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Volume 270, Number 48, Issue of December 1, 1995 pp. 28834-28838
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Ras/Raf Interaction by Anti-oncogenic Mutants of Neurofibromin, the Neurofibromatosis Type 1 (NF1) Gene Product, in Cell-free Systems (*)

(Received for publication, August 7, 1995; and in revised form, September 14, 1995))

Sadao Mori (1),  Takaya Satoh (1),  Hiroshi Koide (1),  Masato Nakafuku (2),  Ernie Villafranca (3),  Yoshito Kaziro (1)(§)

From the  (1)Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan, (2)Institute of Advanced Science and Technology, Nara, Ikoma 630-01, Japan, and (3)Agouron Pharmaceuticals, Inc., San Diego, California 92121-1121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Rasbulletguanosine 5`-O-(3-thiotriphosphate) (GTPS) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward RasbulletGTPS is approximately 5-10 times higher than the wild type. These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.

INTRODUCTION

Mammalian Ras protein acts as a molecular switch regulating intracellular signal transduction. Ras is implicated in various kinds of signaling pathways including proliferation of fibroblast cells, differentiation of pheochromocytoma PC12 cells, T-cell activation, and lymphokine-induced cellular responses in various hematopoietic cell lines. Activity of Ras is controlled by two types of regulators, GDP/GTP exchange factors (GEFs), (^1)and GTPase-activating proteins (GAPs). GEFs stimulate the GDP/GTP exchange reaction that causes the formation of active GTP-bound form, while GAPs enhance the GTPase activity to turn off the signal from Ras. Recently, it has been clarified that mSos-1, a member of GEFs, is involved in a signaling cascade from a receptor tyrosine kinase to Ras. However, the role of GAPs in a signal-dependent modulation of RasbulletGDP/GTP state remains unclear (see (1, 2, 3, 4) for reviews).

The neurofibromatosis type 1 (NF1) responsible gene product, neurofibromin, is a protein consisting of 2,818 or 2,139 amino acids (5, 6, 7, 8, 9) . Neurofibromin belongs to a family of Ras-GAPs; it contains GAP-related domain (GRD), which is found in all mammalian and yeast GAPs(10) . In addition, NF1-GRD by itself has an ability to bind Ras protein, and to stimulate its GTPase activity in a cell-free system (11, 12, 13) . Two GAP-related genes of Saccharomyces cerevisiae, IRA1 and IRA2, were isolated and characterized as genes encoding GTPase stimulators of yeast Ras proteins(14, 15) . Not only GRD itself, but also its flanking regions in Ira1 and Ira2 proteins, share homology with the mammalian NF1 gene product; actually, NF1 protein is capable of interacting with yeast Ras proteins(14, 16) .

By screening a library of NF1-GRD cDNA to which random mutation was introduced by chemical treatment, we have isolated two mutant NF1-GRD clones (designated NF201 and NF204) that suppress the heat shock-sensitive phenotype characteristic of a S. cerevisiae strain carrying an activated mutation (G19V) of Ras, but show no inhibitory effect on the normal growth(17) . In NF201 and NF204, single amino acid substitutions (F1434L for NF201, and K1436R for NF204, respectively) were identified at neighboring positions, suggesting that this surrounding region is important for NF1-GRD/Ras interaction. These mutants exhibit no reduction in their GTPase-stimulating activity, and thus, they are able to complement ira phenotypes of S. cerevisiae. In addition, the obtained NF1-GRD mutants are able to revert the transformation-specific morphology of NIH 3T3-derived Ki-ras-transformed fibroblasts.

In the present paper, we examined the effects of the mutant NF1-GRDs on the interaction of Ras with its effector, Raf, in whole cell and cell-free systems to clarify the mechanism of the anti-oncogenic action of the mutant NF1-GRDs. We found that the affinity of NF1-GRD mutants to the GTP-bound form of Ras is increased by 5-10-fold, suggesting that the mutants tightly bind to RasbulletGTP to form a stable complex and block the interaction of oncogenic Ras with its target.

