From the Department of Molecular Medicine, Cornell
University, Ithaca, New York 14853 and the ** Department of Zoology and
the ¶ Institute of Biochemistry, National Chung Hsing University,
Taichung 402, Taiwan, Republic of China
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ABSTRACT |
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We have previously shown that overexpression of
focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells
promoted their migration on fibronectin. This effect was dependent on
the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to
promote cell migration. In this study, we have examined the role of
PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K
inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted
migration in a dose-dependent manner. Furthermore, a FAK
mutant capable of binding Src but not PI3K was generated by a
substitution of Asp residue 395 with Ala. When overexpressed in CHO
cells, this differential binding mutant failed to promote cell
migration although its association with Src was retained. Together,
these results strongly suggest that PI3K binding is required for FAK to
promote cell migration and that the binding of Src and
p130Cas to FAK may not be sufficient for this event.
Focal adhesion kinase
(FAK)1 is a key component of
integrin-mediated signal transduction (1-3). This 125-kDa nonreceptor
protein-tyrosine kinase (PTK) is localized to focal contacts in
fibroblasts and represents the prototype of a distinct family of
nonreceptor PTKs (4, 5). It rapidly becomes phosphorylated following
cell adhesion to extracellular matrix (ECM) proteins or integrin
clustering by antibodies (6-9). In addition to its role in integrin
signaling, FAK has also been suggested to be a point of convergence of
signaling by other extracellular stimuli (10). The ability of FAK to
transmit signals to downstream targets is dependent on its ability to
interact with several intracellular signaling molecules including Src
family kinases (11, 12), phosphatidylinositol 3-kinase (PI3K; Ref. 13),
Grb2 (14), and p130Cas (15). Tyr-397 has been identified as
the major site of FAK autophosphorylation (16) and the binding site for
the Src-homology (SH)2 domains of Src (11, 12) and PI3K (17). The
binding site for the SH2 domain of Grb2 has been mapped to Tyr-925
(18). The proline-rich region of FAK (residues 712-718) has been
identified as the major binding site for the SH3 domain of
p130Cas (15, 19).
Mounting evidence suggests that FAK plays an important role in
regulating cell migration in response to cell adhesion to ECM proteins
(20, 21). We have shown previously that the overexpression of FAK in
Chinese hamster ovary (CHO) cells promoted migration on fibronectin
(21). We have also demonstrated that p130Cas association
with FAK was required for FAK-promoted cell migration (22).
Furthermore, a substitution of Tyr-397 with Phe abolished FAK-promoted
cell migration (21), implicating a role for Src and/or PI3K in this phenomenon.
PI3K is a heterodimer consisting of an 85-kDa regulatory subunit (p85),
and a 110-kDa catalytic subunit (p110) (23, 24). p85 contains an SH3
domain and two SH2 domains. This kinase phosphorylates the D-3 position
of the inositide ring of phosphatidylinositol and its derivatives,
phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (25). These lipid products are believed to act as
second messengers in a variety of signaling processes including cell
survival (26, 27) and migration (28-30). We have shown that PI3K
associates with FAK in response to cell adhesion (13) or
platelet-derived growth factor (PDGF) stimulation (31) in NIH 3T3
cells. The significance of this interaction in cellular processes is unknown.
In this report, we have found that a FAK mutant capable of binding to
Src, but not to PI3K, failed to stimulate cell migration and that
inhibition of PI3K decreases cell migration. Taken together, these
results strongly implicate a role for PI3K in the facilitation of
FAK-promoted cell migration and suggest that the binding of Src and
p130Cas to FAK may not be sufficient for this process.
Antibodies--
The mouse mAb KT3 (21) and the mouse mAb 12CA5
(anti-HA) (17), which recognize an epitope (KPPTPPPEPET) of the SV40
large T antigen and an epitope (YPYDVPDYA) of the hemagglutinin (HA) protein of the influenza virus, respectively, have been described previously. The rabbit polyclonal anti-FAK (11) and anti-p85 (17) sera
have also been described previously. The anti-Src mAb 327 was purchased
from Calbiochem (San Diego, CA). The anti-Src mAb 2-17 was purchased
from Quality Biotech (Camden, NJ). The rabbit polyclonal
anti-p130Cas (C-20) was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). The anti-phosphotyrosine
(anti-PY) mAb PY20 was purchased from Transduction Laboratories
(Lexington, KY).
