 |
INTRODUCTION |
Cell migration allows for proper immune responses and wound
healing. However, unregulated cell migration leads to pathological processes, including inflammation and tumor cell metastasis.
Identification of molecular signaling mechanisms that positively and
negatively regulate cell migration is critical in understanding these
diseases. Many of the signals regulating motility likely target the
actin cytoskeleton of cells, as movement requires structural
alterations. Migratory cells are characterized by extension of a
leading lamellipodium with membrane ruffles, decreased focal contacts
to the extracellular matrix
(ECM),1 and loss of actin
stress fibers (for review see Ref. 1).
Recent evidence suggests that Abl family kinases regulate the actin
cytoskeleton and influence cell morphology (2-8). However, the
biological significance of this is not clear. c-Abl (Abl) is a member
of the Abelson family of nonreceptor tyrosine kinases, which also
includes Bcr-Abl and the Abl-related gene product (Arg). Abl and Arg
are known to localize to the cell cytoplasm where they bind actin and
associate with focal contacts (9-11). Interestingly, attachment of
cells to fibronectin induces the activation of Abl and the transient
migration of nuclear Abl to the cytoplasm and focal adhesions
(10). Exposure of cells to cytokines also induces Abl activity in the
cell cytoplasm and membrane (12). Direct interaction of Abl family
kinases with the actin cytoskeleton occurs via two conserved domains,
which bind G- and F-actin (9, 13). Coordination of simultaneous binding
of Abl to G- and F-actin is thought to aid in the bundling of actin
filaments (13). The cytoskeletal-associated proteins, amphiphysin-like
protein 1 (ALP1) and Abl/Arg-binding protein 2 (ArgBP2), also bind Abl
family enzymes (5, 6), providing a mechanism of indirect interaction
between Abl family kinases and the cytoskeleton. In addition, fusion of Bcr to Abl creates Bcr-Abl, which primarily localizes to the
cytoskeleton in the cytoplasm and is strongly associated with cell
transformation and leukemia (14, 15). Together, these data indicate
that Abl family kinases are closely associated with the actin
cytoskeleton of cells.
Abl and Arg contain SH2, SH3, and tyrosine kinase domains, which are
highly conserved between the two enzymes (16). Therefore, it is likely
that Abl and Arg share common target proteins. For example, Abl and Arg
kinases contain a proline-rich region that mediates their interactions
with the SH3 domain of c-Crk II (Crk), allowing both Abl and Arg to
tyrosine-phosphorylate Crk (17, 18). Crk belongs to a family of adaptor
molecules containing SH2 and SH3 domains that couple to effector
proteins including p130CAS (CAS), C3G, SOS, Eps15, DOCK180,
Abl, and Arg (17-21). Previous work has shown that the association of
Crk with CAS induces cell migration and enhances invasiveness of
carcinoma cells (22). This provides a molecular switch leading to cell
migration and invasion through a Rac-dependent mechanism.
Crk-CAS coupling depends on the interaction of the Crk SH2 domain with
phosphotyrosine residues present in the substrate domain of CAS (22).
Whereas these findings demonstrate an important role for Crk-CAS
complexes in cell migration, it is not known how the formation of this
molecular complex is regulated in migratory cells. Recent evidence
suggests Crk-CAS coupling may occur through a tightly regulated balance of tyrosine phosphorylation of CAS by Src and/or focal adhesion kinase
and dephosphorylation by phosphatases (19, 23). Previous work has also
shown that phosphorylation of mammalian Crk at tyrosine 221 by Abl
prevents Crk binding to tyrosine-phosphorylated proteins (8, 18). This
suggests an additional mechanism for the regulation of Crk-CAS
complexes in migratory cells. In this case, the phosphorylated tyrosine
at position 221 becomes directly bound by the SH2 domain of Crk,
causing the protein to functionally "fold back" on itself. This
intramolecular folding sterically blocks the SH2 and N-terminal SH3
domains of Crk, thereby preventing the association of Crk with
downstream effector molecules (8, 18, 24). However, the biological
significance of Crk phosphorylation and its coupling to effector
molecules in vivo are poorly defined. Here we investigate the role of Abl family kinases in the regulation of Crk-CAS coupling and the activation of the migration machinery of cells. We provide evidence that Abl family kinases negatively regulate cell migration by
uncoupling CAS-Crk complexes.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Embryonic fibroblasts isolated from
abl
/
arg
/
mice or control mice
(abl+/+ arg+/+) were the generous gift of Dr.
