(Received for publication, January 29, 1997, and in revised form, February 18, 1997)
From the Raf-1 is a major downstream effector of mammalian
Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation.
However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region
(CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to
activate Raf-1. Since these mutations replace Ras residues with those
of Rap1A, we examined if Rap1A lacks the ability to bind to CRR.
Contrary to the expectation, Rap1A exhibited a greatly enhanced binding
to CRR compared with Ha-Ras. Enhanced CRR binding was also found with
Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and
Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with
Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced
binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with
Raf-1 N-terminal region through binding to CRR and Ras-binding domain,
respectively. These results suggest that Rap1A interferes with
Ras-dependent Raf-1 activation by inhibiting binding of Ras
to Raf-1 CRR.
Ras belongs to a family of small GTP-binding proteins playing
essential roles in cell proliferation and differentiation. Mammalian ras genes carrying activating mutations are found in many
types of neoplastic tissue and are able to induce morphological
transformation in vitro when transfected into fibroblast
cell lines. However, the rap1A gene (1), encoding a 21-kDa
GTP-binding protein with high homology to Ras, has been shown to to
induce reversion of the transformed phenotype in
Ki-ras-transformed NIH3T3 cells (2). In addition to the
overall structural homology, Rap1A shares two important structural
features with Ras. One is that Rap1A has an identical effector domain
(amino acids 32-40) to that of Ras. The effector domain of Ras is
essential for the association with and activation of its effectors (3).
The other is that Rap1A undergoes similar post-translational
modification to Ras at its C terminus except that Ras is farnesylated
and Rap1A is geranylgeranylated (4). This modification is essential for
the function of Rap1A as observed for Ras (5, 6).
Raf-1, a serine/threonine kinase regulating the mitogen-activated
protein kinase cascade, is a major mammalian Ras effector and is
thought to play a key role in Ras-induced cellular transformation (7).
Although the precise mechanism of Ras-dependent Raf-1 activation remains unclear, it is known that the effector domain of Ras
interacts with the N-terminal RBD1 (amino
acids 51-131) of Raf-1 and that this interaction is essential for
physical association between these proteins as well as for the
activation of Raf-1 (7). Rap1A, too, has been shown to associate with
Raf-1 N-terminal fragment in vivo (8), and a recent x-ray
diffraction study of the crystal of the complex between Rap1A and Raf-1
RBD has provided evidence for this association at the atomic level (9).
These studies suggest the possibility that the suppression of Ras
function by Rap1A is due to the competitive inhibition of Ras-RBD
interaction (10), although it is unclear why Rap1A cannot activate
Raf-1.
We have recently identified Raf-1 CRR (amino acids 152-184) as another
Ras-binding domain and demonstrated that interaction of Ras with both
RBD and CRR is necessary for the activation of Raf-1 (11). Two
mutations, N26G and V45E, were found to abolish the interaction of
Ha-Ras with CRR and attenuate the activation of Raf-1 by Ha-Ras. The
fact that both of these mutations replaced Ha-Ras residues with
corresponding Rap1A residues prompted us to examine the possibility
that the inability of Rap1A to activate Raf-1 is due to its failure to
interact with Raf-1 CRR. Contrary to the expectation, we found that
Rap1A exhibited a greatly enhanced ability to bind to CRR.