Construction of Expression Plasmids--
The pKH3 plasmids
encoding HA epitope-tagged FAK and Y397F mutants were described
previously (17). The cDNA encoding other FAK mutants (D395A, D396A,
D395/396A, P712/715A) used in this study were generated using
site-directed mutagenesis by overlap extension using the polymerase
chain reaction. The polymerase chain reaction products containing the
desired mutations were treated with restriction enzymes KpnI
and NdeI and then used to replace the corresponding
fragments in the plasmid pGEX-FAK. The desired mutations were confirmed
by dideoxy DNA sequencing. The cDNAs encoding FAK mutants in pGEX2T
vectors were in-frame and transferred to pKH3 vectors using the
BamHI and EcoRI sites. The pKH3 plasmids encoding
FAK and its mutants were used to transiently express FAK proteins in
human embryonic kidney (HEK) 293 cells.
The expression plasmid pCDM8-FAK and pCDM8-FAK P712/715A have been
described previously (21, 22). pKH3-FAK D395A was digested with
MscI and NheI to generate a 1.8-kilobase fragment
containing the D395A substitution. This was then subcloned into
pCDM8-FAK from which the corresponding MscI-NheI
fragment had been excised. The resulting plasmid was designated
pCDM8-FAK D395A and used to transfect CHO cells.
The cDNA encoding the p85 subunit of PI3K that was kindly provided
by Dr. L. C. Cantley (Harvard University) was inserted into pKH3
at the BamHI and EcoRI sites. The pEVX plasmid
encoding c-Src was kindly provided by Dr. D. Shalloway (Cornell University).
Cell Culture and Transfections--
HEK 293 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum (Life Technologies, Inc.). One day after
plating 5 × 105 cells on 60-mm dishes, HEK 293 cells
were transiently transfected with 1 µg of pKH3 plasmids encoding FAK
or its mutants using 10 µl of LipofectAMINE (Life Technologies,
Inc.). In some cases, HEK 293 cells were co-transfected with pKH3-FAK
or its mutants, pKH3-p85, and pEVX-Src. Two days after transfection,
the cells were lysed in 1% Nonidet P-40 lysis buffer containing
protease inhibitors as described previously (8).
CHO cells expressing wild type (WT) FAK, Y397F, and P712/715A have been
described previously (21, 22) and were maintained in F-12 medium with
10% fetal bovine serum and 0.5 mg/ml G418 (Life Technologies, Inc.).
To generate cells stably expressing the D395A FAK mutant, CHO cells
were grown on 10-cm dishes and transfected essentially as described
(21) using LipofectAMINE following the manufacturer instructions.
Clones were selected in G418-containing medium and screened by
immunoblotting using anti-FAK and KT3.
In Vitro Binding Assays--
The plasmids pGEX-Src.SH2 and
pGEX-p85.NSH2 were described previously (17). The cDNA encoding the
SH3 domain of p130Cas (22) was cloned into pGEX2T at the
BamHI and EcoRI sites to generate pGEX-Cas.SH3.
GST fusion proteins were immobilized on glutathione-agarose beads and
then incubated with HEK 293 cell lysates (~10 µg) containing
various HA epitope-tagged FAK proteins in 1% Nonidet P-40 lysis buffer
for 1 h at 4 °C. The complexes were washed four times with 1%
Nonidet P-40 lysis buffer, resolved by SDS-polyacrylamide gel
electrophoresis, and analyzed by immunoblotting with anti-HA.
