From the Department of Physiology II, Kobe University School of
Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
Ras proteins possess multiple downstream
effectors of distinct structures. We and others demonstrated that
Ha-Ras carrying certain effector region mutations could interact
differentially with its effectors, implying that significant
differences exist in their Ras recognition mechanisms. Here, by
employing the fluorescence polarization method, we measured the
activity of effector region synthetic peptides bearing various amino
acid substitutions to inhibit association of Ras with the effectors
human Raf-1 and Schizosaccharomyces pombe Byr2. The effect
of these peptides on association with another effector
Saccharomyces cerevisiae adenylyl cyclase was also examined
by measuring inhibition of the Ras-dependent adenylyl
cyclase activity. The peptide corresponding to the residues 17-44
competitively inhibited Ras association with all the three effectors at
the Ki values of 1~10 µM, and the
inhibition was considerably attenuated by the D38A mutation. The
peptide with the D38N mutation inhibited association of Ha-Ras with
Byr2 but not with the others, whereas that with the P34G mutation
inhibited association of Ha-Ras with Raf-1 and Byr2 but not with
adenylyl cyclase. Thus, the specificity observed with the whole Ras
protein was retained in the effector region peptide. These results
suggest that the effector region residues constitute a major
determinant for differential recognition of the effector molecules,
raising a possibility for selective inhibition of a particular Ras
function.
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INTRODUCTION |
The ras genes are widely conserved from yeasts to
mammals. Their protein products belong to a family of small guanine
nucleotide-binding proteins that operate in key processes of
intracellular signal transduction systems regulating cell growth and
differentiation. In higher eukaryotes including vertebrates, Ras
proteins bind directly and activate Raf (Raf-1, A-Raf, and B-Raf)
proteins, which results in activation of the mitogen-activated protein
kinase phosphorylation cascade (for reviews, see Refs. 1 and 2). In
addition, other mammalian proteins have been shown to associate directly with Ras in a GTP-dependent manner. These include
Ral-guanine nucleotide dissociation stimulator (3-5),
phosphatidylinositol 3-kinase (6), the
isoform of protein kinase C
(7), AF-6 (8), and Rin1 (9). However, the significance of their
interaction with Ras is presently unclear. In the fission yeast
Schizosaccharomyces pombe, its single Ras homologue, Ras1,
is involved in signal transduction from mating pheromone receptors, and
protein kinase Byr2 is its direct downstream effector (10, 11). On the
other hand, in the budding yeast Saccharomyces cerevisiae,
adenylyl cyclase is an immediate downstream effector of a pair of Ras
proteins, Ras1 and Ras2 (for a review, see Ref. 12).
Mutational studies of the effector molecules identified discrete Ras
binding regions. The Ras binding site of mammalian Raf-1 was mapped to
an 81-amino acid segment in its N-terminal regulatory domain (13-15).
Recently another Ras binding site was identified in Raf-1,
corresponding to the cysteine-rich region (16, 17). In yeast adenylyl
cyclase, the leucine-rich-repeat domain, which is composed of tandemly
repeated 23-amino acid leucine-rich motifs (18), was shown to bind
directly to Ras (19). Byr2 contains a Ras binding site within its
N-terminal 206 amino acid residues (11). Also, discrete Ras binding
domains were identified and mapped in other Ras effectors (3-9).
Comparison of the primary structures of these Ras binding domains
revealed very little conservation among them except that those of
Ral-guanine nucleotide dissociation stimulator, AF-6, and Rin1 share a
weakly homologous region called RA domain (20). However, recent studies
on the Ras binding regions of Raf-1 and Ral-guanine nucleotide
dissociation stimulator revealed that they exhibit similar
three-dimensional structures (21-23).
On the other hand, extensive mutational studies on Ras identified amino
acid residues whose substitution abolished the ability to transform
NIH3T3 cells, to induce neurite outgrowth in PC12 cells, to induce
germinal vesicle breakdown in Xenopus laevis oocytes, or to
associate with Raf-1 without affecting the guanine nucleotide binding
properties (24-31). These residues were concentrated in the positions
32-40 of mammalian Ras, so that this region was designated the
effector region. This region almost matches with "switch I," one of
the two regions that take significantly different conformations between
the GTP- and GDP-bound forms of Ras (32, 33). The x-ray
crystallographic study on the complex between a Ras homologue Rap1A and
Raf-1 Ras binding region has provided evidence for their association at
the atomic level (21). In addition, the residues 26-31 and 42-53
flanking the effector region were shown to be critical for effector
activation even though they do not change their conformations upon
GDP/GTP exchange (30, 31, 34). These residues have been proposed to
constitute the "activator region" (31) or "constitutive effector
region" (34), and this region was recently implicated in interaction
with the cysteine-rich region of Raf-1 (17).