Rap1A cDNA was amplified from a human lung
fibroblast cDNA library by polymerase chain reaction (12) using a
pair of primers, 5 MBP fusion proteins of
Raf-1 N-terminal fragments were expressed in Escherichia
coli and immobilized on amylose resin as described (11). Binding
reaction was carried out by incubating 20 µl of the resin carrying
various amounts of MBP-Raf-1 proteins with various amounts of GTP Monolayers of Sf9 cells
(2 × 107 cells) were triply infected with the
recombinant baculoviruses expressing the full-length Raf-1 and
Ha-RasV12, along with that expressing
Ha-RasV12(E31K) or Rap1AV12 (1 × 108 plaque-forming units each). After 72 h
post-infection, the cells were lysed by sonication in 1 ml of buffer B
(20 mM Tris/HCl, pH 7.5, 137 mM NaCl, 1%
Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 1 mM sodium vanadate)
and centrifuged at 13,000 × g for 30 min. Raf-1 was
immunoprecipitated from the supernatant (200 µl) with the anti-Raf-1
antibody C12 (2 µl) (Santa Cruz Biotechnology Inc.) and protein
A-agarose. The Raf-1 activity was determined by incubating the
immunoprecipitates in the presence of GST-MEK (13 µg) and GST-KNERK2
(1 µg) in 30 µl of kinase reaction mixture (20 mM
Tris/HCl, pH 7.5, 10 mM MnCl2, 10 mM MgCl2, 20 mM
In the
previous study, we have shown that Ha-Ras binds to immobilized
MBP-Raf-1(132-206), representing CRR, demonstrating that Raf-1 CRR
acts as another Ras-binding domain independently of RBD (11). This
binding was GTP-independent in contrast to the
GTP-dependent binding to MBP-Raf-1(50-131), representing
RBD. Ha-Ras mutations N26G and V45E abolished binding to CRR without affecting binding to RBD. The fact that these two mutations replaced Ha-Ras residues with those of Rap1A prompted us to examine if Rap1A
lacks the ability to bind to Raf-1 CRR. In the same in vitro binding assay, Rap1A bound to RBD in a GTP-dependent manner
(Fig. 1A, lanes 1 and
2), although the GTP dependence of this binding was less
clear compared with that of Ha-Ras (11). Unexpectedly, Rap1A bound
efficiently to CRR (lanes 3 and 4). As observed
for Ha-Ras, this binding was GTP-independent (lanes 3 and
4) and required an intact zinc finger structure of CRR
because Rap1A bound very poorly to MBP-Raf-1(132-206, C168S) (Fig.
1B). Binding of Rap1A to MBP-Raf-1(48-206) containing both
RBD and CRR was severalfold stronger and less GTP-dependent
than that to RBD (Fig. 1A, lanes 5 and
6). These results suggested that CRR is necessary for
efficient association between Rap1A and Raf-1 and that CRR rather than
RBD plays a major role in this association.
Comparison of the binding properties between Ha-Ras and Rap1A is shown
in Fig. 1C. Ha-Ras bound to CRR yielded roughly 10-fold less
signal than that to RBD even though the amounts of CRR in the binding
reaction were doubled, indicating that the ability of Ha-Ras to bind to
CRR was roughly 20-fold less than that to RBD. This estimation by
immunoblot was consistent with our kinetic measurement of the affinity
of Ha-Ras for RBD and CRR using a competitive inhibition of
Ha-Ras-dependent activation of Saccharomyces cerevisiae adenylyl cyclase (15, 16).2
In a striking contrast, Rap1A bound to CRR yielded stronger signal than
that to RBD under the same condition, indicating that it has the
ability to bind equally to RBD and to CRR (see also Fig. 1A). After taking account of the observation that Rap1A
yielded twice as much signal as Ha-Ras on the equimolar basis (Fig.
1D), the ability of Rap1A to bind to CRR was roughly 10-fold
greater than that of Ha-Ras. In addition, the ability of Rap1A to bind to RBD was about one-half of that of Ha-Ras after the same
normalization. These results indicated that Rap1A has greatly enhanced
rather than impaired activity to bind to CRR.
In our previous report, we found that the binding of Ha-Ras to CRR
required post-translational modification (11). To test if binding of
Rap1A to CRR was also dependent on its post-translational modification,
we incubated RBD and CRR with a lysate of Sf9 cells expressing Rap1A.
As shown in Fig. 1E, RBD bound both modified and unmodified
Rap1A represented by the faster and slower migrating bands,
respectively, on the Western immunoblot as described before (17). In
contrast, CRR bound only the modified form of Rap1A. These results
indicated that binding of Rap1A to CRR also requires its
modification.
The above data are somewhat
contradictory to our previous finding that binding to CRR is necessary
for Raf-1 activation, because Rap1A can bind to CRR better than Ha-Ras
and still cannot activate Raf-1. One possible explanation is that
enhanced binding to CRR is detrimental to the activation of Raf-1. To
test this possibility, we first screened for a Ha-Ras mutant whose
binding to CRR was abnormally enhanced. After testing more than 40 mutants described previously (16), the E31K mutant was found to possess
roughly 10-fold higher activity to bind to CRR compared with wild type (Fig. 1C). Next, we co-expressed Raf-1 with either
Rap1AV12 or Ha-RasV12(E31K) mutant in Sf9
cells. The Raf-1 immunoprecipitates were examined for the activity to
induce phosphorylation of KNERK2 in the presence of MEK (Fig.
