From the a Laboratory of Environmental Biology, Department of Preventive Medicine, Hokkaido University School of Medicine, Sapporo 060-8638, Japan, the e Laboratory of Cellular Oncology, CCR, NCI, National Institutes of Health, Bethesda, Maryland 20892, the d Department of Biochemistry II, Sapporo Medical University School of Medicine, Japan, the f Pharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Co., LTD. 3 Miyahara, Takasaki, Gunma 370-1295 Japan, the i Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan, the j College of Medical Technology, Hokkaido University, Sapporo 060-8638, Japan, the k Department of Hygene, Sapporo Medical University School of Medicine, Sapporo 060-8638, Japan, and the l School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Birmingham, B15 2TT, United Kingdom
Received for publication, October 22, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Searching for proteins in platelets
that can interact with the N-terminal SH3 domain of CrkL (using a
combination of a pull-down assay followed by mass spectrometry), we
have found that human platelets express an ADP-ribosylation factor
(Arf)-specific GTPase-activating protein (GAP), ASAP1, as a
CrkL-binding protein. In spreading platelets, most endogenous
ASAP1 is localized at peripheral focal adhesions. To determine the
physiologic significance of the CrkL-ASAP1 association, we
overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike
endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse
cytoplasmic distribution. However, when co-expressed with wild-type
CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced
focal adhesions. An SH2-mutated CrkL, which cannot localize at
focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus,
CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.
CrkL is a Src homology (SH)21 and SH3 adapter (1-3).
Through its SH2 domain, CrkL binds to
focal adhesions proteins like paxillin and Cas (1-3). CrkL also binds
to a Rap-specific guanine nucleotide exchange factor, C3G, through its
N-terminal SH3 domain and, thus, conveys C3G to focal adhesions. C3G
activates a small GTPase Rap1 and regulates cell adhesion and
spreading, indicating that the CrkL-C3G complex is a critical component
of focal adhesions (4). We previously reported that CrkL is present in
human platelets, and that it is an adapter for WASP, syk, or
phosphorylated STAT5 (5-7).
On the other hand, ADP-ribosylation factors (Arfs) are also members of
the Ras-related small GTPases and function in the regulation of
membrane trafficking and actin cytoskeleton (8, 9). Similar to other
GTPases, the activity of Arfs is regulated positively by GEFs and
negatively by GTPase-activating proteins (GAPs). Recently, several Arf
GAPs have been cloned and characterized and found to have
phosphoinositide-dependent GAP activity toward Arfs (10). ASAP1 (also called DEF-1, for differentiation enhancing factor-1), the prototype of the phosphoinositide-dependent Arf GAP
family, is a multidomain protein with pleckstrin homology,
Arf GAP, ankyrin repeat, proline-rich region, and SH3 domains. ASAP1
binds to phosphatidylinositol (4, 5)P2 through its PH
domain and shows GAP activity toward Arf (11-13). In NIH3T3 cells,
endogenous ASAP1 localizes in focal adhesions and, when overexpressed,
ASAP1 affects cell spreading of NIH3T3 cells on fibronectin (14).
Although the function of Arf in focal adhesions is not clear yet, the
localization of ASAP1 and its effect on cell spreading suggests the
importance of Arf signaling on the dynamics of focal adhesions.
During our continual efforts to clarify the role of CrkL in the
regulation of signal transduction, we found that the SH3 domain of CrkL
binds to ASAP1. The data obtained from studies using platelets and COS7
cells overexpressing ASAP1 revealed that CrkL is a critical lynchpin
between ASAP1 and focal adhesions.