Anthony J. Koleske (3). abl
/
cells were kindly provided
by Dr. Jean M. Lewis and Dr. Jean Y. J. Wang (10). Fibroblasts and
COS-7 cells were maintained in Dulbecco's modified Eagle's medium
(Irvine Scientific) containing 10% fetal bovine serum (Irvine
Scientific), 200 mM L-glutamine, 50 µg
ml
1 gentamicin (Sigma), and 100 mM sodium pyruvate. Cells were incubated at 37 °C with
5% CO2.
Immunoprecipitation and Immunoblotting--
Both
immunoprecipitation and immunoblotting of proteins have been described
previously (22). Briefly, whole cell lysates for immunoblotting were
prepared by washing cells twice with PBS, lysing in 1% SDS lysis
buffer (1% SDS, 2 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor mixture tablet per
40 ml of buffer (CompleteTM, Roche Molecular
Biochemicals)), and immediately boiling for 10 min. Lysates were
prepared for immunoprecipitation by rinsing cells 2× with PBS, lysing
cells in Triton X-100 buffer (50 mM NaCl, 1 mM
EDTA, 50 mM HEPES, 1 mM EGTA, 1% Triton X-100,
2 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 1 protease inhibitor mixture tablet per
40 ml of buffer, 10 mM NaF, pH 8.0), and incubating on ice
for 2 h before clarification of lysates by centrifugation. Lysates
were stored at
80 °C. Generally, 15-25 µg of cellular protein
was separated by SDS-PAGE and immunoblotted according to standard
protocols. However, detection of Arg kinase required 60 µg of total
cellular protein.
Antibodies--
Abl (8E9, BD PharMingen), Crk (Transduction
Laboratories), CAS (Transduction Laboratories), and phosphotyrosine
(Upstate Biotechnology Inc.) HA tag (Roche Molecular Biochemicals),
FLAG tag (Eastman Kodak Co.), vinculin (Sigma) antibodies were
purchased from commercial sources as indicated. Polyclonal sera
recognizing Arg kinase was the generous gift of Dr. Anthony J. Koleske
(3). Polyclonal anti-peptide antibodies recognizing only tyrosine
221-phosphorylated Crk were a gift from Dr. Michiyuki Matsuda (24).
Function-blocking monoclonal antibodies to
1 integrin
subunit (P4C10) and
V
5 (PIF6) have been
described (25).
Immunofluorescence--
Glass coverslips were coated for 2 h at 37 °C with 1 µg/ml human fibronectin. abl+/+
arg+/+ and abl
/
arg
/
fibroblasts were plated on the coverslips and serum-starved overnight
at 37 °C. Cells were then fixed in 4% paraformaldehyde and briefly
permeabilized with 0.1% Triton X-100. The coverslips were blocked for
20 min in 1% bovine serum albumin (fraction V, Sigma) before
incubation with antibodies. Cells were treated with an anti-vinculin
antibody followed by a fluorescein isothiocyanate-labeled goat
anti-mouse secondary antibody and rhodamine-conjugated phalloidin. Cell
fluorescence was analyzed with a laser confocal microscope (model 1024, Bio-Rad) and a Zeiss Axiovert microscope (Thornwood, NY) focused at the cell-substratum interface at a power of × 630. The Abl inhibitor, STI 571, was generously supplied by Dr. Elizabeth Buchdunger (26-28). Cells treated with STI 571 were first serum-starved overnight and then
pretreated with 10 µM STI 571 for 2 h prior to
plating cells on fibronectin-coated coverslips. Cells attached and
spread for 6 h in the continued presence of 10 µM
STI 571 before cells were fixed and processed as described above.
Migration Assays--
Transient transfection of COS-7 cells and
Transwell migration assays were performed as described previously (22).
Briefly, COS-7 cells were transfected with LipofectAMINE and expression vectors encoding wild-type or mutant forms of Abl, wild-type Crk, or
wild-type CAS in addition to a reporter construct encoding
-galactosidase (pCMV·SPORT-
-galactosidase (Life Technologies, Inc.)). The appropriate amounts of empty vector and
-galactosidase expression vector were transfected into control cells. Cells were incubated with LipofectAMINE (Life Technologies, Inc.) and expression vectors for 6-8 h, rinsed, and allowed to recover in media containing 10% fetal bovine serum. Transfected cells were used ~40 h following transfection, allowing for optimal transient expression in these cells.