Recently, we and others found that certain Ras mutants carrying
substitutions in the effector region could discriminate the effector
molecules (35-39). Specifically, more than 50 Ha-Ras mutants were
examined by us for interaction with Raf-1, Byr2, and yeast adenylyl
cyclase, the three effectors whose functions are firmly established by
both genetic and biochemical evidences (36). We found that the D38N
mutant lost the ability to associate with both Raf-1 and adenylyl
cyclase while retaining the activity to associate with and activate
Byr2. Also, the P34G mutation selectively abolished the ability of
Ha-Ras to bind and activate adenylyl cyclase without affecting the
interaction with Raf-1 and Byr2, suggesting that significant
differences exist in the recognition mechanisms by which distinct
effector molecules associate with Ras (36). These observations opened
up a possibility for selective inhibition of the function of a
particular effector by exploiting the differences in the Ras
recognition mechanisms. Here, we demonstrate that short synthetic
peptides corresponding to the effector region can be employed for this
purpose by showing that they retain the ability to differentially
recognize the effectors.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic peptides used in this experiment are
shown in Table I. They were synthesized with a solid-phase method and
purified by C18 reversed-phase high performance liquid chromatography
(purchased from Peptide Institute Inc., Osaka, Japan). The peptides
were dissolved in buffer A (20 mM Tris/HCl (pH 7.4), 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA) or in dimethylformamide and centrifuged at 100,000 × g for 30 min to remove insoluble materials.
Their concentrations were determined spectroscopically by measurement
of absorbance at 293 nm at pH 11.6. An
MBP1 fusion protein of the
Raf-1 N-terminal 206 residues, MBP-Raf-1-(1-206), and a GST fusion
protein of the Byr2 N-terminal 236 residues, GST-Byr2-(1-236), were
described before (11, 17, 36). Yeast strain FS3-1 (MAT
his3
leu2 trp1 ura3 ade8 cyr1-2 ras2::URA3) carrying
plasmids pAD4-GST-CYR1-(606-2026) and YEP-HIS3-ADC1-CAP, which
overexpressed a GST fusion protein of adenylyl cyclase and the
cyclase-associated protein CAP, respectively, under the control of the
ADC1 promoter, was described before (40). The
posttranslationally fully modified and unmodified forms of human Ha-Ras
were extracted from Sf9 cells infected with the baculovirus
carrying Ha-ras and purified as described previously
(41).
Measurement of Fluorescence Polarization--
The purified
unmodified form of Ha-Ras was labeled with fluorescein (*F) at its
N-terminal
-amino group by reaction with fluorescein
succinimidylester (Pan Vera Co., Madison, Wisconsin) at pH 7.0. The
fluorescein-labeled Ha-Ras (*F-Ha-Ras) was separated from the
unincorporated dye by gel filtration chromatography on Sephadex G-25.
One microliter of *F-Ha-Ras (about 105 fluorescence units
or 8 fmol/reaction) loaded with GTP
S or GDP
S was mixed with 1 ml
of buffer A containing various concentrations of the purified
MBP-Raf-1-(1-206) or GST-Byr2-(1-236) in a siliconized glass tube.
The reaction mixture was incubated for 15 min at 30 °C in the
darkness followed by measurement of the fluorescence polarization value
of each tube with BEACON (Pan Vera Co.) at an excitation wavelength of
490 nm and an emission wavelength of 520 nm. For assay of inhibition by
the synthetic peptides, similar measurements were performed with 100 nM MBP-Raf-1-(1-206) or GST-Byr2-(1-236) in the presence
of varying concentrations of the synthetic peptides.
A fluorescence polarization value is given by a difference between the
vertical and horizontal fluorescence intensities divided by a sum of
the vertical and horizontal fluorescence intensities and expressed as
mP units (42, 43). It reflects the molecular size of a fluorescently
labeled molecule (42, 44, 45). There occurs a large increase in the
molecular size of *F-Ha-Ras upon complex formation with the effector
proteins, causing an increase in the fluorescence polarization value.