MATERIALS AND METHODS

Plasmids, Recombinant Proteins, and Antibodies

Mammalian expression plasmids pEF-NF1, pEF-NF201, and pEF-204 have been described elsewhere(17) . Recombinant proteins of the wild-type and mutant NF1-GRDs (amino acid residues from 840 to 1,204) were purified from Escherichia coli. pCMV5-Ras(G12V) and pCMV5-PDGFR were constructed by inserting cDNAs encoding an activated Ras(G12V) protein (18) and mouse beta-type platelet-derived growth factor (PDGF) receptor (19) into pCMV5(20) . E. coli expression plasmids for glutathione S-transferase (GST)-MAP kinase(K57D) and His-MAP kinase kinase were kindly provided by Dr. Eisuke Nishida (Kyoto University, Kyoto, Japan)(21, 22) . Human Ha-Ras proteins were overexpressed in E. coli and purified as described elsewhere (23) . c-RafDeltaC-FH6 protein purified from baculovirus-infected Sf9 cells were kindly provided by Dr. Martin McMahon (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA). Anti-Ras antibody Has 6 was a generous gift from Dr. Takeo Tanaka (Kure National Hospital, Kure, Japan). Human PDGF-BB was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase-labeled anti-mouse IgG antibody (Amersham Corp.), horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham), anti-Ras antibody LA069 (Quality Biotechnology, Inc., Camden, NJ), anti-mouse IgG (Cappel), anti-FLAG antibody M2 (Kodak), and anti-ERK2 antibody (Upstate Biotechnology, Inc.) were commercially purchased.

Preparation of Rat Brain Lysate

Rat brain lysate was prepared essentially according to the method described previously(24) . Briefly, rat brain was homogenized on ice in buffer A (20 mM Hepes-NaOH (pH 7.3), 10 mM MgCl(2), 1 mM EDTA, 1 mM EGTA, 25 mM beta-glycerophosphate, 10 mM NaF, 1 mM Na(3)VO(4), 20 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A) supplemented with 250 mM sucrose. 0.5% (v/v) Triton X-100 and 150 mM KCl were then added to the homogenate, and the lysate was obtained by centrifugation at 15,000 times g for 30 min.

Transient Expression in Human Embryonic Kidney (HEK) 293 Cells

HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. Plasmid DNA was introduced into the cell by a standard calcium phosphate method, and the culture medium was replaced by DMEM containing 1 mg/ml bovine serum albumin on the next day. After additional culture for 1 day, the cells were harvested and lysed into buffer A plus 1% (v/v) Triton X-100 and 150 mM KCl. Cell lysates were obtained by centrifugation at 15,000 times g for 5 min.

Preparation of RasbulletGuanine Nucleotide Complexes

Ras protein was incubated in exchange buffer (50 mM Tris-HCl (pH 7.5), 2 mM MgCl(2), 150 mM NH(4)Cl, 1 mg/ml bovine serum albumin, 5 mM EDTA) in the presence of 1 mM GDPbetaS or GTPS at 30 °C for 20 min. The reaction was terminated by adding 10 mM MgCl(2) and chilling on ice(18) .

MAP Kinase or Extracellular Signal-regulated Kinase (ERK) Kinase (MEK) Kinase Assay

Ras protein complexed with GDPbetaS or GTPS (300 nM) and NF1-GRD at various concentrations were mixed with anti-Ras antibody LA069, anti-mouse IgG, and protein A-Sepharose (Pharmacia Biotech Inc.) suspended within buffer A. Rat brain lysate (1.25 mg/ml) was added to the mixture, followed by incubation at 4 °C for 2 h. Immunoprecipitates were washed twice with wash buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl(2), 150 mM NaCl), once with kinase buffer without phosphatase inhibitors, and suspended in kinase buffer (20 mM Hepes-NaOH (pH 7.3), 5 mM MgCl(2), 0.5 mM MnCl(2), 25 mM beta-glycerophosphate, 10 mM NaF, 1 mM Na(3)VO(4)). GST-MAP kinase (K57D) (100 µg/ml), His-MAP kinase kinase (33.5 µg/ml), and [-P]ATP (50 µM, 37 TBq/mol) were added, and the mixture was incubated at 30 °C for 10 min. The samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. Radioactivity of each band was quantitated by a BAS 2000 image analyzer (Fuji Film).