Immunoprecipitations, Immunoblotting, and in Vitro Kinase
Assays--
Attached cells were lysed in Nonidet P-40 buffer
containing protease inhibitors. To detect the association of FAK with
Src and p85 in HEK 293 cells, lysates (50 µg) from cells
co-transfected with pKH3-FAK, pKH3-p85, and pEVX-Src were incubated
with anti-Src (327) or anti-p85. The co-immunoprecipitation of
transfected WT FAK or mutants with endogenous c-Src in CHO cells was
carried out as described previously using the anti-Src (2-17) (21). To
determine the extent of p130Cas phosphorylation in CHO
cells overexpressing WT FAK or mutants, lysates (800 µg) were
incubated with anti-p130Cas as described (22). The
immunocomplexes were washed, resolved by SDS-polyacrylamide gel
electrophoresis, and analyzed by immunoblotting with anti-HA (1:1000),
KT3 (1:3000), anti-FAK (1:3000), anti-Src (327) (1:500),
anti-p130Cas (1:1000), or anti-PY (1:1000) using the
Amersham Pharmacia Biotech chemiluminescence system for detection. In
some experiments, equal amounts of lysates were analyzed directly
by immunoblotting.
To measure the tyrosine phosphorylation and in vitro
autophosphorylation of ectopically expressed FAK proteins, HA
epitope-tagged FAK proteins were immunoprecipitated with anti-HA from
HEK 293 cell lysates (50 µg) and subjected to immunoblotting with
anti-PY or to an in vitro kinase assay as described earlier
(8).
To measure the PI3K activity associated with ectopically expressed FAK
proteins in CHO cells, epitope-tagged FAK proteins were
immunoprecipitated with KT3 from CHO cell lysates (750 µg) and
subjected to an in vitro PI3K assay as described previously (13).
Cell Migration Assays--
CHO cell migration assays in 48-well
chemotaxis chambers were carried out as described previously (21). For
experiments with the PI3K inhibitors wortmannin and LY294002 (Sigma)
(reconstituted in Me2SO and stored at It is known that PI3K plays an important role in growth
factor-induced cell migration (28-30). To examine the putative role of
PI3K in FAK-promoted cell migration, CHO cells overexpressing WT FAK
and control cells (Neo) were subjected to cell migration assays in the
presence of specific PI3K inhibitors (Fig.
1). Treatment of WT cells with either
PI3K inhibitor resulted in an inhibition of cell migration in a
dose-dependent manner. Furthermore, in the presence of
either 100 nM wortmannin or 75 µM LY294002,
the majority (~80%) of cell migration promoted by FAK overexpression in WT cells was inhibited, with little effect on control cell migration. These results demonstrate that PI3K plays a role downstream of FAK in promoting CHO cell migration on fibronectin. However, from
these results, it was not clear whether FAK-promoted cell migration is
dependent on PI3K, either through direct binding at Tyr-397 of FAK or
through another FAK-promoted signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C), cells
were harvested and washed as described earlier and then resuspended in
F-12 medium containing the indicated concentrations of inhibitor or
Me2SO such that the final concentration of
Me2SO was constant. The cells were pretreated with the
inhibitor at 37 °C and 5% CO2 for 30 min before loading them onto the chemotaxis chamber. They were allowed to migrate on 12 µg/ml fibronectin for 6 h in the presence of the inhibitor and
were then fixed and stained as described previously. For all cell
migration assays, migrated cells were enumerated under a light
microscope at ×200 magnification using the Image-Pro Plus software,
Version 3.0.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FAK-promoted cell migration is inhibited by
the PI3K inhibitors wortmannin or LY294002. CHO cells
overexpressing FAK (WT) or control cells (Neo)
were treated with the indicated concentrations of either wortmannin
(A) or LY294002 (B) for 30 min before subjecting
them to a cell migration assay as described under "Experimental
Procedures." Migrated cells were then fixed, stained, and counted
using a light microscope. Mean cell counts from at least fourteen
fields and three experiments are shown. Relative cell migration was
calculated based on the levels of WT cell migration in the absence of
inhibitor. Error bars represent standard errors.
