Activation of C3G Guanine Nucleotide Exchange Factor for Rap1
by Phosphorylation of Tyrosine 504*
Tamotsu
Ichiba
§,
Yuko
Hashimoto
,
Mie
Nakaya¶,
Yasunobu
Kuraishi§,
Shinya
Tanaka
,
Takeshi
Kurata
,
Naoki
Mochizuki¶, and
Michiyuki
Matsuda¶**
From the
Department of Pathology, National Institute
of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, the
§ Department of Internal Medicine (3), Jikei University
School of Medicine, Nishishinbashi, Minato-ku, Tokyo 105-8461, the
¶ Department of Pathology, Research Institute, International
Medical Center of Japan, Toyama, Shinjuku-ku, Tokyo 162-8655, and the
Department of Pathology, Hokkaido University School of Medicine,
Kita-ku, Sappro 060-8638, Japan
 |
ABSTRACT |
C3G is a guanine nucleotide exchange factor for
Rap1 and is activated by the expression of Crk adaptor proteins. We
found that expression of CrkI in COS cells induced significant tyrosine phosphorylation of C3G. To understand the mechanism by which C3G is
phosphorylated and activated by Crk, we constructed a series of
deletion mutants. Deletion of the amino terminus of C3G to amino acid
61 did not remarkably affect either tyrosine phosphorylation or
Crk-dependent activation of C3G. When C3G was truncated to amino acid 390, C3G was still phosphorylated on tyrosine but was not
effectively activated by CrkI. Deletion of the amino terminus of C3G to
amino acid 579 significantly reduced the Crk-dependent tyrosine phosphorylation of C3G and increased GTP-bound Rap1
irrespective of the presence of CrkI. We substituted all seven tyrosine
residues in this region, amino acids 391-579, for phenylalanine for
identification of the phosphorylation site. Among the substitution
mutants, the C3G-Y504F mutant, in which tyrosine 504 was substituted by
phenylalanine, was remarkably less activated and phosphorylated than
the wild type. All the other substitution mutants were activated and
tyrosyl-phosphorylated by the expression of CrkI. Thus, CrkI activates
C3G by the phosphorylation of tyrosine 504, which represses the
cis-acting negative regulatory domain outside the catalytic region.
 |
INTRODUCTION |
C3G was originally isolated as one of the two major proteins bound
to the SH31 domain of the Crk
adaptor protein (1, 2). C3G consists largely of three regions. The
carboxyl-terminal region, which shares a homologous sequence with
CDC25, catalyzes the guanine-nucleotide exchange reaction for Rap1, a
Ras family protein, but not for the other Ras-family G proteins (3). In
the central part of C3G, three Crk SH3-binding sequences have been
identified (1, 4). Although the function of the amino-terminal region
of C3G remains unknown, recently p130cas, a major Crk
SH2-binding protein, has been shown to bind in this region in
vitro (5).
The human crk gene is translated into two products, CrkI and
CrkII, by alternative splicing (6). CrkII is regulated negatively by
tyrosine phosphorylation, which occurs upon various types of stimulation (7, 8). CrkI, by contrast, lacks the tyrosine phosphorylation site and resembles the v-crk oncogene
product in its structure (6, 9).
Many kinds of stimulation induce binding of the Crk-C3G complex
to a variety of phosphotyrosine-containing proteins, such as receptor
tyrosine kinases (reviewed in Ref. 10). However, the biological
consequences of the activation of C3G are still poorly understood,
mostly because the function of Rap1, the only known effector of C3G, is
obscure. Rap1 competitively suppresses Ras-dependent
transformation and ERK/MAP kinase activation (11, 12). It has been
shown that the sustained activation of Rap1, probably due to the
formation of the Cbl-CrkL-C3G complex, causes T-cell anergy by
suppressing the Ras-MAP kinase pathway (13). In the central nervous
system, however, Rap1, like Ras, activates the ERK/MAP kinase pathway
through the activation of B-raf (14-16). In platelets, an increase in
the intracellular calcium concentration activates Rap1, which in turn
binds to and activates RalGDS (17). Recently we have isolated a novel
guanine nucleotide exchange factor for Rap1, CalDAGI (18). CalDAGI can
be activated by Ca2+ and diacylglycerol; therefore, the
Ca2+-dependent activation of Rap1 appears to be
triggered by CalDAGI, but not C3G.