Co-immunoprecipitation of Ras and c-RafDeltaC-FH6

Ras protein complexed with GDPbetaS or GTPS (300 nM) and NF1-GRD were mixed with anti-Ras antibody LA069, anti-mouse IgG, and protein A-Sepharose suspended within buffer A. c-RafDeltaC-FH6 (60 nM) and rat brain lysate (0.63 mg/ml) was added to the mixture, followed by incubation at 4 °C for 2 h. Immunoprecipitates were washed three times with wash buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl(2), 150 mM NaCl), and subjected to SDS-polyacrylamide gel electrophoresis and subsequent Western blotting using anti-FLAG antibody M2, horseradish peroxidase-labeled anti-mouse IgG, and enhanced chemiluminescence detection reagents.

Measurement of GTPase-stimulating Activity

A competition assay of GAP activity was carried out essentially as described elsewhere(25) . [-P]GTP-bound Ras was prepared by nucleotide exchange reaction in the presence of [-P]GTP (74 TBq/mol). [-P]GTP-bound Ras (5 nM), NF1-GRD (0.7 nM), and various concentrations of RasbulletGTPS were mixed in GTPase buffer (50 mM Tris-HCl (pH 7.5), 15 mM MgCl(2), 150 mM NH(4)Cl, 5 mM EDTA, 1 mg/ml bovine serum albumin), and incubated at 30 °C for 5 min. Radioactivity bound to Ras was measured by a filter binding assay described previously(18) .

RESULTS

Two types of NF1-GRD mutants (NF201 and NF204) inhibit activated Ras-induced phenotypes, but not normal cell growth, in both S. cerevisiae and mouse cells. Since NF1-GRD directly binds to Ras protein and regulates its GTPase activity, it seems likely that the anti-oncogenic action of NF201 and NF204 can be explained by their specific interference with the interaction between oncogenic Ras and its direct effector molecules.

Our previous results indicated that, in Ki-ras-transformed NIH 3T3 cells, the NF1-GRD mutants reversed malignant morphology of the transformed cells without blocking the normal growth(17) . In this study, we utilized transient expression systems to examine whether the mutant NF1-GRDs were capable of inhibiting the function of oncogenic, but not endogenous normal Ras. In various types of mammalian cells, the MAP kinase cascade is known to function downstream of Ras protein, where the activation of MAP kinase depends on the phosphorylation by MAP kinase kinase (MEK), and the phosphorylation of specific residues is known to be sufficient for its activation (see (26) and (27) for reviews). Thus, we introduced expression vectors containing the wild-type and mutant NF1-GRD cDNAs into HEK 293 cells and tested the effects on Ras-mediated hyper-phosphorylation of an endogenous MAP kinase (ERK2) (Fig. 1). In these experiments, phosphorylated ERK2 was detected as a mobility-retarded band blotted by an ERK2-specific antibody. As illustrated in Fig. 1, an activated mutant Ras(G12V) induced the phosphorylation of ERK2, which was diminished by simultaneous transfection of NF201 or NF204, but not of the wild-type NF1-GRD. Immunoblotting using anti-NF1-GRD antibody showed that the amounts of NF1-GRD expressed within each transfectant were equal (data not shown). The results indicate that the mutant NF1-GRDs effectively block the signaling from transforming Ras to MAP kinase in mammalian cells. We also tested the effects of NF201 and NF204 on the PDGF-induced phosphorylation of ERK2 mediated by the endogenous Ras protein. Phosphorylation of ERK2 in HEK 293 cells transfected with an expression plasmid of the PDGF receptor was detected after PDGF treatment for 10 min. Neither NF201 nor NF204, when expressed with the PDGF receptor in HEK 293 cells, caused inhibition of PDGF-promoted MAP kinase phosphorylation, whereas the PDGF-induced phosphorylation of ERK2 disappeared when a dominant-negative mutant Ras (S17N) was expressed, suggesting that the endogenous Ras is implicated in the signaling pathway from the PDGF receptor to MAP kinase (data not shown).

Figure 1: Inhibition of oncogenic Ras(G12V)-induced ERK2 phosphorylation in HEK 293 cells. pEF-NF1 (WT), pEF-NF201 (201), pEF-NF204 (204), or control vector (20 µg each) was introduced into HEK 293 cells with pCMV5-Ras(G12V) (1 µg). Mobility retardation of phosphorylated ERK2 was detected by immunoblot analysis using antibodies specific to ERK2. Arrows indicate the bands of phosphorylated and unphosphorylated ERK2.


