(Received for publication, February 25, 1997, and in revised form, June 5, 1997)
From the Crk is an adaptor protein that consists almost
entirely of SH2 and SH3 domains. We have previously demonstrated, by
using in vivo and in vitro systems, that C3G,
which was identified as a Crk SH3 domain-binding guanine nucleotide
exchange factor, specifically activates Rap1. C3G also binds to other
adaptor proteins, including CrkL and Grb2. In the present study, we
analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway.
Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly
increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3
domains of Crk were required for this activity. However, Crk did not
stimulate the guanine nucleotide exchange activity of C3G for Rap1
in vitro, suggesting that Crk does not activate C3G by an
allosteric mechanism. The requirement of the SH2 domain of Crk for the
enhancement of guanine nucleotide exchange activity for Rap1 could be
compensated for by the addition of a farnesylation signal to Crk,
indicating that Crk enhanced the guanine nucleotide exchange activity
of C3G by membrane recruitment of C3G. These results demonstrate that
Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily
by recruiting C3G to the cell membrane.
It is now widely accepted that several domains consisting of short
consensus motifs play crucial roles in signal transduction. SH2 and SH3
are good examples of such domains (1-3). The SH2 domain binds
sequences containing phosphotyrosine (4, 5), whereas the SH3 domain
forms a complex with polyproline type II helices (6). A group of
proteins, including Grb2, Crk, CrkL, and Nck, consist mostly of the SH2
and SH3 domains and are now known as adaptor proteins (7, 8).
c-Crk protein was isolated as a cellular homolog of v-Crk, which was
originally identified as an oncoprotein of a chicken retrovirus, CT10
(9). Alternative splicing of the human crk gene generates
two Crk proteins, designated as 28-kDa CrkI and 40/42-kDa CrkII (10).
CrkII is composed of one SH2 domain and two SH3 domains, whereas CrkI
lacks the carboxyl-terminal SH3 domain. The SH2 domain of Crk binds
epidermal growth factor receptor and Shc, which are phosphorylated by
EGF1 stimulation (5, 11).
Integrin stimulation also induces tyrosine phosphorylation of
p130cas and paxillin, resulting in binding to Crk (12, 13).
Cbl, which was originally identified as a cellular counterpart of the v-Cbl oncogene product (14), is tyrosine-phosphorylated upon various
extracellular stimulations and binds to Crk (15, 16). Therefore, Crk is
implicated in the signal transduction pathway of both growth factor and
cell adhesion.
Cellular targets of the SH3 domain of Crk include C3G, Sos, DOCK180,
c-Abl, and EPS15 (17-21). C3G was isolated by screening of expression
libraries with the amino-terminal SH3 domain of Crk as a probe. The
carboxyl-terminal region of C3G has homology to the catalytic domain of
CDC25Mm, which is a guanine nucleotide exchange factor for
Ras. Using both in vivo and in vitro systems, we
demonstrated that the target GTPase of C3G is Rap1 (22).
Rap1, also known as smg p21 or Krev-1, was
originally identified as an anti-oncogenic protein which efficiently
reverses the morphologic transformation of the
v-Ki-ras-expressing NIH 3T3 cell line, DT (23-26). Studies
on chimeras of Rap1 and H-Ras have suggested that Rap1 antagonizes Ras
through the competition of the effector proteins of Ras (27). This is
also supported by the observation that the activation of the
c-fos promoter/enhancer from Ras, but not from the activated
Raf-1, which is an immediate downstream effector of Ras, is inhibited
by Rap1 (28). Furthermore, constitutively activated Rap1 efficiently
inhibits Ras-dependent activation of mitogen-activated
protein kinase by lysophosphatidate or EGF (29).
Rap1 is regulated mostly by two groups of proteins. The first one is
the GTPase-activating protein, which stimulates the intrinsic GTPase
activity of Rap1. Two proteins have been shown to possess GTPase-activating protein activity for Rap1 (30, 31). The second are
guanine nucleotide exchange factors for Rap1, which include
smg GDS and C3G (22, 32). smg GDS also stimulates the nucleotide dissociation of Ki-Ras, Rho, Rac, and mCdc42 (33). In
contrast, C3G specifically stimulates the guanine nucleotide exchange
reaction of Rap1 among various Ras-family G proteins (22).