It has been reported that C3G activates JNK through SEK (19).
Mixed-lineage kinases, MLK3 and DLK, appear to be involved in this
pathway (20). Because constitutively active Rap1 does not activate JNK
and because dominant-negative Rap1 does not inhibit the
C3G-dependent activation of JNK, it has been proposed that another G protein may transduce signals to JNK from C3G.
Activation of C3G occurs primarily through membrane recruitment (10,
21). Expression of CrkI in COS cells activates guanine nucleotide
exchange activity of C3G, concomitant with its translocation to the
cell membrane (22). An SH2 mutant of CrkI, which is not translocated to
the membrane, cannot activate C3G; however, addition of a farnesylation
signal to this SH2 mutant restored the CrkI-dependent activation of C3G partially. Deletion of the amino-terminal third activates C3G to the level of C3G co-expressed with Crk. These observations strongly suggest the presence of a cis-acting
negative regulatory domain in the noncatalytic domain of C3G. In the
present paper, we delineate the negative regulatory domain and the
tyrosine phosphorylation site of C3G.
 |
EXPERIMENTAL PROCEDURES |
Expression Plasmids--
pCAGGS-CrkI encodes the wild-type human
CrkI (21). CrkI-R38V and CrkI-W169L are SH2 and SH3 mutants,
respectively (21). pCAGGS-C3G and pCAGGS-C3G-F express the wild-type
and the farnesylated form of C3G, respectively (3). pEBG-Rap1 and
pEBG-C3G encode GST-tagged Rap1 and C3G, respectively (2). Full-length
and truncated C3G cDNAs were obtained by polymerase chain reaction and subcloned into the pCAGGS-His expression vector, which contains the
polyhistidine-tag sequence derived from pBlueBacHisA (Invitrogen) at
the 5' end of the cloning site. pCAGGS-His-C3G-d61 encodes C3G from
amino acid 62 to the carboxyl end; -d390 from 391; and -d579 from 580. In other C3G expression vectors, a tyrosine codon(s) was substituted
into that of phenylalanine by polymerase chain reaction-based
mutagenesis. Substituted tyrosines are indicated by superscripts; the
vectors are pCAGGS-C3G-Tyr504,
pCAGGS-C3G-Tyr554,561,570, and
pCAGGS-C3G-Tyr478,485,579.
Cell Culture and Transfection--
COS1 (CCL 1651, ATCC), 3Y1
rat fibroblast (0734, Japan Cell Resource Bank (JCRB)),
v-crk-transformed 3Y1 cells, Crk-3Y1 (23), SR-3Y1
v-src-transformed 3Y1 cells (0742, JCRB), and HR-3Y1
v-Ha-ras-transformed cells (0734, JCRB) were cultured in
Dulbecco's modified Eagle's medium (Nissui, Tokyo) supplemented with
10% fetal calf serum. COS1 cells were transfected by the DEAE-dextran method.
Antibody--
Antibodies against C3G and Crk were developed in
our laboratory (2, 6). Anti-Crk monoclonal antibody and horseradish peroxidase-conjugated anti-phosphotyrosine antibody RC20 were purchased
from Transduction Laboratories, Lexington, KY; anti-C3G polyclonal
antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA.
Analysis of Guanine Nucleotide Exchange Activity of C3G for Rap1
in COS1 Cells--
Guanine nucleotides bound to Rap1 were analyzed
essentially as described previously (24). Briefly, 105 COS1
cells plated on 35-mm dishes were transfected with 0.2 µg of
expression plasmids by the DEAE-dextran method. Forty-eight hours after
transfection, cells were labeled for 2 h with 0.05 mCi of
32Pi in 0.5 ml of phosphate-free medium.
GST-fused Rap1 was collected by glutathione-Sepharose 4B (Amersham
Pharmacia Biotech). Guanine nucleotides bound to Rap1 were separated by
thin layer chromatography and quantitated with a Molecular Imager
(Bio-Rad). For analysis of protein expression, cells were processed
similarly without isotopic labeling. Cleared lysates were separated by
SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed
with antibodies, and detected by an ECL chemiluminescence system
(Amersham Pharmacia Biotech).