Blood from healthy volunteers, after obtaining written informed
consent, was drawn by venipuncture into a one-tenth volume of 3.8%
(w/v) trisodium citrate and gently mixed. Alternatively, buffy coat,
provided by the Hokkaido Red Cross Blood Center (Sapporo, Japan), was
used instead of whole blood. Washed human platelets were prepared as
described previously (7) and suspended in a modified Hepes-Tyrode
buffer (129 mM NaCl, 8.9 mM NaHCO3,
0.8 mM KH2PO4, 2 mM
KCl, 0.8 mM MgCl2, 5.6 mM dextrose,
and 10 mM Hepes, pH 7.4) at a concentration of 3 × 108 cells/ml with apyrase (2 units/ml) at 37 °C.
Specific affinity-purified anti-ASAP1 antibody (642) was prepared as
previously described (12). Anti-ASAP1, anti-phospho-FAK, and
anti-paxillin (cross-reactive with Hic-5) monoclonal antibodies were
from Transduction Laboratories (Jackson, KY). Anti-CrkL and anti-C3G
polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). An anti-CrkL monoclonal antibody was from Upstate Technologies
(Lake Placid, NY). An anti-FLAG monoclonal antibody, thrombin, and
other reagents were from Sigma.
Immunoprecipitation and Immunoblotting--
Platelets in
suspension (0.5 ml) were lysed by the addition of an equal amount of
lysis buffer (15 mM Hepes, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM EGTA, 1 mM sodium orthovanadate, 0.8 µg/ml leupeptin, 2% Triton
X-100 (v/w), pH 7.4). Immunoprecipitation and immunoblotting using the
enhanced chemiluminesence (ECL) methods were performed as previously
described (7).
GST Binding Assays--
GST-CrkL N-terminal SH3 fusion protein
was a gift from Dr. Brian J. Druker (Oregon Health Sciences
University). Production of GST fusion proteins and binding experiments
using cell lysates were performed as previously described (7). GST
fusion proteins were isolated from sonicated bacterial lysates using
glutathione-Sepharose beads. Coomassie Brilliant Blue-stained gels were
used to normalize the expression of the various GST fusion proteins.
MALDI-TOF/MS--
From platelet lysates, we
precipitated proteins that bind to the N-terminal SH3 domain of CrkL.
Following digestion by trypsin, the bound proteins were analyzed by
MALDI-TOF/MS using a Voyager-DE/STR (Applied Biosystems, Foster City,
CA). The proteins were identified by comparison between the molecular
weights determined by MALDI-TOF/MS and the theoretical peptide masses
from the proteins registered in NCBInr.
Isolation of Platelet Cytoskeleton--
The Triton
X-100-insoluble cytoskeleton from thrombin-stimulated platelets was
isolated as previously described (15). An equal amount of lysis buffer
was added to the platelet suspensions to solubilize the platelets.
After 5 min on ice, the lysate was centrifuged at 10,000 × g. The resulting pellet was washed twice in washing buffer.
For one-dimensional SDS electrophoresis, the Triton X-100-insoluble
pellets were solubilized in SDS sample buffer. The supernatant was
diluted with an equal volume of 2× concentrated SDS sample buffer.
Ectopic Expression in COS7--
The full-length cDNA of a
murine ASAP1 Localization of ASAP1 in Spread Platelets and COS7
Cells--
Platelets from diluted platelet-rich plasma were allowed to
spread on glass coverslips for 1 h at 37 ° or COS7 cells grown on glass coverslips were fixed in 3.7% paraformaldehyde. Following treatment with 100 mM Tris-HCl (pH 7.4) for 15 min, the
cells were permeabilized in 0.15% Triton X-100 for 1 min and blocked with Block Ace (Snow Brand, Tokyo) for 30 min. The samples were incubated with primary and secondary antibodies, and subjected to
extensive washing. Alexa-labeled secondary antibodies and
Alexa-conjugated phalloidin were purchased from Molecular Probes
(Eugene, OR). Images were taken with an inverted confocal
laser-scanning microscope (Zeiss LSM 510) with a ×100 oil objective
lens and processed by Adobe Photoshop version 7.0.