Our aim in this study was to analyze the effect of adaptor proteins
that bind to C3G on the C3G-Rap1 pathway. We found that Crk enhances
the guanine nucleotide exchange reaction of C3G, and that the
enhancement occurs mostly by promoting of the translocation of C3G
to the membrane.
cDNAs of CrkI, CrkII, CrkI-R38V,
and CrkI-W169L (34) were subcloned into the pCAGGS eukaryotic
expression vector, generating pCAGGS-CrkI, pCAGGS-CrkII,
pCAGGS-CrkI-R38V, and pCAGGS-CrkI-W169L, respectively (Fig.
1). Similarly, pCAGGS-Grb2 and
pCAGGS-CrkL were constructed by ligating cDNA fragments of Grb2 and
CrkL. pCAGGS-C3G, encoding the authentic C3G, was described previously (35). The cDNA fragment corresponding to the catalytic domain of
C3G (amino acids 619-1077) was amplified by polymerase chain reaction
and subcloned into pCAGGS after the addition of the Myc-tag sequence to
its 5
Cos1 cells and human
embryonic kidney 293T cells were cultured in Dulbecco's modified
Eagle's medium (Nissui, Tokyo) supplemented with 10% fetal calf
serum. Cos1 cells were transfected by DEAE-dextran method as described
previously (22). Human embryonic kidney 293T cells were transfected by
calcium phosphate method.
Anti-Crk antibody was obtained from Transduction
Laboratories. Production of the anti-C3G antibodies was described
previously (35). Anti-C3G 1A/1B and anti-C3G-C sera were raised against the central region (amino acids 285-676) and the carboxyl terminus region (amino acids 1065-1077) of C3G, respectively. The epitope of
anti-C3G monoclonal antibody SN21 was in the region between amino acids
393-676. Anti-GST rabbit antibody was prepared in our laboratory.
Guanine nucleotides bound to Rap1 were analyzed
essentially as described previously (22). 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 (Pharmacia).
Guanine nucleotides bound to Rap1 were separated by thin-layer
chromatography and quantitated with a PhosphorImager analyzer, BAS2000
(Fuji Film). For the analysis of protein expression, cells were
similarly processed without isotopic labeling. The cleared lysates were
separated by SDS-PAGE, transferred to a nylon membrane, probed with
various antibodies, and detected by ECL chemiluminescence system
(Amersham Corp.).
The guanine nucleotide exchange activity of C3G for Rap1
was measured as described previously (22). Expression and purification of recombinant Rap1, C3G, and CrkI were described previously (22, 34).
C3G preincubated with GST or GST-CrkI was combined with [3H]GDP-loaded Rap1 and an excess of cold GTP. The
radioactivity of [3H]GDP remaining on Rap1 was measured
after a 20-min incubation at 30 °C.
Cell fractionation was performed as
described previously (36). Briefly, 105 Cos1 cells
transfected with expression plasmids were washed twice with
phosphate-buffered saline, disrupted by freeze-thawing in liquid
nitrogen, suspended with 150 µl of detergent-free W buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2). The soluble and the insoluble
fraction were separated by centrifugation at 15,000 × g for 10 min. Proteins in the insoluble fraction were solubilized with 37.5 µl of lysis buffer (0.5% Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2). Extracts were centrifuged at
15,000 × g for 10 min, and the supernatants were used
as a particulate fraction. 2 µl of soluble and 4 µl of particulate fraction were subjected to SDS-PAGE and immunoblotting using anti-C3G 1A/1B sera.
293T cells were plated at a density of
2.0 × 104 cells in a 35-mm diameter dish and
transfected with expression plasmids. Thirty-six hours after
transfection, cells were starved for 14 h in Dulbecco's modified
Eagle's medium containing 0.5% bovine serum albumin and stimulated
with 100 ng/ml EGF (Takara Biochemicals, Kyoto) at 37 °C for 3 min.