Immunoprecipitation--
Cells were lysed in lysis buffer (0.5%
Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 5 mM MgCl2) and cleared by
centrifugation. Equal amounts of lysates were incubated with various
antibodies at 4 °C. The immune complexes were collected by protein
A-Sepharose FF (Amersham Pharmacia Biotech, Tokyo). In some
experiments, SDS was added at 1%, incubated at 95 °C for 3 min, and
diluted 10-fold with lysis buffer, followed by immunoprecipitation by
the use of anti-C3G antibody and protein A-Sepharose FF. The immune
complexes were separated by SDS-PAGE and analyzed by Western blotting
as described (21).
 |
RESULTS |
Activation of Farnesylated C3G by CrkI--
Previously, we
proposed that CrkI activates C3G primarily by recruiting C3G to the
membrane fraction (21). We examined whether the translocation to the
membrane is sufficient for the activation of C3G by use of a C3G
mutant, C3G-F, which localizes mainly in the membrane fraction due to
farnesylation (Fig. 1, A and
B). The wild-type C3G and C3G-F increased GTP-Rap1 from 8%
to 17% and to 16%, respectively. As we discussed previously (21), the protein level of C3G-F is always significantly lower than that of the
wild type (Fig. 1C). Considering the protein expression level, the specific activity of the farnesylated C3G was 4.5-fold higher than that of the wild-type C3G (Fig. 1D).
Co-expression of CrkI enhanced Rap1 activation by C3G-F as well as the
wild-type C3G, suggesting that membrane recruitment is not the sole
mechanism of C3G activation by CrkI. In these experiments, we found
that the activation of C3G was accompanied by its tyrosine
phosphorylation (Fig. 1C). Because the farnesylated C3G also
became tyrosine-phosphorylated by CrkI expression, we speculated that
tyrosine phosphorylation plays a major role in the activation of
C3G.

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Fig. 1.
Activation and phosphorylation of C3G by the
expression of CrkI. A, COS1 cells were transfected with
the expression vectors encoding GST-Rap1 and the proteins indicated at
the bottom. After 32Pi-labeling,
guanine nucleotides bound to Rap1 were analyzed as described in the
text. B, the radioactivity of each spot in A was
quantitated. Mean values obtained from three experiments are shown with
standard deviations. C, in parallel experiments, total cell
lysates and anti-C3G immunoprecipitates without
32Pi labeling were analyzed by immunoblotting
with either anti-C3G sera or anti-Crk monoclonal antibody.
D, the specific activity of C3G was calculated from the data
of panels B and C. The GTP/(GTP + GDP)
on Rap1 divided by the amount of C3G was shown as -fold increase from
the value of C3G without CrkI expression.
|
|
Correlation of C3G Tyrosine Phosphorylation with Its
Activation--
To examine further the role of tyrosine
phosphorylation of C3G, we used two CrkI mutants, CrkI-R38V and
CrkI-W169L. The SH2 mutant CrkI-R38V and the SH3 mutant CrkI-W169L are
unable to activate C3G (21). The wild-type and mutant CrkI proteins
were expressed to a similar level and did not affect the expression of
C3G (Fig. 2A); however, only
the wild-type CrkI induced detectable tyrosine phosphorylation of C3G
(Fig. 2B). The CrkI-W169L did not bind to CrkI due to its
SH3 mutation, and CrkI-R38V could not induce tyrosine phosphorylation
of C3G due to its SH2 mutation (Fig. 2C). To confirm that
the 135-kDa protein detected by the anti-phosphotyrosine antibody was
C3G, we used GST-tagged C3G (Fig. 2C). In the cells expressing GST-C3G, we observed a molecular shift of the
tyrosine-phosphorylated protein to the 160-kDa region, as expected from
the molecular mass, 28 kDa, of GST.

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Fig. 2.