2C). The results showed that
Rap1AV12 and Ha-RasV12(E31K) indeed could not
activate Raf-1 (lanes 1-4). Further, triple expression of either of them along with Ha-RasV12 and Raf-1
was found to suppress the activation of Raf-1 by Ha-RasV12
(lanes 5 and 6). These data suggested that the
enhanced binding to CRR is indeed detrimental to the activation of
Raf-1. They also suggested the involvement of this enhanced binding in
the suppressive action of Rap1A in Ras-dependent Raf-1
activation, which is examined in the following section.
We reasoned that the greatly enhanced
ability of Rap1A to bind to CRR found here may also be involved in
competitive inhibition of Ras-Raf-1 association by Rap1A. Rap1A would
inhibit association of Ras with a Raf-1 N-terminal region containing
both RBD and CRR much more efficiently than that with RBD alone. To
test this idea, we first incubated MBP-Raf-1(50-131) with a fixed
amount of Ha-Ras and increasing amounts of Rap1A. As expected, the
amount of bound Ha-Ras was found to be reduced by increasing amounts of
Rap1A, whereas that of bound Rap1A was increased (Fig.
3A). We then tested MBP-Raf-1(48-206) in the
same experiment. To our surprise, the binding of Ha-Ras to
MBP-Raf-1(48-206) was found enhanced to some extent even when the
amounts of bound Rap1A were increased (Fig. 3B).
The only possible explanation for these results would be that Rap1A and
Ha-Ras co-associate with the same Raf-1 N-terminal molecule through
their independent binding to CRR and RBD, respectively, and that these
associations mutually stabilize each other. To test this possibility,
we immobilized GST-Ha-Ras fusion protein onto glutathione-Sepharose and
incubated it with Rap1A in the absence or the presence of MBP-Raf-1
fusion proteins. As shown in Fig. 3C, no Rap1A was found to
be associated with GST-Ha-Ras when they were incubated in the absence
of MBP-Raf-1 (lane 1). Incubation in the presence of
MBP-Raf-1(50-131) did not result in association of Rap1A with
GST-Ha-Ras either, whereas MBP-Raf-1(50-131) was associated with
GST-Ha-Ras (lane 2). Remarkably, when incubated in the
presence of MBP-Raf-1(48-206), not only MBP-Raf-1(48-206) but also
Rap1A was found to be associated with GST-Ha-Ras (lane 3).
These results indicated that Rap1A and Ha-Ras co-associate with Raf-1
N-terminal region only when it contains both RBD and CRR. Similar
results could be obtained if association of GST-Ha-Ras with
MBP-Raf-1(48-206) induced dimerization of MBP-Raf-1(48-206), leading
to subsequent binding of Rap1A to the second MBP-Raf-1(48-206) molecule. However, induced dimerization of MBP-Raf-1(48-206) was not
the case because incubation of GST-Ha-Ras and untagged Ha-Ras in the
presence of MBP-Raf-1(48-206) (the same experiment as Fig. 3C, lane 3, except that Ha-Ras was used instead
of Rap1A) did not result in any association between GST-Ha-Ras and
Ha-Ras (data not shown).
The current model of Ras-suppressive action of Rap1A involves
competitive inhibition by Rap1A of the interaction between Ras and
Raf-1 RBD (10). However, we found in this study that Rap1A possessed
greatly enhanced ability to bind to CRR, another Ras-binding domain
identified by us (11) and others (18-20). This strong binding even
resulted in the co-association of Rap1A and Ha-Ras with Raf-1
N-terminal region through their independent binding to CRR and RBD,
respectively, and these bindings might mutually stabilize each other.
Because we have previously shown that binding of Ras to both CRR and
RBD is necessary for Raf-1 activation (11), it is very likely that this
triple complex formation will result in impairment of Raf-1 activation
due to the failure of Ha-Ras to bind to CRR. The previous observations
that unmodified Rap1A cannot suppress Ras function (5, 6) is also
consistent with this, because we found that modification of Rap1A is
essential for its binding to CRR.