Migration assays were performed using Boyden chambers containing polycarbonate membranes (tissue culture treated 6.5 mm diameter, 10 µm thickness, 8 µm pores, Transwell® (Costar) or QCM kit (Chemicon International Inc.)) as described (22). Membranes were coated on the
bottom only with 10 µg/ml of either rat tail collagen type I (Upstate
Biotechnology, Inc.) or human fibronectin (Oxford Biomedical Products)
for 2 h at 37 °C. Prior to haptotaxis migration, cells were
serum-starved overnight. Following 3-5 h of migration, cells were
fixed with ethanol and stained with crystal violet, or transfected cells were fixed in
-galactosidase fixative and stained using X-gal
as a substrate as described previously (22).
Transfection of COS-7 and abl
/
arg
/
Cells--
For
transient transfections of COS-7 cells, 800,000 cells per 100-mm dish
were plated overnight. LipofectAMINE (20 µl) was preincubated with
DNA (3.5 µg total) in 1 ml of transfection media (Dulbecco's
modified Eagle's medium) at room temperature for 45 min. For
transfections of abl
/
arg
/
and control
wild-type fibroblasts only 250,000 cells were plated overnight, and the final concentration of DNA used for transfection was 4 µg. The volume
was brought up to 6 ml and layered over cells for 6-8 h at 37 °C.
Cells were used within 48 h of transfection subsequent to serum
starvation overnight. Expression vectors for Abl constructs were the
generous gift of Dr. Martin A. Schwartz and Dr. Jean Y. J. Wang.
Vectors included an HA-tagged wild-type Abl (29), mutant dominant
active (DA) Abl, and mutant kinase dead (KD) forms of Abl (30).
Previously described constructs encoding wild-type Crk (14) and pSSR
expression vector containing wild-type CAS (22, 31, 32) proteins were
also used.
Wound Assays--
abl+/+ arg+/+ and
abl
/
arg
/
fibroblasts were plated at
150,000 cells per well on a glass coverslip in media containing 0.5%
fetal bovine serum. Cells were incubated overnight before the monolayer
of cells was "wounded" by scraping with a clean pipette tip. Wells
were rinsed 3 times with PBS and recovered with media containing 0.5%
serum. Wound assays were incubated at 37 °C for 18 h before
slides were rinsed with PBS and fixed in 4% paraformaldehyde. Cells
were lightly stained with crystal violet for contrast before
photography at × 100.
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RESULTS |
Abl Is a Negative Regulator of Haptotaxic Cell Migration--
Abl
directly associates with actin and regulates signaling cascades
involved in organization of the cytoskeleton, suggesting a function for
Abl in cell migration. To investigate this possibility, COS-7 cells
were transiently transfected with either a mutationally inactive,
kinase dead (KD) Abl or a mutant dominant active (DA) Abl along with a
-galactosidase reporter vector to detect transfected cells. Control
cells (MOCK) were transfected with equivalent amounts of the empty
expression vector in addition to a
-galactosidase reporter
construct. Transfected cells were allowed to migrate by haptotaxis in
Boyden chambers coated underneath only with either collagen type I or
human fibronectin (Fig. 1A).
To ensure that only transfected cells were included for statistical
analysis, cells expressing
-galactosidase were counted and control
wells were included to determine any differences attributable to cell loading or differential transfection efficiencies. Overall transfection efficiencies varied less than 10-15% between experiments and among the different expression vectors in the same cell type (data not shown). Expression of KD Abl enhanced COS-7 cell migration, on both
collagen and fibronectin, whereas transfection of cells with DA Abl
inhibited cell migration. Similarly, expression of wild-type Abl in
cells, which also leads to its constitutive activation (29), inhibited
cell migration (illustrated below, Fig.s 3 and 5). Importantly, cell
viability and attachment of an aliquot of transfected cells was checked
in each experiment. c-Abl expression did not influence cell attachment
to the ECM (Fig. 1B), spreading, or viability (data not
shown). The abilities of DA Abl to inhibit cell migration and of KD Abl
to enhance cell migration both suggest that Abl is a negative regulator
of cell migration.

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Fig. 1.
Expression of kinase dead Abl enhances cell
migration, whereas kinase-activated Abl inhibits cell migration.