Cells were washed twice with TBS-V buffer (20 mM Tris-HCl,
pH 7.5, 150 mM NaCl, and 1 mM
Na3VO4) and lysed in lysis buffer (10 mM Tris (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 0.5% Triton X-100, 1 mM
Na3VO4). Cell lysates were cleared by
centrifugation and immunoprecipitated with anti-EGF receptor polyclonal
antibody (Amersham, Little Chalfont, UK). The immune complex was
analyzed by SDS-PAGE and immunoblotting using anti-EGF receptor
monoclonal antibody (MBL, Nagoya, Japan) or anti-C3G 1A/1B sera.
Cos1 cells were transfected
with expression vectors encoding GST-tagged Rap1, C3G, and/or adaptor
proteins. After 48 h, guanine nucleotides bound to GST-Rap1 were
analyzed by thin-layer chromatography (Fig. 2, A and
B). As reported previously
(22), expression of C3G increased the ratio of GTP/(GDP + GTP) on Rap1.
Expression of CrkI or CrkII remarkably enhanced the
C3G-dependent increase of GTP-bound Rap1, whereas CrkI or
CrkII alone did not affect the ratio of GTP/(GDP + GTP) on Rap1.
Neither CrkI mutant with nonfunctional SH2, CrkI-R38V, nor that with
nonfunctional SH3, CrkI-W169L, enhanced this C3G-dependent
Rap1 activation. We confirmed that similar amounts of C3G and the
wild-type and mutant Crk proteins were expressed in Cos1 cells (Fig.
2C). These results demonstrated that both the SH2 and SH3
domains of Crk were required for the enhancement of the guanine
nucleotide exchange activity of C3G. We observed a similar enhancement
of C3G activity by the other Crk-like protein, CrkL (Fig. 3,
A and B). Another
adaptor protein, Grb2, also increased the ratio of GTP to GDP on Rap1.
However, the increase in GTP-bound Rap1 was less than that by CrkI,
CrkII, or CrkL. In these experiments, coexpression of the adaptor
proteins did not alter the level of C3G expression (Fig.
3C).
To understand the mechanism by which Crk enhances the guanine
nucleotide exchange activity of C3G, we constructed a plasmid, C3G-CD,
that encoded only the catalytic domain of C3G. To our surprise, C3G-CD
activated Rap1 more efficiently than did the authentic C3G (Fig. 4,
A and B). We
examined the expression of C3G-CD and the wild-type C3G by an antibody
against the carboxyl-terminal region of C3G. As shown in Fig.
4C, the amount of C3G-CD was comparable to that of the
wild-type C3G (Fig. 4C). Therefore, the amino-terminal region of C3G appears to regulate negatively the guanine nucleotide exchange activity for Rap1. Moreover, the catalytic activity of this
C3G-CD mutant was enhanced no further by the co-expression of CrkI.
This result strongly suggests that direct binding of Crk to the
amino-terminal region of C3G is required for the up-regulation of
C3G.
To examine whether Crk activates C3G through an
allosteric mechanism, we measured the guanine nucleotide exchange
activity of C3G in vitro in the presence or absence of CrkI.
As shown in Fig. 5A, addition
of GST-fused CrkI did not enhance the catalytic activity of C3G for
Rap1. The binding of the recombinant CrkI to C3G in vitro
was confirmed by immunoblotting (Fig. 5B). We concluded that
binding of Crk to C3G is not sufficient to enhance the catalytic
activity of C3G in vitro.
The SH2 domain
of Crk binds to tyrosine-phosphorylated proteins such as EGF receptor
(5, 11), p130cas (37), Shc (17), and paxillin (13). These
proteins are localized to the plasma membrane either constitutively or
transiently after various stimulations. It is also known that
overexpression of v-Crk or CrkI induces tyrosine phosphorylation of
these Crk SH2-binding proteins either by the activation of tyrosine
kinases or by the competitive inhibition of protein-tyrosine
phosphatases (9-11). Thus, it has been postulated that at least one of
the functions of the SH2 domain of Crk is to recruit the SH3-binding
proteins to the plasma membrane. To test this possibility, we analyzed the subcellular distribution of C3G in the presence or absence of CrkI.
As shown in Fig. 6, co-expression of CrkI
increased the amount of C3G in the particulate fraction.
