(Received for publication, August 7, 1995; and in revised form, September 14, 1995))
From the
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 Rasguanosine 5`-O-(3-thiotriphosphate)
(GTP
S) 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 Ras
GTP
S 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.
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), ()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 Ras
GDP/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 RasGTP to form a
stable complex and block the interaction of oncogenic Ras with its
target.
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 GDP
S-bound Ras as a control was almost
undetectable compared to the activity associated with
Ras
GTP
S, suggesting that the MEK kinase activity was
precipitated through the interaction with the effector domain of
Ras
GTP. 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 RasGTP
S by NF1-GRD mutants. The
activity of MEK kinase immunoprecipitated with Ras
GTP
S or
Ras
GDP
S (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-RafC-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 Ras
GDP
S did not
contain any detectable amount of c-Raf
C-FH6, whereas the
GTP
S-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-RafC-FH6 with Ras
GTP
S by NF1-GRD mutants.
c-Raf
C-FH6 immunoprecipitated with Ras
GTP
S, or
Ras
GDP
S (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-Raf
C-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 RasGTP
S 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 GTP
S-bound Ras exist as competitive
inhibitors against
P-labeled Ras
GTP. As shown in Fig. 4, GTPase-stimulating activities of the mutant NF1-GRDs
were decreased at lower concentrations of Ras
GTP
S compared
with the wild type, indicating that the affinity of NF201 and NF204 to
Ras
GTP
S 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 RasGTP
S. GAP activities of the wild-type and
mutant NF1-GRDs were measured by a filter binding assay using
Ras
[
-
P]GTP as a substrate. Various
concentrations of Ras
GTP
S were added to the reaction as
competitive inhibitors. Relative GAP activity in comparison with the
value without Ras
GTP
S as 100% (wild type,
; NF201,
; NF204,
) are shown as mean values of two or three
independent experiments.
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-RafC-FH6 co-immunoprecipitated with
Ras
GTP
S 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
Ras
NF1-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 RasGTP 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.