Requirement of the SH2 and SH3 domains of
CrkI for phosphorylation of C3G. COS1 cells were transfected with
expression plasmids encoding the indicated proteins (top)
and analyzed as in Fig. 1C. A, total cell lysates
were analyzed by immunoblotting with anti-C3G or anti-Crk monoclonal
antibody. B and C, anti-C3G immunoprecipitates
were analyzed by immunoblotting with anti-C3G antibody,
anti-phosphotyrosine monoclonal antibody, or anti-Crk monoclonal
antibody.
|
|
Tyrosine Phosphorylation of C3G in Transformed Cells--
We next
searched for conditions in which the endogenous C3G is phosphorylated
on tyrosine. We examined a series of 3Y1 rat fibroblasts transformed by
oncogenes for the tyrosine phosphorylation of C3G. As shown in Fig.
3A, we found a prominent
tyrosine-phosphorylated 135-kDa protein in the anti-C3G immune
complexes from 3Y1 cells transformed by v-crk or
v-src, but not from parent 3Y1 cells and Ha-Ras-transformed
3Y1 cells. Several proteins of some 130 kDa are known to be
phosphorylated on tyrosine in cells transformed by v-Crk or v-Src (25).
To remove these proteins from anti-C3G immune complexes, we denatured
the cell lysates before immunoprecipitation. Under this condition,
most, if not all, phosphotyrosine-binding domains such as SH2 cannot
refold.2 Even with this
treatment, the 135-kDa phosphotyrosine-containing protein was
precipitated with the anti-C3G antibody (Fig. 3B). Thus, we
concluded that C3G is phosphorylated on tyrosine in cells transformed
by v-Crk or v-Src.

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Fig. 3.
Tyrosine phosphorylation of C3G in
transformed cells. A, cells used are parent 3Y1;
Crk-3Y1, 3Y1 transformed by v-Crk; HR-3Y1, v-Ha-Ras-transformed 3Y1;
SR-3Y1, v-Src-transformed 3Y1. Equal amounts of cell lysates
were immunoprecipitated with anti-C3G and analyzed by immunoblotting
with anti-C3G or anti-phosphotyrosine monoclonal antibody.
B, cell lysates were heat-denatured in the presence of 1%
SDS, diluted with lysis buffer, and analyzed as in A.
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Delineation of the Phosphotyrosine-containing Region--
We
constructed C3G mutants and examined them for their
CrkI-dependent tyrosine phosphorylation. Only
representative examples are shown in Figs.
4 and 5.
C3G-d390 was the shortest among the deletion mutants that were
phosphorylated on tyrosine under the presence of CrkI. C3G-d579,
deleted to amino acid 579, almost completely lost its tyrosine
phosphorylation. C3G-d579 retained its binding to CrkI, as expected
from the presence of the major CrkI-binding site, amino acids 588-617
(4). Our data showed 7 tyrosine residues between amino acids 391 and
579 as candidates for tyrosine phosphorylation site(s). We constantly
observed a 120-kDa phosphotyrosine-containing protein
co-imunoprecipitated with C3G. It did not react with
anti-p130cas antibody and remained unidentified at this
moment.

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Fig. 4.
Schematic representation of C3G mutants.
Closed boxes represent three Crk-binding sites.
The catalytic domain is shown by the hatched box.
Tyrosine residues between amino acids 391 and 579 are depicted with
circles above the bar. In each mutant,
closed circles denote the substitution from
tyrosine to phenylalanine.
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Fig. 5.
Phosphorylation and binding of the C3G
mutants to CrkI. 293T cells expressing the proteins indicated on
the top were lysed and immunoprecipitated with either
anti-Crk monoclonal antibody 3A8 or anti-C3G antibody and probed with
antibodies as indicated on the bottom. Arrows
indicate the positions of the wild-type and C3G-d390 mutant
proteins.