To examine whether membrane translocation of C3G can account
for the activation of C3G by Crk, we constructed plasmids encoding CrkI-F and CrkI-R38V-F that have the Ki-Ras-derived CAAX box
at the carboxyl termini of CrkI and CrkI-R38V, respectively. The CAAX box was shown to be farnesylated and required for the
membrane-targeting of Ki-Ras protein. As shown in Fig. 7, A
and B, CrkI-R38V-F enhanced the activation of Rap1 by C3G to a level similar to that by CrkI-F. The
expression level of C3G was not influenced by the expression of the
wild-type and mutant CrkI proteins. This result clearly demonstrated
that the SH2 domain of CrkI was not required when CrkI was localized to
the plasma membrane by the farnesylation. Enhancement of the activation
of C3G by CrkI-F was always less efficient than that by the wild-type
CrkI, although CrkI-F was expressed more abundantly than the wild-type
CrkI (Fig. 7C). Expression of these wild-type and mutant
CrkI did not alter the amount of C3G expressed in Cos1 cells (Fig.
7C). We also confirmed that the addition of the
farnesylation signal did not alter the amount of Crk bound to C3G in
immunoprecipitation experiments (data not shown).
It has
been reported that Crk binds to EGF receptor upon EGF stimulation (11).
We examined whether the EGF stimulation also recruits C3G to the EGF
receptor in 293T cells. As shown in Fig. 8, C3G bound to EGF receptor upon EGF
stimulation in a manner dependent on the expression of CrkI. This
result strongly suggests that Crk recruits C3G to the plasma membrane
upon physiological stimulation.
In the present study, we have demonstrated that the expression of
Crk enhances the guanine nucleotide exchange activity of C3G to Rap1
in vivo. Crk did not enhance the guanine nucleotide exchange
activity of C3G in vitro, arguing against the allosteric activation of C3G by Crk. Analysis by use of SH2 and SH3 mutants of Crk
demonstrated that both the SH2 and SH3 domains of Crk are required for
the enhancement. In contrast to CrkI-R38V, a CrkI mutant with a
non-functional SH2 domain, the mutant that has a membrane localization
signal on its carboxyl terminus, CrkI-R38V-F, enhanced the catalytic
activity of C3G to a level similar to that of CrkI-F, the wild-type
CrkI with a membrane localization signal. The result indicates that the
function of the SH2 domain is essentially to recruit Crk to the plasma
membrane.
However, we should point out the difference between the membrane
recruitment of C3G by CrkI and that by the farnesylation. The
enhancement of C3G activity by CrkI-F was less efficient than that by
the wild-type CrkI (Fig. 7). Thus, CrkI-F may not recruit C3G to the
place where the optimum activation of C3G occurs. In the cells
overexpressing v-Crk or CrkI, we and others observed the increase of
several phosphotyrosine-containing proteins including paxillin and
p130cas, to which v-Crk and CrkI bind (9, 10). C3G may be
activated most efficiently in the close vicinity of these Crk-binding
proteins, which is known to localize at the focal adhesions (8).
It is unknown why membrane recruitment of C3G by CrkI enhances guanine
nucleotide exchange activity of C3G. It is possible that the membrane
targeting of C3G simply facilitates its interaction with the substrate,
Rap1. Alternatively, C3G may be modified at the membrane and activated
enzymatically. To address this question, we have to measure the
specific activity of C3G from the cells with or without the expression
of CrkI.
We previously reported that farnesylated C3G activated Rap1 in Cos1
cells less efficiently than did the wild-type C3G (22). This
observation appears to argue against our proposal that CrkI enhances
guanine nucleotide exchange activity of C3G by membrane recruitment. We
have found that the amount of the farnesylated C3G expressed in Cos1
cells is significantly less than that of the wild-type; therefore, we
cannot conclude that the farnesylated C3G was not as active as the
wild-type C3G. Moreover, only the farnesylated C3G but not the
wild-type C3G could revert the transformation of NIH 3T3 cells
expressing Ki-Ras (22). This finding supports our proposal that
membrane-targeted C3G is more active than the wild-type C3G.
The significance of the translocation of the guanine nucleotide
exchange factor to the membrane has been suggested for Sos, a guanine
nucleotide exchange protein of Ras (38-41). In this case, another
adaptor protein, Grb2, recruits Sos to the membrane upon various
stimulations, as we have observed for CrkI and C3G. It has also been
reported that the binding of Grb2 to Sos does not activate Sos in
vitro (42).