A, COS-7 cells were transiently transfected with either the
empty vector (MOCK), mutant FLAG-tagged KD or DA forms of Abl kinase
along with a reporter construct encoding -galactosidase. Cells were
serum-starved overnight and allowed to migrate in a haptotaxis assay
using Boyden chambers coated on the bottom surface only with collagen
type I (10 µg/ml) or human fibronectin (10 µg/ml). Cells were
allowed to migrate for 4-5 h at 37 °C. Cells that had migrated to
the underneath surface of membranes and stained blue with X-gal
substrate were counted per × 100 field. Results shown reflect the
mean and S.D. of three replicate experiments. B, equivalent
numbers of cells transfected as described in A were allowed
to attach to collagen or fibronectin-coated wells (10 µg/ml) for
3 h and then fixed and stained with X-gal. C, lysates
were prepared from transfected cells treated the same as those used in
migration and attachment experiments above. Immunoprecipitation
(IP) and immunoblotting were performed using either anti-Abl
or anti-FLAG tag antibodies as indicated.
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Abl Is a Negative Regulator of Insulin-induced Cell
Migration--
Growth factors and cytokines potentiate cell movement
on extracellular matrix proteins (for review see Refs. 33 and 34), and
Abl kinase activity is also regulated by cytokines (12). Therefore, the
ability of insulin to influence migration of cells expressing KD or DA
Abl was examined. Transfected COS-7 cells were allowed to migrate
toward collagen in the presence or absence of an insulin gradient (Fig.
2A). Expression of KD Abl
enhanced the migratory effect of insulin about 2-fold relative to
mock-transfected cells, whereas DA Abl expression impeded cell
migration. Cell attachment to the extracellular matrix was not
significantly influenced by Abl expression or the presence of insulin
(Fig. 2B). COS-7 cell adhesion and migration on collagen was
dependent on
1 integrins, because an
anti-
1 antibody, but not an
anti-
V
5 antibody, could block these
processes (Fig. 2C). These results indicate that Abl negatively regulates insulin-induced cell migration but not cell attachment to the ECM.

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Fig. 2.
Expression of kinase dead Abl enhances cell
migration, whereas kinase-activated Abl inhibits cell movement in
response to insulin. A, COS-7 cells were transfected
with either the empty expression vector (MOCK), KD Abl, or DA Abl in
addition to a -galactosidase reporter vector. Cells were
serum-starved overnight and allowed to migrate toward collagen type 1 (10 µg/ml) or human fibronectin (10 µg/ml) for 3 h in the
presence (+) or absence ( ) of an insulin gradient (10 µg/ml). Cells
that had migrated to the underneath surface of membranes and stained
blue with X-gal substrate were counted per × 40 field. Results
are mean and S.D. of three replicate experiments. B,
equivalent numbers of cells transfected as described in A
were allowed to attach to collagen or fibronectin-coated wells (10 µg/ml) for 3 h, then fixed and stained with X-gal. Blue staining
cells were counted per × 100 field. C, cells
transfected as in A were allowed to attach to
collagen-coated wells in the presence of function-blocking antibodies
to either V 5 or 1
integrins present on these cells. Adherent cells expressing
-galactosidase protein were counted per × 100 field. Results
are the mean and S.D. from three replicate experiments.
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Abl Inhibits Cell Migration by Preventing Crk-CSA
Coupling--
Previous work has shown that the molecular coupling of
Crk and CAS is a necessary signaling event for migration (22, 35). Crk-CAS coupling depends on the interaction of the Crk SH2 domain with
phosphotyrosine residues present in the substrate domain of CAS (36).
That Abl and Arg are known to tyrosine-phosphorylate Crk preventing Crk
SH2 interactions with effector proteins (8, 17, 18) suggests a
mechanism for Abl inhibition of cell migration, through the regulation
of Crk-CAS coupling. To investigate this possibility, endogenous
Crk-CAS complexes were examined in cells expressing either DA or KD
forms of Abl (Fig. 3A). KD Abl
expression resulted in an increase in Crk-CAS complexes, whereas the
expression of DA Abl reduced Crk-CAS coupling. Associated with the
uncoupling of Crk-CAS complexes by DA Abl was an increase in Crk
phosphorylation seen as a decrease in mobility by SDS-PAGE (Fig.
3A) and positive phosphotyrosine blotting (data not shown).