To determine whether the direct binding of PI3K to FAK is a
prerequisite for its function in FAK-promoted cell migration, a FAK
mutant deficient only in PI3K binding was generated by a substitution
of Asp-395 with Ala (see below). Songyang et al. (32)
suggested that residues both N- and C-terminal to the phosphotyrosine may contribute to the specific binding of the SH2 domain to its cognate
phosphotyrosine and showed that the optimal binding sequence for the
p85 N-terminal SH2 domain is EEDpYVEM. An examination of FAK sequences
revealed that sequences flanking Tyr-397 (TDDpYAEI) conformed well with
the binding motif for the p85 N-terminal SH2 domain. Based on these
observations, two Asp residues upstream of Tyr-397 were mutated either
singly or in combination to generate a PI3K binding-defective FAK
mutant. These FAK mutants were transiently expressed in HEK 293 cells,
and their ability to bind the SH2 domains of Src and p85 was examined
in vitro (Fig. 2A).
Similar to the mutation in Tyr-397, the mutation converting Asp-396 (or in combination with Asp-395) to Ala abolished FAK binding to the SH2
domains of Src and p85. Interestingly, a mutation in Asp-395 inhibited
FAK binding to the SH2 domain of p85 but not Src. To examine if the FAK
D395A mutant also selectively binds to Src but not p85 in intact cells,
HEK 293 cells were transiently co-transfected with expression plasmids
encoding HA epitope-tagged FAK or its mutants, c-Src, and p85. Two days
after transfection, cells were lysed, and the association of FAK with
Src or p85 was detected by co-immunoprecipitation (Fig. 2B).
Consistent with the results from in vitro binding
experiments, the mutation in Asp-396 abolished FAK association with
both Src and p85, and importantly, the mutation in Asp-395 only
prevented FAK association with p85 but not Src.
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To examine the effects of FAK mutations in Asp-395 and Asp-396 on its
tyrosine phosphorylation and in vitro autophosphorylation, HA epitope-tagged WT FAK or its mutants were transiently expressed in
HEK 293 cells, immunoprecipitated with anti-HA, and subjected to
immunoblotting with anti-PY or to an in vitro kinase assay (Fig. 3A). The tyrosine
phosphorylation and in vitro autophosphorylation of FAK
mutants containing a substitution of Asp-396 with Ala were as low as
those of the Y397F mutant, indicating that Asp-396 is essential for the
phosphorylation event upon Tyr-397. This also reflects the inability of
the FAK D396A mutant to bind the SH2 domains of Src and p85. In
contrast, the tyrosine phosphorylation and in vitro
autophosphorylation of FAK D395A mutant remained comparable with the
WT. These results indicated that the inability of the D395A mutant to
bind PI3K was most likely not because of an overall conformational
change. To exclude the possibility that the mutation in Asp-395 causes
a subtle change in protein conformation and thereby affects the binding
of FAK to other proteins, the capacity of FAK D395A to bind to the SH3
domain of p130Cas was examined in vitro (Fig.
3B). The result clearly showed that the ability of FAK to
bind the SH3 domain of p130Cas was not affected by mutating
Asp-395. Together, these data suggest that the inability of FAK D395A
to bind p85 is indeed because of the preference of the p85 SH2 domain
for an aspartic acid at residue 395 in FAK.
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To determine whether PI3K binding is required for FAK-promoted cell
migration, stable CHO cell lines overexpressing FAK D395A were
established. The expression of FAK D395A was examined by immunoblotting
with anti-FAK or KT3 (Fig.
4A). The expression levels of
ectopic WT FAK and D395A mutant in CHO cells were comparable and
severalfold higher than that of endogenous FAK. To examine the
association of endogenous PI3K with WT and mutant FAK in CHO cells,
ectopically expressed FAK proteins were immunoprecipitated by mAb KT3,
and the immunocomplexes were subjected to an in vitro PI3K
activity assay (Fig. 4B). Approximately 10% of total
cellular PI3K activity were found to associate with ectopic WT FAK
(data not shown). As expected, the levels of PI3K activity associated with WT FAK and P712/715A mutant were similar. Surprisingly,
approximately 15% of PI3K activity that WT FAKs had were
co-precipitated with Y397F and D395A mutants. The cell migration assays
indicated a marked increase (~2.5-fold) in cell migration for WT
cells compared with both Y397F and control (Neo) cells, which were
similar in their rates of migration. Interestingly, both D395A clones
failed to promote cell migration (Fig. 4C). These results
together suggest that the residual binding of PI3K to FAK Y397F or
D395A mutant in CHO cells is not sufficient for promoting migration.