Mass Spectrometry Following a Pull-down Assay--
Platelet
proteins were precipitated by the N-terminal SH3 domain of CrkL,
expressed as a GST fusion protein, and subjected to SDS-PAGE analysis
followed by Coomassie Brilliant Blue staining. A 140-kDa band was
observed, which was consistently precipitated by GST-CrkL but not GST
alone. A mass spectrometry analysis following trypsin digestion
revealed that the 140-kDa band contained several peptides derived from
the human homologue of mouse ASAP1 (KIAA1249 clone) (Fig.
1A). The anti-ASAP antibody
consistently recognized the 140-kDa protein precipitated by GST-CrkL
(Fig. 1B, left panel). Furthermore, ASAP1
was also specifically immunoprecipitated from platelet lysates by
anti-CrkL antibody, indicating the in vivo interaction of
CrkL with ASAP1 (Fig. 1B, right panels).
Translocation of ASAP1 Following Platelet Activation--
We next
examined whether ASAP1 translocates to the Triton X-100-insoluble
pellets in activated platelets, which represent the operationally
defined actin cytoskeleton (15, 16), as reported for CrkL (5). We found
that ASAP1 translocated to the thrombin-activated actin cytoskeleton
depending upon platelet aggregation (Fig. 1C). We also
confirmed and extended our previous observations that CrkL also
translocates to aggregation-dependent Triton X-100-insoluble
pellets (Fig. 1C). These data suggest that the ASAP1-CrkL
complex may be involved in the organization of the actin cytoskeleton
following platelet aggregation.
The Localization of ASAP1 in Spreading Platelets--
In
glass-activated spreading human platelets, most of ASAP1 was localized
in the areas where stress fibers terminate (Fig. 2B). The ASAP1-rich structures
were also enriched with CrkL, Hic-5, and phosphorylated FAK, suggesting
that, in platelets, ASAP1 is accumulated at focal adhesions (Fig. 2,
A, C, and D). This result is
consistent with the previous report of ASAP1 localization in NIH3T3
cells (12). It should be noted that platelets express Hic-5, a focal
adhesions protein, instead of paxillin (17). As the spreading of
platelets on glass coverslips is also an
Ectopic Expression of CrkL and ASAP1 in COS7 Cells--
Wild-type
CrkL and ASAP1 were overexpressed in COS7 cells. CrkL was purified from
cell lysates by immunoprecipitation. The immunoprecipitates were
subjected to Western blotting following SDS-PAGE analysis. FLAG-tagged
CrkL and ASAP1 were present in the CrkL immunoprecipitate, and the
immunizing peptide for the CrkL antiserum strongly inhibited the
precipitation of both bands (Fig.
3A), suggesting that CrkL and
ASAP specifically associate in COS7 cells. Both wild-type and
SH2-mutated CrkL were coprecipitated with ASAP1, suggesting that the
intact SH2 domain is not necessary for CrkL-ASAP1 interaction (Fig.
3B). Next, we examined the effects of CrkL overexpression on
the localization of both endogenous ASAP1 and CrkL. In the cells where
CrkL was not overexpressed, both CrkL and ASAP1, expressed at low
levels, diffusely distributed as small dots, which were relatively
enriched at the peripheral portion of the cells (Fig.
4A). However, on uncoated
glass coverslips, CrkL overexpression led to the formation of
peripheral focal adhesions, presumably through the
CrkL-mediated increased avidity of integrin (21, 22) to
adhesive proteins in bovine serum adsorbed on glass coverslips. Both
CrkL and endogenous ASAP1 exactly colocalized at these focal adhesions
(Fig. 4B). It was reported that the overexpression of ASAP1
led to destruction of focal adhesions in NIH3T3 cells cultured on
fibronectin-coated glass coverslips (14). Interestingly, the
overexpression of ASAP1 and CrkL did not inhibit the formation of focal
adhesions or colocalization of these proteins (Fig. 4B). CrkL is known to accumulate at focal adhesions through its interaction with Cas or paxillin, via its SH2 domain (1-3). In agreement with the
critical role of the SH2 domain, the SH2-mutated CrkL did not induce
the formation of peripheral focal adhesions or accumulation of ASAP1 at
the structures (Fig. 4C). The overexpression of ASAP1 alone
did not induce peripheral focal adhesions and it was diffusedly
distributed (Fig. 4D) as was reported previously (14). It
appeared that ASAP1 does not disrupt focal adhesions if enough CrkL is
present to accommodate ASAP1. In other words, overexpressed ASAP1 may
remove other CrkL ligands from focal adhesions. The prime candidate of
such molecules is C3G (4), which is a ligand for CrkL and accumulates
at focal adhesions, through its interaction with CrkL.