To further assess the hypothesis that the NF1 mutants suppress the function of Ras(G12V) by binding more strongly than the wild-type NF1-GRD, we next compared the ability of the wild-type and mutant NF1-GRDs to inhibit the interaction of Ras and its effector in cell-free systems. Specific association of GTP-bound Ras and Raf serine/threonine kinases has been shown by coprecipitation and affinity chromatography(24, 28, 29, 30, 31) . Furthermore, Ras/Raf interaction was detected in intact yeast cells utilizing the two-hybrid system(29, 31, 32) . These results strongly suggest that Raf is a direct target of Ras in the signal transduction of mammalian cells, although the regulatory mechanism of Raf kinase activity following the binding of Ras has not been fully understood (see (26) and (27) for reviews). On the other hand, Raf phosphorylates and subsequently activates MAP kinase kinase (MEK), the activator of MAP kinase, in various cell types. Thus, the interaction of Ras and the effector can be quantitated by measuring MEK kinase activity associated with Ras protein. Fig. 2shows dose-dependent inhibition by the wild-type and mutant NF1-GRDs of co-immunoprecipitation of MEK kinase activity in rat brain lysate with recombinant GTPS-bound Ras protein. In this experiment, MEK kinase activity within Ras immunoprecipitates was quantitated by incorporation of P into E. coli-produced kinase-negative MAP kinase as a substrate in the presence of recombinant MEK. MEK kinase activity in the immunoprecipitate of GDPbetaS-bound Ras as a control was almost undetectable compared to the activity associated with RasbulletGTPS, suggesting that the MEK kinase activity was precipitated through the interaction with the effector domain of RasbulletGTP. Although there are several subtypes of Raf protein responsible for Ras-dependent MEK kinase activity, B-Raf seems predominant in terms of the activity in rat brain lysate ((33) , and data not shown). As illustrated in Fig. 2, both NF201 and NF204 completely abolished the coprecipitation of MEK kinase activity at 10 nM, while the inhibitory effects were detected only at the concentrations more than 50 nM in the case of the wild-type NF1-GRD. Radioactivity incorporated into each band was quantitated, and IC values were calculated as 3.5 nM for NF201 and NF204, and 8 nM for the wild type, respectively, from the data of three independent experiments.

Figure 2: Inhibition of the association of MEK kinase activity with RasbulletGTPS by NF1-GRD mutants. The activity of MEK kinase immunoprecipitated with RasbulletGTPS or RasbulletGDPbetaS (as a control) by anti-Ras antibody (LA069) was measured by in vitro kinase assay using GST-MAP kinase(K57D), His-MAP kinase kinase, and [-P]ATP as substrates. The wild-type and mutant NF1-GRDs were included during the immunoprecipitation at various concentrations. Bands corresponding to the phosphorylated GST-MAP kinase(K57D) are shown.


Association of Ras and Raf can be assessed also by Western blotting analysis following co-immunoprecipitation of Raf with Ras. We used recombinant Ha-Ras protein and the N-terminal fragment of c-Raf-1 consisting of 324 amino acids tagged with FLAG sequence (designated c-RafDeltaC-FH6) because the C-terminal catalytic region of c-Raf-1 protein is not required for the binding to Ras. Fig. 3shows the inhibitory effect of NF1-GRD on the association of Ras and c-Raf-1. In this case also, the immunoprecipitate of RasbulletGDPbetaS did not contain any detectable amount of c-RafDeltaC-FH6, whereas the GTPS-bound Ras associated with the Raf fragment. At the concentration of 200 nM, both NF201 and NF204 were able to compete against the association of Ras and Raf, while the wild-type NF1-GRD could not diminish the interaction at this condition.

Figure 3: Inhibition of the association of c-RafDeltaC-FH6 with RasbulletGTPS by NF1-GRD mutants. c-RafDeltaC-FH6 immunoprecipitated with RasbulletGTPS, or RasbulletGDPbetaS (as a control) by anti-Ras antibody (LA069) was measured by immunoblot analysis using anti-FLAG antibody M2. The wild-type and mutant NF1-GRDs were included during the immunoprecipitation at various concentrations. Bands corresponding to c-RafDeltaC-FH6 are shown.