Previous work has shown that this slower migrating band results from
the tyrosine phosphorylation of Crk (20, 24). Consistent with this,
Crk-CAS complexes were increased in cells expressing KD Abl, which
appears to inhibit the phosphorylation and mobility shift of Crk. These
findings suggest that Abl activation promotes Crk phosphorylation and
prohibits the interaction of Crk with CAS. In fact, Abl activation
blocked cell migration induced by the expression of exogenous Crk and CAS (Fig. 3B). Importantly, inhibition of Abl activity did
not influence extracellular signal-regulated kinase activity in these cells, which is a separate signaling pathway capable of promoting cell
migration (35) (Fig. 3C). This suggests that the negative regulation of cell migration by Abl results from the ability of Abl to
inhibit endogenous Crk and CAS interactions.

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Fig. 3.
c-Abl inhibits cell migration through the
disruption of Crk-CAS complexes. A, COS-7 cells were
transiently transfected with empty vector (MOCK), KD Abl, or DA Abl.
Endogenous Crk protein was immunoprecipitated (IP) with
anti-Crk antibodies and then immunoblotted with antibodies recognizing
CAS or Crk. Tyrosine-phosphorylated Crk protein (Crk-P)
can be distinguished from unphosphorylated Crk (Crk) by its
decreased mobility by SDS-PAGE (20, 24). B, COS-7 cells were
transfected with either the empty expression vector (Mock),
HA-tagged Abl (Abl), Crk and CAS (Crk/CAS), or
HA-tagged Abl together with Crk and CAS (Crk-CSA +Abl)
expression vectors. All cells were cotransfected with a
-galactosidase reporter vector. Cells were serum-starved overnight
and then added to Boyden chambers coated on the bottom only with
collagen type I (10 µg/ml). Following a 4-5-h incubation, the number
of transfected cells migrating was determined by counting blue cells
coexpressing the -galactosidase vector on the underside of the
membrane per × 200 field. Results are the mean and S.D. from a
representative of three independent experiments. An aliquot of cells
treated as described for the migration experiment was lysed in
detergent and immunoblotted with anti-HA (Abl), anti-CAS, or anti-Crk
antibodies (lower panel). C, COS-7 cells were
transfected with empty expression vector (Mock), KD Abl, or
DA Abl and serum-starved overnight. Lysates were immunoblotted with
antibodies to either the phosphorylated/activated forms of Erk1
(Erk1-P) and Erk2 (Erk2-P). Blots were then
stripped and reprobed with antibodies to total Erk2 protein.
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abl
/
arg
/
Cells Demonstrate an Enhanced Migratory Phenotype
That Is Reversed by Reconstitution with Abl Kinase--
To examine
directly the influence of Abl on cell migration and Crk-CAS coupling in
nontransfected cells, the ability of abl
/
cells (29) to
migrate on the ECM was determined. Surprisingly, we found that cells
devoid of Abl showed no difference in cell migration (Fig.
4A), and the relative amounts
of Crk-CAS complexes in these cells were not significantly different
from wild-type cells with Abl (abl+/+) (Fig. 4B).
In addition, a similar level of phosphorylated Crk protein could be
detected in both abl
/
cells and abl+/+ cells
(Fig. 4B). These results are likely explained by the
presence of the Abl-related protein, Arg, which is expressed in these
cells (18). Arg has been reported to interact with and phosphorylate
Crk in vitro (17), but the significance of this interaction
is not known. Recently, double null mice lacking Abl and Arg proteins
(abl
/
arg
/
) were developed. Although these animals die early during embryogenesis, fibroblasts could be
isolated from early stage embryos (3). These cells provided a unique
opportunity to examine directly the role of endogenous Abl as well as
Arg in the regulation of cell migration. As shown in Fig.
5A, abl
/
arg
/
cells exhibit an enhanced ability to migrate
relative to cells isolated from wild-type littermate animals. Loss of
Abl and Arg function in these cells did not appear to influence cell
attachment or overall spreading on the ECM (Fig. 5, B and
C). However, although both abl
/
arg
/
and wild-type cells readily attach and spread on
fibronectin to the same degree, wild-type cells exhibit a flat circular
appearance, whereas abl
/
arg
/
cells
produce numerous filopodia and are polarized, indicative of a migratory
cell type (18) (Fig. 5C). As expected, reconstitution of
abl
/
arg
/
cells with Abl kinase reduced
the ability of cells to migrate on fibronectin (Fig.
5D).

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Fig. 4.