Thus, the change in fluorescence polarization of *F-Ha-Ras is
proportional to the amount of the complex formed with the effector
molecule.
Adenylyl Cyclase Inhibition Assay--
Adenylyl cyclase was
solubilized from the crude membrane fraction of the yeast FS3-1
carrying pAD4-GST-CYR1-(606-2026) and YEP-HIS3-ADC1-CAP with buffer C
(50 mM Mes (pH 6.2), 0.1 mM MgCl2, 0.1 mM EGTA, 1 mM
-mercaptoethanol)
containing 1% LubrolPX, 0.6 M NaCl, and 1 mM
phenylmethylsulfonyl fluoride. After centrifugation at 100,000 × g for 1 h at 4 °C, the resulting supernatant was used for the measurement of adenylyl cyclase activity in the presence of varying concentrations of the GTP
S-bound form of
posttranslationally fully modified Ha-Ras as described before (19, 40).
For the measurement of inhibition of adenylyl cyclase activities by the synthetic peptides, varying amounts of the peptides were added to the
reaction mixtures before commencing the reaction.
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RESULTS |
Binding of Fluorescently Labeled Ha-Ras to N-terminal Regions of
Raf-1 and Byr2--
We employed the fluorescence polarization method
to quantitatively analyze the association of Ha-Ras with its various
effectors. The change in fluorescence polarization value of *F-Ha-Ras
is proportional to the amount of a complex formed with its effector molecule. In this experiment, *F-Ha-Ras was loaded with GTP
S or
GDP
S and examined for association with various concentrations of
MBP-Raf-1-(1-206) (Fig. 1A)
or GST-Byr2-(1-236) (Fig. 1B). *F-Ha-Ras alone exhibited an
mP value of approximately 100, and the complex formation of *F-Ha-Ras
with the effector polypeptides was detectable at the effector
concentration as low as 10 nM. The binding was almost
saturated at 100 nM for MBP-Raf-1-(1-206) or at 500 nM for GST-Byr2-(1-236) with the increase of the mP value
by approximately 30 units. The extent of increase was consistent with
that predicted from the increase in molecular size upon the complex
formation with the effector polypeptides based on the observation that
the reciprocals of the mP values correlate linearly with the reciprocal
values of the molecular weights of the fluorescently labeled proteins
(44, 45). This suggested that the binding reactions occurred
stoichiometrically. The maximal bindings were reached within 10 min of
incubation, and the values remained stable for the following 30 min
(data not shown). The binding reactions were dependent on the GTP-bound
configuration of *F-Ha-Ras, and almost no binding was observed for MBP
or GST only (Fig. 1, A and B). Since the
*F-Ha-Ras concentration used was very low at 8 pM,
essentially all of MBP-Raf-1-(1-206) or GST-Byr2-(1-236) added to the
reactions could be regarded as in a free form. When reciprocals of the
fluorescence polarization increase values were plotted against
reciprocals of the effector concentrations, a straight line could be
drawn with an intersect with the horizontal axis giving
Kd values of 35 nM for
MBP-Raf-1-(1-206) and 90 nM for GST-Byr2-(1-236) (Fig.
1C). These values are comparable although somewhat higher
than those estimated from the inhibition profiles of
Ras-dependent adenylyl cyclase activity by the same effector fragments (11, 19).

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Fig. 1.
Measurement of association of Ha-Ras with
Raf-1 and Byr2 by fluorescence polarization method. A, eight
fmol each of *F-Ha-Ras were loaded with GTP S and examined for
association with increasing concentrations of MBP-Raf-1-(1-206) ( )
or MBP (×) by using the fluorescence polarization method as described
under "Experimental Procedures." Shown in the longitudinal axis are
the increases in the fluorescence polarization values (mP) over that
obtained with *F-Ha-Ras alone (approximately 100 mP units). Similar
experiments were carried out with *F-Ha-Ras loaded with GDP S for
association with MBP-Raf-1-(1-206) ( ). The data represent the mean
and S.E. of a representative result of duplicate assays that were
repeated three times, yielding equivalent results. B,
association of the GTP S-loaded *F-Ha-Ras with GST-Byr2-(1-236)
( ) or GST (×) and that of the GDP S-loaded *F-Ha-Ras with
GST-Byr2-(1-236) ( ) were measured, and the results were shown as
described in A. C, double reciprocal plots of the
data on the association of the GTP S-loaded *F-Ha-Ras with
MBP-Raf-1-(1-206) ( ) or GST-Byr2-(1-236) ( ).