Moreover, adhesion assays indicated that all clones had similar
adhesive strength to fibronectin (data not shown), indicating that the decrease in migration observed in D395A clones was not because of an
alteration in cell adhesion.
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We have previously demonstrated that phosphorylation of Tyr-397 in FAK
is required for binding the SH2 domains of Src and PI3K (17). Thus, it
is unclear whether the inability of FAK Y397F to promote cell migration
is because of its inability to bind Src and/or PI3K. To clarify this,
the association of FAK D395A with c-Src in CHO cells was examined. Both
WT FAK and FAK D395A but not FAK Y397F co-precipitated with c-Src in
CHO cells (Fig. 5A). We have
previously demonstrated that the association of p130Cas
with FAK and FAK-promoted phosphorylation of p130Cas by Src
are critical for FAK-promoted migration (22). We examined the
phosphorylation state of p130Cas in FAK D395A cells as an
indicator of a FAK/Src-depending cell migration pathway as well as an
indirect indicator of FAK/p130Cas association. Consistent
with prior experiments (22), WT FAK exhibited a promotion of
p130Cas phosphorylation compared with the FAK P712/715A
(Fig. 5B), which is unable to bind p130Cas or
promote p130Cas phosphorylation (22). FAK D395A was able to
promote p130Cas phosphorylation at an estimated 2-fold
level above WT FAK, indicating that the FAK/Src specific pathway is
still functioning in the D395A mutant. Taken together, these results
strongly suggest that PI3K binding to FAK is required for FAK to
promote cell migration and that FAK:Src association alone may be
insufficient for this cellular function.
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DISCUSSION |
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In this report, we examined the role of PI3K in FAK-promoted cell migration. First we showed that two PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Our results are consistent with other work showing the importance of PI3K in increased cell motility stimulated by growth factors such as PDGF and hepatocyte growth factor (28-30). However, these data do not indicate whether PI3K association with FAK is required for increased cell migration and cannot rule out the involvement of PI3K in a FAK-independent migration pathway. To address this, we next established stable cell lines overexpressing FAK and a mutant defective in PI3K binding. Using this system, we showed that the PI3K binding-deficient mutant failed to promote cell migration toward fibronectin. These results strongly suggest that FAK:PI3K association is required for FAK-promoted cell migration.
We have previously identified that the autophosphorylation site Tyr-397 is the primary site for PI3K binding (17) and is required for the function of FAK in promoting cell migration (21). We show here that the introduction of a mutation in Asp-395 completely abolishes FAK association with PI3K SH2 domain but not Src SH2 domain in vitro. The D395A mutant transiently expressed in HEK 293 cells also loses its ability to bind to intact p85 but not Src. However, in CHO cells some residual binding of PI3K to Y397F and D395A mutants can be detected, indicating that other mechanisms such as SH3 interaction may also be involved. This result is consistent with the previous report that the SH3 domain of p85 could bind to a proline-rich region (amino acids 875-884) of FAK in vitro (33) and suggest that an SH3 interaction besides SH2 interaction might also participate in FAK:PI3K interactions in response to certain stimuli in different cell types. Furthermore, together with our observation that FAK D395A mutant retained its tyrosine phosphorylation and in vitro phosphorylation to the same level as WT FAK, it appears that Asp-395 makes a major contribution for specific binding of the SH2 domains of PI3K to FAK. This finding also supports the notion that residues N-terminal of phosphotyrosine are critical for specific binding to certain SH2 domain-containing proteins.