Overexpression of C3G Inhibits the ASAP1-CrkL Association--
C3G
binds to CrkL through specific interaction of the proline-rich domain
of C3G with the N-terminal SH3 domain of CrkL (4). If ASAP1 binds to
the N-terminal SH3 domain of CrkL in vivo, then the
overexpression of C3G should compete with ASAP1 for binding to CrkL.
The results shown in the Fig. 5 supported
this hypothesis. The overepression of C3G inhibited co-precipitation of
ASAP1 and CrkL (Fig. 5), suggesting that the binding of both proteins
to CrkL may be mutually exclusive.
Despite the recognized importance of Arf in cell biology (8, 9),
the role of Arf in platelet function has received surprisingly little
attention. Our finding that ASAP1 is a novel CrkL ligand, which may be
involved in The mechanisms of accumulation of ASAP1 at focal adhesions were
unclear. Our data suggest that CrkL is one of the carriers of ASAP1 to
focal adhesions, not ruling out the possibility that adapters like Crk
(1-3) have similar roles in other situations. Recently, Liu et
al. (25) reported that ASAP1 directly binds to FAK and suggest
that ASAP1 may be a component of multimolecular complexes at focal
adhesions. The overexpression of wild-type ASAP1 reportedly retarded
the spreading of REF52 cells plated on fibronectin (25). The
overexpression of a truncated variant of ASAP1 unable to bind to FAK
resulted in a less pronounced inhibition of cell spreading (25). These
data suggest that while FAK is a major scaffolding protein for ASAP1,
there must be focal adhesions proteins other than FAK responsible for
localization of ASAP1. As CrkL binds to paxillin or Cas, and locates at
focal adhesions (1-3), the adapter may contribute to
"FAK-independent" functions of ASAP1 at focal adhesions.
Interestingly, it was recently reported that the overexpression of
ASAP1 inhibited spreading of NIH3T3 cells, and the effect was
independent of its Arf GAP activity (26). It is possible that physical
replacement of C3G by ASAP1 is involved in such an effect. More
recently, a RalBP1-binding protein was shown to interact with mouse
ASAP1 (27). In the same report, the association of PAG2 (a human
homologue of ASAP1) and POB1 was demonstrated. It was suggested that
POB1 interacts with PAG2 through its proline-rich motif, similar to
FAK, thereby regulating cell migration. Taken together, CrkL is a
critical regulator of localization of ASAP1, likely through its
interaction with focal adhesions proteins like paxillin or Cas, and,
through cooperation with FAK and POB1, the adapter may be critically
involved in the regulation of the cytoskeleton at focal adhesions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expression plasmid was constructed in the pCI (FLAG
tag) vector. Human CrkL cDNA, a gift from John Groffen (Children's
Hospital, Los Angeles, CA), was subcloned into pCXN2 with FLAG
tag and designated as pCXN2-Flag-CrkL. SH2 mutant of CrkL, which was
mutated on codon 38, CGC (Arg) to GTC (Val), was generated by PCR,
subcloned into pCXN2 vector with FLAG tag, and designated as
pCXN2-Flag-CrkLR38V. The expression vector of FLAG tag C3G (a gift from
Dr. Michiyuki Matsuda) was described previously (4). To obtain cell
lysates, COS7 cells were transfected with 2 µg of recombinant
plasmids using FuGENE 6 (Roche Diagnostics, Indianapolis, IN). The
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. After 24 h, the cells
were harvested with SDS sample buffer and sonicated for SDS-PAGE
analysis. Alternatively, the cells were harvested with the lysis buffer
as described above for immunoprecipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
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Fig. 1.