Taken together with the above results, it is suggested that the affinity of the mutant NF1-GRDs toward Ras protein in its GTP-bound conformation is increased. Then, we measured the affinity between the wild-type and mutant NF1-GRDs and RasbulletGTPS by a competition assay of the GTPase-stimulating activity. Both the wild-type and mutant NF1-GRDs possess similar levels of GTPase-stimulating activity ((17) , and data not shown). Suppression of the GTPase-stimulating activity occurs when excess amounts of GTPS-bound Ras exist as competitive inhibitors against P-labeled RasbulletGTP. As shown in Fig. 4, GTPase-stimulating activities of the mutant NF1-GRDs were decreased at lower concentrations of RasbulletGTPS compared with the wild type, indicating that the affinity of NF201 and NF204 to RasbulletGTPS was higher than that of the wild type. IC was calculated as 4 nM for NF201, 7 nM for NF204, and 35 nM for the wild-type NF1, respectively.

Figure 4: Competitive inhibition of GAP activity of NF1-GRD by RasbulletGTPS. GAP activities of the wild-type and mutant NF1-GRDs were measured by a filter binding assay using Rasbullet[-P]GTP as a substrate. Various concentrations of RasbulletGTPS were added to the reaction as competitive inhibitors. Relative GAP activity in comparison with the value without RasbulletGTPS as 100% (wild type, circle; NF201, ; NF204, ) are shown as mean values of two or three independent experiments.


DISCUSSION

In a previous study(17) , we demonstrated that a single amino acid substitution can confer a strong anti-oncogenic activity specific to activated Ras protein on the NF1 gene product. Furthermore, the mutant NF1-GRDs exhibited no inhibitory effect on normal cell growth regulated by endogenous Ras proteins. In this study, we further analyzed the molecular mechanism of their anti-oncogenic action. We assumed that the mutant NF1-GRDs tightly bind to transforming Ras with higher affinity than the wild-type NF1-GRD blocking the interaction between Ras and its target molecules. In contrast, the signal transduction through endogenous normal Ras is not affected because the normal Ras is rapidly converted to an inactive GDP-bound form, to which the NF1-GRDs can no longer bind. This hypothesis is based on the following observations; 1) NF1-GRD directly binds to the effector domain of Ras, which is crucial also for association with an effector, for instance c-Raf-1; and 2) the NF1-GRD mutants suppressed the activity of transforming Ras mutants both in S. cerevisiae and mammalian cells although the direct targets of Ras are different between these organisms.

First, we found that the NF1-GRDs inhibited Ras(G12V)-induced, but not PDGF receptor and endogenous normal Ras-mediated, MAP kinase phosphorylation in a transient expression system using HEK 293 cells. The results support the assumption that the mutant NF1-GRDs block the signaling by binding and sequestering oncogenic Ras because the MAP kinase cascade functions at immediate downstream of Ras. Then, we reconstituted the association of Ras and its effector, Raf, in cell-free systems and analyzed the effects of NF201 and NF204. MEK kinase activity as well as the amounts of recombinant c-RafDeltaC-FH6 co-immunoprecipitated with RasbulletGTPS were decreased in the presence of the mutant NF1-GRDs at lower concentrations than the wild type. Binding affinities of NF201 and NF204 to Ras calculated by a competition assay of the GAP activity were 5-9 times higher than the wild-type NF1-GRD. Initially, we attempted to measure the dissociation constants of RasbulletNF1-GRD complex more directly using the purified recombinant proteins by biophysical procedures such as tryptophan fluorescent quenching. However, these attempts have not been successful.

Neurofibromin has been postulated as a tumor suppressor gene product because RasbulletGTP level is constitutively high in malignant Schwannoma cells from NF1 patients although Ras and GAP are functionally normal (34, 35) . However, in other types of cells, for example melanoma and neuroblastoma cell lines, growth-inhibitory function of neurofibromin seems independent of GTPase-enhancing activity(36) . Furthermore, neurofibromin displays tumor-suppressive properties in v-ras-transformed NIH 3T3 cells(37) , in favor of the possibility that the anti-tumor function of neurofibromin is independent of GAP activity. Our observations presented in this paper support a presumable mechanism, the specific inhibition of Ras/Raf interaction, which may also explain the GTPase stimulation-independent function of neurofibromin, although the full-length neurofibromin diminishes also normal cell growth in contrast to NF201 and NF204(17, 37) . In mammalian cells, GAP(2) , phosphatidylinositol 3-kinase(38) , ral guanine nucleotide dissociation stimulator(39, 40, 41) , and Rin1(42) , in addition to Raf family proteins, are reported to interact with the effector region of Ras. Therefore, it is likely that several kinds of signaling pathways are controlled by Ras, although, in this paper, we described the inhibitory action of the NF1 mutants only to Ras/Raf interaction. It may be interesting to compare the effect of the NF1 mutants on different effectors in future experiments.