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Inhibition of Ras Effector Association by Synthetic
Peptides--
We next examined the ability of synthetic peptides
encompassing the Ha-Ras effector region with various single amino acid substitutions (Table I) to interfere with
the Ha-Ras-Raf-1 and Ha-Ras-Byr2 associations. The peptide
corresponding to the residues 17-44 was chosen because this peptide
had been used successfully in inhibition of the Ras binding to Raf-1 or
to protein kinase C
(7, 46). In addition, similar experiments were
carried out with synthetic peptides SII and AD, corresponding to the
switch II region (residues 58-76) of Ha-Ras and to a part of the
activator region (residues 39-51), respectively (Table I). Fig.
2 shows inhibition profiles of the
association of *F-Ha-Ras with MBP-Raf-1-(1-206) (Fig. 2A)
or with GST-Byr2-(1-236) (Fig. 2B) by increasing
concentrations of the various peptides. Wild-type effector region
peptide effectively inhibited the Ras association with both of the
effectors with 70% inhibition for Raf-1 and 55% inhibition for Byr2
at the concentration of 10 µM, whereas that carrying the
D38A mutation was much less effective. In addition, the specificities
of inhibition by the various peptides were clearly different between
the two effectors. Although P34G substitution did not notably affect
the inhibitory activity of the peptide against both Raf-1 and Byr2,
D38N substitution selectively impaired the inhibitory activity against
complex formation of *F-Ha-Ras with Raf-1-(1-206) but not with
Byr2-(1-236). Both SII and AD peptides had no inhibitory effect up to
the concentration of 10 µM. Kinetic analyses of the
inhibition patterns were carried out as follows to determine
Kd values of each peptide for the two effectors.
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Table I
Amino acid sequences of Ha-Ras peptides used in this study
Wild type peptide is derived from Ha-Ras; P34G, D38N, or D38A mutant
peptides cover the same residues as that of wild type. Identical
residues are shown by dashes.
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Fig. 2.
Inhibition of Ha-Ras associations with Raf-1
and Byr2 by synthetic peptides. A, association of *F-Ha-Ras
(8 fmol) with 100 nM MBP-Raf-1-(1-206) was subjected to
inhibition by increasing concentrations of the effector region peptides
(residues 17-41) wild type ( ), P34G ( ), D38N ( ), and D38A
( ) or of SII ( ) and AD ( ) peptides. B, similar
inhibition experiments by the peptides were carried out for 100 nM GST-Byr2-(1-236). The symbol representing each peptide
is the same as in A. The results shown in A and
B are representative of three independent experiments
yielding equivalent results. C,
Y/Yp was plotted against P for
inhibition of association with MBP-Raf-1-(1-206) by the peptides wild
type ( ), P34G ( ), D38N ( ), or D38A ( ), and the
Kd values of the peptides were calculated from the
intersect values on the horizontal axis as described in the text.
D, similar plotting and calculation of Kd
were carried out for inhibition of association with GST-Byr2-(1-236)
by the same set of peptides. The symbol representing each peptide is
the same as in C.