Identification of ASAP1 as a CrkL ligand in
human platelets. A, the partial amino acid sequence of
human ASAP1. The sequence was predicted from an incomplete cDNA
sequence of the KIAA1249 clone. The underlined peptides were
identified by a mass spectrometry analysis of the trypsin-digested
140-kDa protein co-precipitated with GST-CrkL. B,
in vitro and in vivo interaction of CrkL with
ASAP1. Left panel, platelet proteins co-precipitated
with GST-CrkL were separated by SDS-PAGE (7.5-15% polyacrylamide).
Western blot analysis was conducted with an anti-ASAP1 polyclonal
antibody (642). Right panels, platelet lysates were
prepared using the detergent buffer as described under "Experimental
Procedures," immunoprecipitated with anti-CrkL monoclonal antibody
and separated by SDS-PAGE (7.5-15% polyacrylamide). The
CrkL-immunizing peptide (30 µg/ml) was added during
immunoprecipitation as indicated. Western blot analysis was conducted
with an anti-ASAP1 monoclonal antibody (upper panel) or an
anti-CrkL (lower panel) polyclonal antibody.
HC, heavy chains of the anti-CrkL antibody, used for
immunoprecipitation. C, translocation of ASAP1 to the
cytoskeletal fraction upon activation of platelets by thrombin (1 unit/ml). Platelets were lysed with Triton X-100-EGTA buffer before
(lanes 1 and 4) or after stimulation by thrombin
with (lanes 2 and 5) or without (lanes
3 and 6) stirring. Lysates were separated by high-speed
centrifugation into soluble fractions (lanes 1-3) and
insoluble (lanes 4-6) pellets. Proteins from each fraction
were separated by SDS-PAGE and immunoblotted with anti-ASAP1 monoclonal
or anti-CrkL polyclonal antibodies as indicated.
IIb
3 integrin-dependent
process (18-20), the data further suggest that the ASAP1-CrkL complex
is involved in reorganization of the cytoskeleton following ligation of
IIb
3 integrin. As we cannot express
proteins in platelets, we further investigated the physiologic
significance of the CrkL-ASAP1 interaction in ectopic expression
systems.
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Fig. 2.
Colocalization of ASAP1 with CrkL,
phosphorylated-FAK, and Hic-5 at focal adhesions. Platelets in
diluted platelet-rich plasma were allowed to attach and spread on glass
coverslips, and were fixed and stained for ASAP1, CrkL,
phosphorylated FAK, Hic-5, and F-actin as indicated.
View larger version (20K):
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Fig. 3.
Specific association of CrkL with ASAP1 in
COS7 cells. A, ASAP1 and wild-type CrkL were
overexpressed in COS7 cells. CrkL was immunoprecipitated from the
lysates with an anti-CrkL polyclonal antibody. The CrkL-immunizing
peptide (30 µg/ml) was added during precipitation as indicated.
Replicate samples (± the immunizing peptide) were separated by
7.5-15% SDS-PAGE, transferred to nitrocellulose membranes, and then
immunoblotted with an anti-FLAG monoclonal antibody (upper
panel) or with an anti-CrkL polyclonal antibody (lower
panel). B, wild-type CrkL (lane 1) or
SH2-mutated CrkL (lane 2) was overexpressed with ASAP1 in
COS7 cells. CrkL was purified from the soluble extracts by
immunoprecipitation, and the denatured samples were separated by
7.5-15% SDS-PAGE, transferred to nitrocellulose membrane, and then
immunoblotted with an anti-FLAG monoclonal antibody.
View larger version (22K):
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Fig. 4.
