medline] [Order article via Infotrieve]
  40. Quilliam, L. A., Hisaka, M. M., Zhong, S., Lowry, A., Mosteller, R. D., Han, J., Drugan, J. K., Broek, D., Campbell, S. L., Der, C. J. (1996) J. Biol. Chem. 271, 11076-11082[Abstract/Free Full Text]
  41. Mistou, M.-Y., Jacquet, E., Poullet, P., Rensland, H., Gideon, P., Schlichting, I., Wittinghofer, A., and Parmeggiani, A. (1992) EMBO J. 11, 2391-2397[Abstract]
  42. Polakis, P., and McCormick, F. (1993) J. Biol. Chem. 268, 9157-9160[Abstract/Free Full Text]
  43. Casey, P. J., Solski, P. A., Der, C. J., Buss, J. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8323-8327[Abstract]
  44. Hancock, J. F., Magee, A. I., Childs, J. E., Marshall, C. J. (1989) Cell 57, 1167-1177[Medline] [Order article via Infotrieve]
  45. Clarke, S., Vogel, J. P., Deschenes, R. J., Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647[Abstract]
  46. Guttierez, L., Magee, A. I., Marshall, C. J., Hancock, J. F. (1989) EMBO J. 8, 1093-1098[Abstract]
  47. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S., and Rine, J. (1989) Science 245, 379-385[Medline] [Order article via Infotrieve]
  48. Shou, C., Wurmser, A., Ling, K., Barbacid, M., and Feig, L. A. (1995) Oncogene 10, 1887-1893[Medline] [Order article via Infotrieve]


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