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Guanine Nucleotide Exchange Activity of the C3G Deletion
Mutants--
The activity of the guanine nucleotide exchange reaction
for Rap1 was also examined for C3G deletion mutants with or without the
expression of CrkI (Fig. 6). The guanine
nucleotide exchange activities of C3G-d61 and C3G-d390 were similar to
those of the wild-type C3G, and both were activated by the
co-expression of CrkI. In contrast, C3G-d579 showed the highest basal
guanine nucleotide exchange activity to the level of the wild-type C3G
co-expressed with CrkI. As shown in Fig. 5, the expression level of
C3G-d579 was similar to that of the wild-type C3G; therefore, C3G-d579 appears to be a catalytically active mutant. The activity of C3G-d579 was not enhanced further by the expression of CrkI. These results demonstrate that both the tyrosine phosphorylation site(s) and the
negative regulatory region of C3G are between amino acids 391 and
579.

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Fig. 6.
Guanine nucleotide exchange activity of C3G
truncation mutants. A, COS1 cells were transfected with
pEBG-Rap1 and the C3G mutant expression vectors as indicated at the
bottom. After 32Pi labeling, guanine
nucleotides bound to Rap1 were analyzed by thin layer chromatography.
B, the radioactivity of each spot was quantitated and is
shown as bars. Mean values obtained from three experiments
are shown with standard deviation.
|
|
Requirement of Tyrosine 504 for Both Tyrosine Phosphorylation and
CrkI-dependent Activation--
We substituted all of the
seven tyrosine residues for phenylalanine to identify the tyrosine
residue that is critical for phosphorylation and
CrkI-dependent activation (Figs. 4 and
7). Results for three representative
mutants, among several mutants tested, are shown in Fig. 7. Two
mutants, C3G-Y554F/Y561F/Y570F and C3G-Y478F/Y485F/Y579F, which have
three amino acid substitutions, were tyrosine-phosphorylated by the
expression of CrkI, as was the wild-type C3G. However, tyrosine
phosphorylation of C3G-Y504F was increased significantly by CrkI (Fig.
7A). The activation of the guanine nucleotide exchange for
Rap1 by these C3G mutants correlated with the tyrosine phosphorylation
(Fig. 7, B and C). Both C3G-Y554F/Y561F/Y570F and
C3G-Y478F/Y485F/Y579F were activated by the expression of CrkI, as was
the wild-type C3G. In contrast, the activation of the C3G-Y504F mutant
by CrkI was marginal. These data demonstrate that tyrosine 504 is the
most critical tyrosine residue for the phosphorylation and
CrkI-dependent activation of the guanine nucleotide
exchange for Rap1.

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Fig. 7.
Phosphorylation and guanine nucleotide
exchange activity of C3G amino acid substitution mutants.
A, COS1 cells were transfected with pEBG-Rap1 and the
expression vectors indicated on the top. After 48 h,
the cells were lysed and immunoprecipitated with anti-C3G serum,
followed by immunoblotting with anti-C3G serum, anti-Crk monoclonal
antibody, or anti-phosphotyrosine antibody. B, in parallel
experiments, cells were labeled with 32Pi, and
guanine nucleotides bound to Rap1 were analyzed by thin layer
chromatography. C, the radioactivity of each spot was
quantitated and is shown as bars. Mean values obtained from
three samples are shown with standard deviations.
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|
 |
DISCUSSION |
We have shown that the increase in the catalytic activity of C3G
correlated with its tyrosine phosphorylation. Amino acid substitution
of Tyr-504 to Phe renders C3G refractory to CrkI-dependent activation and phosphorylation. Removal of the amino-terminal region,
including Tyr-504, generated a C3G mutant that was fully active
irrespective of the presence of CrkI; therefore, we concluded that
CrkI-induced phosphorylation of Tyr-504 represses the
cis-acting negative regulatory domain of C3G.
It should be noted, however, that even the C3G-Y504F mutant was also
activated slightly by CrkI. Because Y504F was still phosphorylated weakly on tyrosine, tyrosine phosphorylation of other residue(s) may
replace the function of Y504F phosphorylation. Alternatively, C3G may
be activated by CrkI via a phosphorylation-independent mechanism.
Recently, p130cas, crk-associated
substrate, has been shown to bind directly to C3G (5).
Pro-267 and Pro-270 of C3G are essential for the binding of the SH3
domain of p130cas. When we deleted this region in the C3G-d390
mutant, C3G could not be activated effectively by the expression of
Crk, even though C3G-d390 is tyrosyl-phosphorylated to a similar extent
as the wild-type C3G (Fig. 6). Thus, binding of p130cas may be
required for optimal activation of C3G after the phosphorylation of
Tyr-504.