A study with Ras/Rap1A chimera indicated that transforming potential of
Ras requires both of the two regions, residues 21-31 and 45-54. On
the other hand, Rap1A anti-oncogenicity requires mainly residues
21-31, although residues 45-54 is also required for full activity to
suppress transformation (21). Disregarding residues that are changed
conservatively, that are variable among the Ras family, and that are
not exposed on the protein surface, three residues, 26, 31, and 45, have been postulated to determine whether the protein is oncogenic or
anti-oncogenic (3, 21-24). In fact, replacements of residues 26 (or 26 plus 27), 31 (or 30 plus 31), or 45 of activated Ras with those of
Rap1A resulted in attenuation of transforming activity (22, 24).
However, it remained unclear which of these residues plays the most
critical role.
Residues 26-28 (including 26) and 42-49 (including 45) have been
proposed to constitute a contiguous domain on the surface of Ras
protein (3, 25). The domain, termed "activator domain," was
suggested to play an important role for activation of effectors through
some physical interaction with them. In the previous study, we found
that Ha-Ras proteins carrying mutations N26G and V45E failed to bind to
Raf-1 CRR (11). These mutants were also incapable of activating Raf-1
in Sf9 cells. Based on these findings, we proposed that the activator
domain interacts with Raf-1 CRR and that this interaction is essential
for Raf-1 activation. This predicts that the inability of Rap1A to
activate Raf-1 may result from its failure to bind to CRR, because
Rap1A contains residues Gly26 and Glu45, which
abolished binding to CRR in the context of Ha-Ras. Contrary to this
expectation, we found here that Rap1A exhibited greatly enhanced rather
than reduced binding to CRR.
One explanation for this apparent discrepancy is that side chains of
the activator domain residues such as 26 and 45, which are divergent
between Ras and Rap1A, might not interact directly with CRR. Instead,
the activator domain residues such as Phe28 and
Lys42, which are conserved between Ras and Rap1A, might
participate in direct interaction with CRR. Consistent with this idea,
F28A and K42A mutations have been shown to impair activities of Ras (3,
25). In the context of Ha-Ras, both Asn26 and
Val45 might be necessary for these interacting residues to
assume their functional conformation, which are destroyed by the N26G
and V45E mutations. On the other hand, in the context of Rap1A, such
effects of Gly26 and Glu45 might be masked by
conformational effects of other flanking residues that are not
conserved with Ha-Ras. In this regard, residue Lys31 of
Rap1A might play a critical role because we found here that E31K
mutation alone enhanced CRR binding in the context of Ha-Ras.
A support for the conformational role of the residue 31 over the
activator domain also comes from a study of Rap1A mutants. Nassar
et al. recently solved the structure of Rap1A(E30D,K31E) double mutant complexed with RBD by x-ray crystallography (26). Comparison of this structure with that of wild-type Rap1A complexed with RBD indicated that the mutation led to a movement by more than 1.2 Å of a loop containing residues 44-50, which overlapped with the
activator domain. Thus, it is likely that residues 30 and 31 of Ras
influence the conformation of the activator domain residues. In
addition, Glu31 of Rap1A(E30D,K31E) was found to interact
directly with Lys84 of Raf-1 RBD, suggesting that E31 of
Ras takes part in the same ionic interaction. Nassar et al.
also showed that Rap1A(E30D,K31E) and Rap1A(K31E) acquired an activity
to stimulate transcription from a Ras-dependent promoter
in vivo, albeit to a small extent, and argued that this is
accounted for by a large increase in the affinity of Rap1A for RBD due
to the newly created ionic interaction. However, the result is also
consistent with our finding that the identity of the residue 31 affects
CRR binding. The observed conformational change of the activator domain
of Rap1A might have brought about a reduction in the affinity of the
Rap1A activator domain for CRR to a level appropriate for Raf-1
activation, contributing to the acquisition of the Ras-like activity.
Although the solution structure of CRR has been solved (27), further
understanding of the mechanism of CRR binding should await the
structural analysis of the complex between CRR and Ras or Rap1A.
We thank A. Kikuchi at Hiroshima University
School of Medicine for providing GST-MEK, GST-KNERK, and pV-IKS, K. Kishi at Kyoto University School of Medicine for providing human lung
fibroblast cDNA library, and X.-H. Deng for skillful technical
assistance. We also thank A. Seki and A. Kawabe for help in preparation
of this manuscript.