Fluorescence micrographs showing the
localization of ASAP1 and CrkL in COS7 cells. COS7 cells were
allowed to attach and grow on glass coverslips overnight and were
transfected with expression vectors. After a further overnight
incubation, the cells were fixed and stained for CrkL and ASAP1 as
indicated. A, wild-type CrkL was overexpressed.
B, both wild-type CrkL and ASAP1 were overexpressed.
C, both SH2-mutated CrkL and ASAP1 were overexpressed.
D, only ASAP1 was overexpressed and identified by a
monoclonal anti-FLAG antibody.
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Fig. 5.
Overexpression of C3G inhibits the
association of CrkL with ASAP1. Wild-type CrkL was co-expressed
with C3G (lane 1), ASAP1 (lane 2), or both
(lane 3). The Triton X-100-soluble extracts (20 µg/ml/lane) were denatured and subjected to SDS-PAGE followed by
immunoblotting. Five separate membranes were prepared and treated with
anti-FLAG, anti-C3G, anti-CrkL, or anti-ASAP1 antibodies as indicated.
B, CrkL was purified from cell lysates separated by
7.5-15% SDS-PAGE, transferred to nitrocellulose membranes, and the
membranes were then immunoblotted with anti-ASAP1, anti-C3G, or with
anti-CrkL antibodies as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3 signaling, should set
the molecular basis for further studies on Arf and its GAPs in
platelets. Mass spectrometry analyses following two-dimensional
electrophoresis of platelet protein were performed, and the presence of
a number of hitherto undescribed platelet proteins was reported (23, 24). Our approach confirms that mass spectrometry is indeed a powerful
approach to identify platelet proteins. Furthermore, our approach also
employs a pull-down assay before mass spectrometry, thus enabling us to
identify a novel protein-to-protein interaction.
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ACKNOWLEDGEMENTS |
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We thank Drs. Brian J. Druker, Michiyuki Matsuda, and John Groffen for generously providing reagents. We appreciate the technical assistance of Kazutoshi Nagao and Asuka Watanabe.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Science and Technology of Japan (to I. W., H. F., S. T., K. M.), CREST (to I. W., K. N.), Human Frontier Science Program (to A. O., L. M. M.), Mitsui Insurance Welfare Foundation (to A. O.), Daiwa Health Foundation (to A. O.), Mochida Memorial Foundation (to A. O.), Kanehara Ichiro Foundation (to A. O.), Takeda Science Foundation (to A. O.), Iijima Foundation for Food Science (to A. O.), Mitsubishi Pharma Research Foundation (to A. O.), Ssakawa Scientific Research Grant (to C. N.), and the Clark Foundation (to A. O., C. N.).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.
b To whom correspondence should be addressed: Laboratory of Environmental Biology, Dept. of Preventive Medicine, Hokkaido University School of Medicine, N15W7, Kita-ku, Sapporo, 060-8638, Japan. Tel.: 81-11-706-5066; Fax: 81-11-706-7819; E-mail: aoda@med.hokudai.ac.jp.
c Both authors contributed equally to this work.
g Present address: R&D Center, Production Dept., Pharmaceutical Division, Kirin Brewery Co., LTD., 1-2-2 Souja, Maebashi, Gunma 371-0853 Japan.
h Present address, Dept. of Biology, School of Education, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku, Tokyo 169-8050, Japan.
m These authors contributed equally to this work.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M210817200
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ABBREVIATIONS |
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The abbreviations used are: SH, Src homology domain; Arfs, ADP-ribosylation factors; FAK, focal adhesions kinase; GAP, GTPase-activating protein; ASAP1, ARF GAP-containing SH3, ANK repeat, and pleckstrin homology domains; DEF-1, differentiation enhancing factor-1; ECL, enhanced chemiluminesence; GST, glutathione S-tranferase; STAT, signal transducer and activator of transcription; MALDI-TOF/MS, matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry.
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