Previous results from our laboratory showed that a FAK mutant (P712/715A) deficient in p130Cas binding failed to promote cell migration (22). In this report, we showed that FAK D395A deficient in PI3K binding also failed to increase cell migration. Although a residual binding of PI3K to D395A mutant was detected in CHO cells, it was apparently not sufficient for promoting cell migration. In fact, an analogous situation has also been described that a residual binding of p130Cas to FAK P712/715A mutant possibly mediated by a second proline-rich region (amino acids 875-884) on FAK does not allow for FAK-promoted migration (22). These results together suggest that thresholds of signals transmitted independently through the associations of p130Cas and PI3K with FAK have to be reached for enhanced cell migration. Lack of either one will not turn on an "intracellular machinery" for the induction of cell migration. Another possible interpretation is that the inability of FAK D395A to promote cell migration is because of a defect in p130Cas binding. This defect may result from a subtle conformation change by the D395A substitution, or a mutually dependent binding of PI3K and p130Cas to FAK. The latter possibility was excluded by our observation that the D395A mutant retained its ability to bind the SH3 domain of p130Cas in vitro and promote p130Cas phosphorylation in vivo.
The tyrosine phosphorylation of p130Cas and its subsequent association with the adapter protein Crk have been shown to play an important role in promoting cell migration (34). In addition, the tyrosine phosphorylation of p130Cas has also been shown to be a result of binding to FAK (22). Here, we have shown that expression of FAK D395A increased p130Cas tyrosine phosphorylation at a roughly 2-fold level above WT FAK in CHO cells. Although the reason for this elevation in p130Cas phosphorylation is unclear, this result supports our conclusion that p130Cas binding and phosphorylation is necessary but not sufficient for FAK-promoted cell migration. Furthermore, several studies have shown that Src is important for phosphorylation of p130Cas (35, 36). Because FAK D395A still binds to Src, our results also suggest that FAK:Src association alone is not sufficient for the enhancement of cell migration. However, we cannot exclude the necessity of Src in FAK-promoted cell migration because of its function, at least, in phosphorylating p130Cas. Therefore, it is likely that FAK-promoted cell migration depends on the coordinate regulation of a number of these signaling events involving PI3K, p130Cas, and Src.
We have previously shown that FAK:PI3K association is enhanced by cell
adhesion to ECM proteins (13) or PDGF stimulation (31). In this report,
we have shown that this association is likely to be involved in cell
migration upon cell adhesion to ECM proteins. However, it is not clear
if this interaction is also involved in PDGF-stimulated cell migration.
Furthermore, we do not know if FAK generally participates in growth
factor-induced cell motility using a mechanism similar to that in ECM
protein-induced cell migration. It is known that the tyrosine
phosphorylation of FAK is stimulated by a number of growth factors (31,
37). Although the mechanisms by which growth factor receptors and
integrins regulate FAK phosphorylation may be different (38), it is
possible that phosphorylated FAK recruits the same set of intracellular signaling molecules to facilitate cell migration. Answers to these questions will be interesting, and experiments are now in progress to
test these possibilities.
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ACKNOWLEDGEMENTS |
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We thank Dr. L. C. Cantley for the cDNA encoding the p85 subunit of PI3K and Dr. D. Shalloway for the expression vector encoding c-Src. We also thank Dr. D. C. Han for the construction of pGEX-Cas.SH3 and P.-C. Chan for the critical reading of this manuscript.
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FOOTNOTES |
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* This research was supported by National Science Council, Taiwan, Grant NSC88-2311-B005-027 (to H.-C. C.) and grants from the National Institutes of Health (to J.-L. G).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.
§ These authors contributed equally to this work.
Is an established investigator of the American Heart Association.
To whom correspondence should be addressed. Tel.:
886-4-2854922; Fax: 886-4-2851797; E-mail: hcchen{at}nchu.edu.tw.
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ABBREVIATIONS |
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The abbreviations used are: FAK, focal adhesion kinase; PTK, protein-tyrosine kinase; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; SH, Src-homology; CHO, Chinese hamster ovary; PDGF, platelet-derived growth factor; HA, hemagglutinin; HEK, human embryonic kidney; anti-PY, anti-phosphotyrosine; WT, wild-type; GST, glutathione S-transferase; mAb, monoclonal antibody.
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