Department of Physiology II,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Expression and Purification of Rap1A and Ha-Ras
Proteins
-CGGGATCCGATATGCGTGAGTACAAGCTAG-3
and
5
-AACTGCAGCAGCTAGAGCAGCAGACATGATTTC-3
. After cleavage with
BamHI and PstI in the primer sequences, it was
cloned into matching cleavage sites of the baculovirus transfer vector
pBlueBac III (Invitrogen Inc., San Diego, CA). The cDNA for an
activated Rap1A, Rap1AV12, was prepared by
oligonucleotide-directed mutagenesis (13) and cloned into pBlueBac III
as for the wild-type cDNA. pV-IKS, another baculovirus transfer
vector for expressing proteins as GST fusions, was provided by Dr. D. Midra (University of California, San Francisco, CA) through Dr. A. Kikuchi (Hiroshima University, Hiroshima, Japan) (14). For expression
of Ha-Ras fused to GST, Ha-Ras cDNA was amplified by polymerase
chain reaction using a pair of primers,
5
-CGCGTCTAGAATGACGGAATATAAGCTGGTG-3
and
5
-GCCGGAATTCTCAGGAGAGCACACACTTG-3
. After digestion with
XbaI and EcoRI in the primer sequences, it was
cloned into matching cleavage sites of pV-IKS. Structures of the
constructs were confirmed by DNA sequence analysis. Preparation of
recombinant baculoviruses expressing Ha-Ras, Rap1A, and their mutants
and the purification of the post-translationally modified proteins from
infected Sf9 cells were carried out as described (15, 16).
S-
or GDP-bound Ha-Ras or Rap1A in a total volume of 100 µl of buffer A
(20 mM Tris/HCl, pH 7.4, 40 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM
MgCl2, and 0.1% Lubrol PX) as described (11). After
incubation at 4 °C for 2 h, the resin was washed, and the bound
proteins were eluted with buffer A containing 10 mM maltose
and subjected to SDS-PAGE followed by Western immunoblot detection with
anti-Ras monoclonal antibody Y13-259 (Oncogene Science Inc., Manhasset,
NY) or anti-Rap1A polyclonal antibody (Santa Cruz Biotechnology Inc.,
Santa Cruz, California). Both anti-Ha-Ras and anti-Rap1A antibodies
exhibited little cross-reactivity to Rap1A and Ha-Ras, respectively
(data not shown). The assay for competitive inhibition of Ras binding
to the MBP-Raf-1 fusion proteins by Rap1A were carried out by including
a fixed amount of Ha-Ras and various amounts of Rap1A in the same
binding reaction. For the in vitro co-association of Rap1A
with GST-Ha-Ras, GST-Ha-Ras in Sf9 cell lysate was first immobilized on
glutathione-Sepharose and then loaded with GTP
S. The resin was then
incubated with GTP
S-bound Rap1A in the absence or the presence of
purified MBP-Raf-1(51-131) or MBP-Raf-1(48-206). The binding
condition was the same as described above except that the bound
proteins were eluted with 10 mM glutathione in buffer
A.
-glycerophosphate, and 50 µM
[
-32P]ATP (4,000 cpm/pmol)) for 30 min at 25 °C.
After the incubation, proteins in the reaction mixture were
fractionated by SDS-PAGE and subjected to autoradiography to detect
phosphorylation of GST-KNERK2 as described (11, 14).
Rap1A Has Enhanced Binding Activity to Raf-1 CRR
Fig. 1.
Association of various Raf-1 N-terminal
fragments with wild-type Rap1A, Ha-Ras, and Ha-Ras(E31K). The
amounts of Rap1A, Ha-Ras, and Ha-Ras(E31K) proteins bound to various
MBP-Raf-1 fusion proteins immobilized on amylose resin were measured by
Western immunoblotting with the anti-Ras and anti-Rap1A antibodies.