Many of the guanine nucleotide exchange factors for the Ras family are
also regulated negatively by the domain(s) outside the catalytic
region. The in vitro activity of the full-length CDC25Mm/Ras-GRF was 25 times lower than that of the
catalytic domain consisting of the carboxyl-terminal 285 amino acid
residues (26). It has also been reported that
CDC25Mm/Ras-GRF is activated either by phosphorylation or
by calmodulin binding (27, 28). Therefore, it is likely that
phosphorylation and/or calmodulin binding represses the negative
regulatory domain of CDC25Mm/Ras-GRF. The nature of the
phosphorylation of Ras-GRF was not reported previously; however, it
does not seem to be tyrosine phosphorylation, because protein
phosphatase 1 effectively inhibited Ras-GRF (27).
Similarly to the situation for CDC25Mm/Ras-GRF, deletion of
either the amino-terminal or carboxyl-terminal regions of Sos, the catalytic domain of which resides in the center, promotes the guanine
nucleotide reaction of Sos more efficiently than does the full-length
Sos protein (29, 30); thus, both the amino and carboxyl termini of Sos
function as cis-acting negative regulatory domains. It has
also been shown that the amino- and carboxyl-terminal domains inhibit
allosterically the association of the catalytic domain of Sos with the
Ras protein (30).
Altogether, the general scheme can be deduced that the catalytic
activity of the guanine nucleotide exchange proteins for Ras-family
proteins is regulated by one or more cis-acting negative regulatory elements. The negative regulation is repressed by various mechanisms such as phosphorylation, calmodulin binding, calcium binding, or diacylglycerol binding.
The physiologic relevance of the tyrosine phosphorylation of C3G awaits
further study. However, we observed tyrosine phosphorylation of C3G in
cells transformed by v-crk or v-src. Because C3G
enhances the v-crk-dependent transformation, it
is likely that the v-Crk-dependent tyrosine phosphorylation
of C3G plays a significant role in the oncogenic process (19).
Compared with the number of proteins known to be phosphorylated on
tyrosine residues upon various types of stimulation, the number of the
enzymes that can be activated by tyrosine phosphorylation are not
numerous except for the tyrosine kinases themselves. Phospholipase C-
appears to be the first enzyme that was shown to be activated upon in vitro tyrosine phosphorylation (31). Vav, which is a member of the Dbl family of proteins and promotes the guanine nucleotide exchange of Rac, is recruited to tyrosine kinases and is
activated by tyrosine phosphorylation (32). C3G has become the first
example of guanine nucleotide exchange factor for the Ras family
proteins that can be activated by tyrosine phosphorylation.
While we were revising this manuscript, the cloning of
Drosophila C3G was reported (33). Amino acid sequence of the
central part of human C3G, which includes Crk-binding sites and the
tyrosine 504, does not show significant homology with that of
Drosophila C3G; while the carboxyl-terminal catalytic domain
and the amino-terminal region represent high sequence homology. Even
so, we have found that the sequence surrounding tyrosine 504, Lys-Pro-Tyr-Ala, is conserved between the human and
Drosophila C3Gs. This finding suggests that the
Drosophila C3G may also be regulated by tyrosine phosphorylation.
 |
ACKNOWLEDGEMENTS |
We thank S. Hattori, T. Gotoh, and J. Miyazaki for materials and technical advice. We also thank Robert
Ingham for his critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Health and Welfare, Ministry of Education, Science, and Culture, the Science and Technology Agency, and the Human Science Foundation, Japan.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: Dept. of Pathology,
Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo, 162-8655, Japan. Tel.: 81-3-3202-7181 (Ext.
2833); Fax: 81-3-3205-1236; E-mail: mmatsuda{at}ri.imcj.go.jp.
2
Y. Hashimoto, M. Nakaya, and M. Matsuda,
unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
SH3, Src
homology 3;
SH2, Src homology 2;
GST, glutathione
S-transferase;
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated protein;
JNK, c-Jun N-terminal kinase;
PAGE, polyacrylamide gel electrophoresis.
 |
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