It has been reported that lysine 1423, which is conserved among mammalian, Drosophila, and yeast GAPs, is crucial for the function of NF1 protein. Somatic mutations of this residue were found in various types of cancers(43) . Biochemical studies have shown that mutation of NF1-GRDs at this position resulted in the loss of their GTPase-stimulating activity (43, 44, 45) as well as the thermal stability (46) , suggesting that lysine 1423 plays an important role in interacting with Ras. Further investigation by Poullet et al.(45) has revealed that the second mutation at position 1434 from phenylalanine to serine rescued the mutation at lysine 1423. Phenylalanine 1434 may also be involved in Ras/NF1-GRD interaction because NF201(F1434L) gains higher affinity to Ras as described in this paper. The mutant NF1-GRD(F1434S) of Poullet et al.(45) , like NF201, is also capable of suppressing the heat shock-sensitive phenotype of S. cerevisiae caused by Ras2(G19V). However, as previously discussed by Poullet et al.(45) , it is possible that, in contrast to the case of NF201(F1434L), the effects of NF1-GRD(F1434S) may be independent of Ras interaction because no significant increase of the affinity toward Ras was detected in NF1-GRD(F1434S). Moreover, NF1-GRD(F1434S) is growth-inhibitory in S. cerevisiae like the full-length NF1 protein in NIH 3T3 cells. From these observations, it is possible that an additional mechanism to reduce the transforming Ras function may exist also in the case of NF201 and NF204. Interestingly, a 56-amino acid fragment of NF1-GRD(1441-1496) without any GAP activity, which is located in close vicinity to, but outside, the above mutation sites, is also able to abolish malignant phenotypes of v-ras-transformed NIH 3T3 cells(47, 48) . Although the precise mechanism of anti-oncogenic action of this fragment remains unclear at present, it may function as a competitive inhibitor of a target of Ras like NF201 and NF204.

Inhibitors against the lipid modification of Ras protein are considered promising anti-cancer reagents. The farnesylation inhibitors do not affect normal Ras function since unmodified endogenous Ras accumulates in cytoplasm as a GDP-bound form. On the other hand, unmodified oncogenic Ras, which accumulates in cytoplasm as a GTP-bound form, may act as a dominant-negative inhibitor. NF201 and NF204 also show strong anti-oncogenicity in spite of their innocuous properties to normal Ras-mediated signal transduction. Hence, it is possible, in future, that these molecules may be useful tools for the gene therapy of human cancers, and for this purpose, it is desirable to isolate a stronger mutant of NF1-GRD. We made a NF1-GRD carrying double mutations (F1434L/K1436R) by site-directed mutagenesis, and tested whether it exhibited more severe effects on transforming Ras. However, the double mutant suppressed heat shock sensitivity of S. cerevisiae, carrying Ras(G19V) only to an extent similar to that in NF201 and NF204 (data not shown). Probably, it is necessary to make a mutation within a region distinct from the domain including phenylalanine 1434 and lysine 1436 to see an additive effect.

FOOTNOTES

*
Work at the Tokyo Institute of Technology was supported by Schering-Plough Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226, Japan. Tel.: 81-45-924-5745; Fax: 81-45-924-5822.

(^1)
The abbreviations used are: GEF, GDP/GTP exchange factor; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; GAP, GTPase-activating protein; GDPbetaS, guanosine 5`-O-(2-thiodiphosphate); GRD, GTPase-activating protein-related domain; GST, glutathione S-transferase; GTPS, guanosine 5`-O-(3-thiotriphosphate); HEK, human embryonic kidney; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase or ERK kinase; NF1, neurofibromatosis type 1; PDGF, platelet-derived growth factor.

ACKNOWLEDGEMENTS

We are grateful to Martin McMahon, Eisuke Nishida, and Lewis T. Williams (University of California, San Francisco, CA) for providing us with recombinant proteins and expression plasmids.

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