A, 10 pmol of GTPS-bound (T) or GDP-bound
(D) Rap1A were incubated with 20 pmol of MBP-Raf-1(50-131)
(RBD), MBP-Raf-1(132-206) (CRR), and
MBP-Raf-1(48-206) (RBD+CRR). B, 10 pmol of
GTP
S-bound Rap1A were incubated with 20 pmol of MBP-Raf-1(132-206)
(CRR) and its mutant MBP-Raf-1(132-206, C168S)
(C168S). C, 10 pmol of GTP
S-bound Rap1A,
Ha-Ras(E31K), and Ha-Ras were incubated with either 50 pmol of
MBP-Raf-1(50-131) (RBD) or 100 pmol of MBP-Raf-1(132-206) (CRR). D, equal amounts (1 pmol) of Ha-Ras
(Ha-Ras) and Rap1A (Rap1A) were detected by
anti-Ras antibody and anti-Rap1A antibody, respectively. E,
GTP
S-loaded Sf9 cell lysates containing roughly equal amounts of
post-translationally modified and unmodified Rap1A were incubated with
50 pmol of MBP-Raf-1(50-131) (RBD) and 50 pmol of
MBP-Raf-1(132-206) (CRR). Experiments shown were repeated at least three times yielding similar results.
[View Larger Version of this Image (35K GIF file)]
Fig. 2.
Suppression of Ha-Ras-dependent
activation of Raf-1 by Rap1A and Ha-Ras(E31K). Sf9 cells were
infected with the recombinant baculovirus expressing indicated
proteins. A, the amount of Raf-1 present in Nonidet P-40
extract of the infected Sf9 cells was measured by Western
immunoblotting with the anti-Raf-1 antibody. The arrow
indicates the position of Raf-1 on the blot. B, the amount of
Ha-RasV12 or Rap1AV12 proteins in the same
extract was measured by Western immunoblotting with the mixture of the
anti-Ras and anti-Rap1A antibodies. The arrow indicates the
position of Ha-RasV12 or Rap1AV12 on the blot.
C, Raf-1 was immunoprecipitated from the same extract by the
anti-Raf-1 antibody and examined for its activity to induce phosphorylation of GST-KNERK2 in the presence of GST-MEK. The arrow indicates the position of phosphorylated GST-KNERK2 in
the autoradiograph. The results are representative of three experiments giving similar results.
[View Larger Version of this Image (33K GIF file)]
Fig. 3.
Coassociation of Ha-Ras and Rap1A with the
Raf-1 N-terminal region containing both RBD and CRR. A, the
binding assay was done as described in the legend to Fig. 1 except that
various amounts of GTPS-bound Rap1A were included in the binding
reaction together with 10 pmol of GTP
S-bound Ha-Ras and 10 pmol of
MBP-Raf-1(50-131). The numbers indicate the amounts of
Rap1A used in pmol. The blot was first probed with the anti-Ras
antibody (lower panel) and then reprobed with the anti-Rap1A
antibody (upper panel). B, the same as in
A except that MBP-Raf-1 (48-206) was used instead of MBP-Raf-1(50-131). C, 10 pmol of immobilized GST-Ha-Ras
were loaded with GTP
S and incubated with 10 pmol of GTP
S-bound
Rap1A in the absence (lane 1) or the presence of 10 pmol of
MBP-Raf-1(50-131) (lane 2) or MBP-Raf-1 (48-206)
(lane 3). The eluted proteins were resolved by SDS-PAGE and
subjected to Western immunoblotting. Top panel, immunoblot
detection of GST-Ha-Ras with the anti-Ras antibody; middle
panel, detection of associated MBP-Raf-1 fusion proteins with the
anti-MBP antibody; bottom panel, detection of associated
Rap1A with the anti-Rap1A antibody. Similar results were obtained in
three independent experiments.
[View Larger Version of this Image (26K GIF file)]
*
This investigation was supported by grants-in-aids for
cancer research and for scientific research from the Ministry of
Education, Science, and Culture of Japan and by grants from the
Ciba-Geigy Foundation (Japan) for the Promotion of Science and from the
Suntory Institute for Bioorganic Research.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.
To whom correspondence should be addressed. Tel.:
81-78-341-7451 (Ext. 3230); Fax: 81-78-341-3837; E-mail:
kataoka{at}icluna.kobe-u.ac.jp.
1
The abbreviations used are: RBD, Ras-binding
domain; CRR, cysteine-rich region; MBP, maltose-binding protein;
GTPS, guanosine 5
-O-(3-thiotriphosphate); GST,
glutathione S-transferase; MEK, mitogen-activated protein
kinase kinase/extracellular signal-regulated kinase kinase; KNERK, a
kinase negative mutant of ERK2; PAGE, polyacrylamide gel
electrophoresis.
2
C.-D. Hu, K. Kariya, and T. Kataoka, unpublished
results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.