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The fluorescence polarization value (Y) is proportional to
the amount of *F-Ha-Ras bound to the effector and hence formulated as
Y = Ymax · X/(KRas + X), where
Ymax, X, and
KRas represent the maximal polarization value,
the effector concentration, and the Kd value of
*F-Ha-Ras for the effector, respectively. Assuming that the association
with *F-Ha-Ras is competitively inhibited by a peptide whose
concentration (P) is in a vast excess over X, the
concentration of the effector sequestered by the peptide is
X · P/(Kp + P),
where Kp represents the Kd value
of the peptide for the effector. Thus, the free effector concentration
available for association with *F-Ha-Ras is given by X · (1
P/(Kp + P)). The
fluorescence polarization value (Yp) in the presence
of the peptide (P) can be formulated as
Yp = Ymax · X
· (1
P/(Kp + P))/(KRas + X · (1
P/(Kp + P))). From the
two equations is derived Y/Yp = (KRas/Kp · (KRas + X)) · P + 1. When Y/Yp was plotted against
P for inhibition by the various peptides of association with
Raf-1 (Fig. 2C) or with Byr2 (Fig. 2D), a
straight line can be drawn for either case that converged on the
longitudinal axis at Y/Yp = 1. The
Kp value can be calculated as
KRas · P0/(KRas + X),
where P0 represents the P value at
the intersect on the horizontal axis (Fig. 2, C and
D). Thus, the Kp values for
MBP-Raf-1-(1-206) of wild-type, P34G, D38N, and D38A peptides were
calculated as 2.1, 4.4, 28, and 28 µM, respectively,
given that KRas is 35 nM and
X is 100 nM. Similarly, the
Kp values of the same set of peptides for
GST-Byr2-(1-236) were calculated as 3.8, 2.0, 4.7, and 39 µM, respectively.
Inhibition of Ha-Ras-dependent Adenylyl Cyclase
Activity by Synthetic Peptides--
The effects of the various
synthetic peptides on association between Ha-Ras and yeast adenylyl
cyclase were examined by measuring inhibition of
Ha-Ras-dependent adenylyl cyclase activity (Fig. 3). Wild-type peptide inhibited adenylyl
cyclase activity obtained by the 8 nM post-translationally
modified form of Ha-Ras by a half at approximately 21 µM
(Fig. 3A). A notable difference from Raf-1 and Byr2 was
observed in the effect of P34G substitution, which was found totally
ineffective in inhibition of adenylyl cyclase activity. Assuming that
the inhibitory associations of the peptides are competitive with that
of Ha-Ras, the inhibition constants (Ki) for the
peptides can be derived from a conventional equation V = Vmax · X/(Km · (1 + I/Ki) + X), where
V, Vmax, X,
Km, and I represent the
adenylyl cyclase activity, the maximal adenylyl cyclase activity, the
concentration of Ha-Ras, the Kd value for Ha-Ras,
and the inhibitor peptide concentration, respectively. Rearranging the
equation according to Lineweaver and Burk gives 1/V = 1/Vmax + Km · (1 + I/Ki)/Vmax · X.

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Fig. 3.
Inhibition of Ha-Ras-dependent
adenylyl cyclase activity by synthetic peptides. A, adenylyl
cyclase activity dependent on 8 nM GTP S-bound Ha-Ras was
subjected to inhibition by increasing concentrations of the effector
region peptides wild type ( ), P34G ( ), D38N ( ), or D38A ( )
or of SII ( ) and AD ( ) peptides. The results shown are
representative of three independent experiments yielding equivalent
results. B, adenylyl cyclase activities dependent on various
concentrations of Ha-Ras were subjected to inhibition by varying
concentrations of the wild-type effector region peptide, and a
reciprocal of the activity was plotted against a reciprocal of the
Ha-Ras concentration in the presence of the peptide concentration: 0 ( ), 10 µM ( ), 20 µM ( ), or 40 µM ( ). C, a similar plotting as
B was carried out for inhibition of
Ha-Ras-dependent adenylyl cyclase activity by the D38N
peptide with the concentration of 0 ( ), 10 µM ( ),
20 µM ( ), or 40 µM ( ). D,
a similar plotting as B was carried out for inhibition of
Ha-Ras-dependent adenylyl cyclase activity by the D38A
peptide with the concentration of 0 ( ), 10 µM ( ),
20 µM ( ), or 40 µM ( ). E,
reciprocals of the values at the intersections on the horizontal axis
in B, C, and D, apparent
Km, were plotted against the concentrations
(I) of inhibitor peptides wild type ( ), D38N ( ), and
D38A ( ). The Ki values for the peptides could be
obtained from the intersects on the horizontal axis as described in the
text.
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Adenylyl cyclase activities dependent on various concentrations of
Ha-Ras were subjected to inhibition by varying concentrations of the
peptides, and a reciprocal of the activity was plotted against a
reciprocal of the Ha-Ras concentration. This gave a series of straight
lines that converged on the longitudinal axis for each concentration of
the peptide, wild type (Fig. 3B), D38N (Fig. 3C),
and D38A (Fig. 3D), as predicted from the above equation. The intersect of each line on the horizontal axis corresponds to
1/Km · (1 + I/Ki).