Rap1 is phosphorylated by protein kinase A at Ser169,
located at the carboxyl-terminal basic region that is critical for the interaction with smg GDS (43). This phosphorylation
stimulates the guanine nucleotide dissociation of Rap1 by
smg GDS (44). However, we could not observe any difference
in the phosphorylation level of Rap1 in the presence or absence of CrkI
(data not shown). Therefore, it is unlikely that Crk enhances the
guanine nucleotide exchange reaction of C3G through the phosphorylation
of Rap1.
Crk and CrkL enhance the guanine nucleotide exchange activity of C3G
more efficiently than does Grb2. It is possible that Grb2 cannot alter
C3G localization efficiently because Grb2 does not bind to C3G as
strongly as do Crk and CrkL (45). Alternatively, because the SH2
domains of Crk and CrkL bind similar sets of proteins (45, 46), both
may recruit C3G to a position closer to Rap1 than does Grb2.
Experiments on SH2/SH3 chimeras of Crk/CrkL and Grb2 would be
useful.
C3G-CD, which lacks the amino-terminal region of C3G, catalyzed the
guanine nucleotide exchange reaction for Rap1 more efficiently than did
the authentic C3G. Although the in vitro study neglected the
allosteric activation of C3G by Crk, it is still possible that
expression of Crk can counteract the negative regulation of C3G by the
amino-terminal region in vivo. A similar phenomenon is known
for Sos, a guanine nucleotide exchange factor for Ras. The deletion of
the carboxyl-terminal Grb2-binding region sites enhances the guanine
nucleotide exchange activity and the transforming activity of the
farnesylated Sos protein (41). Although the exact role of the
amino-terminal region of C3G has yet to be elucidated, it is possible
that it is involved in the down-regulation of the C3G-Rap1 signaling
pathway.
We thank Y. Hashimoto and E. Kiyokawa for
critical reading of this manuscript, and B. J. Mayer and T. Gotoh
for materials and technical advice.
Department of Pathology,
Department of
Pathology,
Section of Molecular
Carcinogenesis, Department of Pathology, Children's Hospital,
Los Angeles, California 90027
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Expression Plasmids
end. The resulting vector was designated as pCAGGS-Myc-C3G-CD.
pEBG-Rap1 encodes Rap1 fused to GST (22). For the expression of
farnesylated CrkI protein, the cDNA fragment corresponding to the
CAAX box of Ki-Ras was fused to the carboxyl-terminal region
of CrkI cDNA, as described previously (22). The fused CrkI-CAAX box cDNA was subcloned into pCAGGS, generating
pCAGGS-CrkI-F.
Fig. 1.
Schematic representation of adaptor proteins,
C3G, and their mutants used in this study. CAAX denotes
the farnesylation signal of Ki-ras.
[View Larger Version of this Image (40K GIF file)]
Expression of Adaptor Proteins Enhanced the Guanine Nucleotide
Exchange Activity of C3G for Rap1
Fig. 2.
CrkI and CrkII enhance the guanine nucleotide
exchange activity of C3G for Rap1. A, Cos1 cells were
transfected with pEBG-Rap1 and the expression vectors encoding the
proteins indicated on the bottom. After 48 h, the transfected
cells were labeled with 32Pi for 2 h.
GST-tagged Rap1 was collected by glutathione-Sepharose from the cell
lysates. Guanine nucleotides bound to Rap1 were analyzed by thin-layer
chromatography. B, the radioactivity of each spot was
measured by a PhosphorImager, and the ratio of GTP to GTP + GDP was
calculated. Mean values obtained from three samples are shown with S.D.
C, in parallel experiments, cells without 32Pi labeling were analyzed by SDS-PAGE
followed by immunoblotting with either anti-Crk monoclonal antibody or
anti-C3G 1A/1B sera.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
CrkL and Grb2 also enhance the
guanine-nucleotide exchange activity of C3G. A, Cos1 cells
transfected with the expression plasmids encoding the proteins
indicated on the bottom were labeled with 32Pi
as described in the legend to Fig. 2. B, the radioactivity of each spot was measured by PhosphorImager for calculation of the
ratio of GTP to GTP + GDP. Mean values obtained from three samples are
shown with standard deviations. C, in parallel experiments, cells without 32Pi labeling were analyzed by
SDS-PAGE followed by immunoblotting with anti-C3G 1A/1B sera.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Crk-binding sites of C3G are required for
enhancement of guanine nucleotide exchange activity by Crk.