Thus, Ki for each peptide could be determined by
plotting reciprocals of the intersect values against I (Fig.
3E). The Ki values could be estimated
from intersects of the straight lines on the horizontal axis and was
determined to be 10, 56, and 68 µM for wild-type, D38N, and D38A peptides, respectively.
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DISCUSSION |
Synthetic peptides encompassing the Ras effector region, in
particular the one corresponding to the residues 17-44, were used successfully to competitively inhibit association of Ras with its
effector proteins including Raf-1 (46) and the
isoform of protein
kinase C (7). Contact epitope scanning using a series of smaller
peptides suggested that many residues across the residues 17-51 may be
involved in direct interaction with Raf-1 N-terminal fragment (47). In
the present study, we have shown for the first time that introduction
of certain single amino acid substitutions can confer the ability to
discriminate Ras effectors, human Raf-1, fission yeast Byr2, and
budding yeast adenylyl cyclase upon the effector region peptide. The
association of the wild type and its mutant peptides with the three
effectors was quantitatively examined by measuring competitive
inhibition of the binding of the effectors to Ha-Ras, which was
monitored by the fluorescence polarization method (42-44). The use of
this method enabled us to measure the equilibrium binding constants of
the effector molecules for Ras in solution and to determine the
Kd values for association with the peptides from
kinetic analyses of the inhibition profile. This is certainly
advantageous over other nonequilibrium procedures such as an
enzyme-linked immunosorbent assay and an affinity precipitation of the
complex formed, both of which require the physical separation of free
and bound ligands considering that the binding nature of Ras to Raf-1,
for example, is substoichiometric and the dissociation rate is very
high (48).
The results clearly demonstrated selectivity in binding of the peptides
to the distinct effectors. Specifically, D38N substitution attenuated
the affinity of the effector region peptide for both Raf-1 and adenylyl
cyclase by about 10-fold without affecting that for Byr2. The
importance of Asp-38 of Ras for interaction with Raf-1 had been
indicated from extensive mutational and structural studies on the whole
Ras protein and its homologue Rap1A (21, 24-33). In contrast, P34G
substitution almost abolished the interaction with adenylyl cyclase but
did not appreciably affect that with Raf-1 or Byr2. These
specificities, summarized in Table II,
are in good coincidence with those observed with the whole Ha-Ras protein (36), indicating that residues 17-44 are sufficient to assume
a specific conformation that interacts selectively with a particular
set of the effectors depending on a single amino acid substitution. The
results strongly suggest that the effector region residues by
themselves constitute a major determinant for differential recognition
of the effector molecules, although we cannot exclude the possibility
that residues outside of the effector region may contribute to this
recognition process. Since other Ras effector molecules such as
Ral-guanine nucleotide dissociation stimulator and phosphatidylinositol
3-kinase were also known to possess distinct requirements for the
effector region residues from Raf-1, Byr2, or adenylyl cyclase and from
one another (37, 38), it may be possible to find a synthetic peptide
carrying certain amino acid substitutions that specifically recognize
any member of the multiple Ras effector molecules.
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Table II
Selective interaction of Ha-Ras mutant proteins and effector region
synthetic peptides with Ras effectors
Unless otherwise indicated, data on the interaction of Ha-Ras proteins
with effectors are from Ref. 36.
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Our results have also shown that it is possible to achieve selective
inhibition of the association of Ras with a particular effector
molecule without significantly affecting those with other effectors by
employing the antagonist peptide with a relatively small molecular size
around 3 kDa. This may open up a possibility to design a compound that
mimics a specific conformation of the effector region peptide bearing
the particular substitution, which is in theory deducible from the
x-ray crystallographic or nuclear magnetic resonance spectroscopic
analysis. From a viewpoint of effectiveness in cancer drug therapy, it
is possible that the effector-specific inhibition, which could
potentially be achieved by administration of such a compound, is
superior to the total inhibition of Ras functions by, for example, a
compound that directly inactivates Ras. In addition, the effector
region peptides carrying a series of substitutions might be found
useful as effector-specific inhibitors for analysis of individual
cellular functions of the distinct effector molecules.
We thank X.-H. Deng for skillful technical
assistance and T. Okada, A. Seki, and A. Kawabe for help in preparation
of this manuscript.