A, Cos1 cells were transfected with the expression plasmids
encoding the proteins indicated on the bottom and analyzed by
thin-layer chromatography as described in Fig. 2. C3G-CD consists only
of the catalytic region of C3G. B, the radioactivity of each
spot was measured by PhosphorImager for calculation of the ratio of GTP
to GTP + GDP. Mean values obtained from three samples are shown with
S.D. C, in parallel experiments, cells without
32Pi labeling were analyzed by immunoblotting
with the anti-C3G-C serum, which was raised against the
carboxyl-terminal region of the C3G (35).
[View Larger Version of this Image (26K GIF file)]
Fig. 5.
Crk does not enhance the catalytic activity
of C3G in vitro. A, Rap1 was loaded with
[3H]GDP as described in the text. The guanine nucleotide
exchange reaction was started by the addition of C3G preincubated with GST or GST-CrkI. After 20-min incubation at 30 °C, Rap1 was absorbed on a nitrocellulose membrane, and the radioactivity was measured. B, C3G was incubated with either GST or GST-CrkI on ice for
10 min. C3G was immunoprecipitated anti-C3G 1A/1B sera and protein A-Sepharose. GST or GST-CrkI was collected by glutathione-Sepharose 4B.
The proteins collected on the beads were analyzed by SDS-PAGE and
immunoblotting by either anti-C3G 1A/1B sera or anti-GST
antibody.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Crk recruits C3G to the particulate
fraction. Cos1 cells transfected with either pCAGGS-C3G alone or
pCAGGS-C3G and pCAGGS-CrkI were subjected to cell fractionation as
described in the text. The soluble (sup.) and the
particulate (ppt.) fractions were analyzed by SDS-PAGE and
immunoblotting using anti-C3G 1A/1B sera or anti-Crk antibody.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
Requirement of the SH2 domain of Crk for
enhancement of C3G activity is complemented by the addition of a
farnesylation signal to Crk. A, Cos1 cells were transfected
with the expression plasmids encoding the proteins indicated on the
bottom and analyzed by thin-layer chromatography as described in Fig.
2. CrkI-F and CrkI-R38V-F have a farnesylation signal on their carboxyl
terminus. B, the radioactivity of each spot was measured by
PhosphorImager for calculation of the ratio of GTP to GTP + GDP. Mean
values obtained from three samples are shown with standard deviations. C, in parallel experiments, cells without
32Pi labeling were analyzed by immunoblotting
with either anti-C3G 1A/1B sera or anti-Crk antibody.
[View Larger Version of this Image (30K GIF file)]
Fig. 8.
C3G bound to EGF receptor upon EGF
stimulation in a manner dependent on CrkI. 293T cells were
transfected with expression plasmids and stimulated with EGF for 3 min.
Cells were lysed and immunoprecipitated with anti-EGF receptor
(EGFR) polyclonal antibody. The immune complex was analyzed
by SDS-PAGE and immunoblotting using either anti-EGF receptor
monoclonal antibody or anti-C3G 1A/1B sera.
[View Larger Version of this Image (26K GIF file)]
*
This work was supported in part by grants from the Ministry
of Education, Science, and Culture, by grants from the Ministry of
Health and Welfare, and by grants from 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.
¶
Research Resident of Japan Research Development
Corporation.
§§
To whom correspondence and reprint requests should be addressed:
Dept. of Pathology, Research Institute, International Medical Center of
Japan, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan. Tel.:
81-3-5285-1111 (ext. 2624); Fax: 81-3-5285-1189; E-mail: mmatsuda{at}nih.go.jp.
1
The abbreviations used are: EGF, epidermal
growth factor; SH2, Src homology 2; SH3, Src homology 3; GST,
glutathione S-transferase; PAGE, polyacrylamide gel
electrophoresis.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.