Cells devoid of c-Abl do not illustrate
significant changes in cell migration or Crk-CAS complexes.
A, either wild-type (abl+/+) or
abl / 3T3 fibroblast cells were serum-starved overnight
and allowed to migrate in Boyden chambers coated on the bottom only
with human fibronectin (10 µg/ml) for 3 h in Boyden chambers.
Migrating cells were fixed and stained with crystal violet and counted
per × 200 field. Results are the mean and S.D. of a
representative of at least three experiments. B, Crk-CAS
complexes were examined from abl+/+ and abl /
3T3 cells. Cells were serum-starved overnight on culture dishes and
then lysed in detergent. Lysates were immunoprecipitated with anti-Crk
antibody and immunoblotted for CAS and Crk. Note the presence of both
phosphorylated (Crk-P) and unphosphorylated Crk
(Crk) proteins as illustrated earlier (Fig. 3).
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Fig. 5.
Embryonic fibroblast cells isolated from
abl /
arg / mice show
increased cell migration, which is reversed by reconstitution with Abl
kinase. A, abl / arg /
(A/A / ) or control wild-type fibroblasts
(A/A+/+) were serum-starved overnight and then allowed to
migrate in Boyden chambers coated on the bottom only with human
fibronectin (10 µg/ml). After 3 h, migrating cells were fixed
and stained with crystal violet and counted per × 200 field.
Results are means and S.D. from a representative of multiple
experiments. Whole cell lysates were prepared and immunoblotted using
antibodies recognizing Abl or Arg proteins. B, cells were
allowed to attach to fibronectin (10 µg/ml)-coated wells for 3 h
and rinsed, and then attached cells were stained with crystal violet
and counted per × 200 field. C, phase contrast
photomicrographs of abl+/+ arg+/+ and
abl / arg / cells plated on fibronectin (10 µg/ml)-coated coverslips and allowed to attach for 30 or 90 min.
Cells were then fixed and photographed at × 100. Arrows indicate the presence of filopodia. D,
abl / arg / cells were transiently
transfected with empty expression vector (Mock) or HA-tagged
Abl (Abl) expression vectors along with -galactosidase
reporter vectors. Cells were serum-starved overnight and allowed to
migrate in a haptotaxis assay using Boyden chambers coated on the
bottom surface only with human fibronectin (10 µg/ml). Following a
3-h incubation, cells that had migrated to the underneath surface of
membranes and stained blue with X-gal substrate were counted per × 200 field. Results shown reflect the mean and S.D. of a
representative of multiple experiments. An aliquot of cells treated as
described for the migration experiment was lysed in detergent and
immunoblotted with anti-HA antibody.
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To examine further the capacity of abl
/
arg
/
cells to migrate, wound assays were performed that
provide an additional method to examine changes in cell motility (37).
In this experiment, a confluent monolayer of either wild-type or
abl
/
arg
/
cells was wounded with a
sterile pipette tip. Cells were then allowed to migrate into the wound
for 18 h. As shown in Fig. 6,
abl
/
arg
/
cells readily migrate out of
the margin and into the wound relative to wild-type control cells.
Interestingly, treatment of wild-type cells with the pharmacological
inhibitor of Abl kinase activity, STI 571 (26-28), enhanced their
ability to migrate into the wounded area. The treatment of
abl
/
arg
/
cells with STI 571 had no
apparent effect on cell migration nor did it impact cell adhesion or
viability of wild-type or abl
/
arg
/
cells (data not shown). Associated with the increased migration of STI 571-treated wild-type cells were fewer focal contacts and a more polarized shaped, similar to abl
/
arg
/
cells (Fig. 7). Conversely, untreated
wild-type cells illustrated numerous, densely staining focal contacts
associated with thick actin bundles located at the cell periphery. The
capacity of STI 571 to alter abl+/+ arg+/+ migratory and morphological characteristics provides further evidence for the ability of Abl to regulate the cell cytoskeleton and motility. Thus, inhibition of Abl and Arg kinase activity with KD Abl, STI 571, or gene disruption all increase cell migration without impacting cell
adhesion or spreading on the ECM.

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Fig. 6.
Abl and Arg negatively regulate cell
migration in wound assays. abl / arg /
and wild-type (abl+/+ arg+/+) cells were plated
at high density on coverslips in 0.5% serum and allowed to form a
confluent monolayer overnight. A wound was then introduced with a
sterile pipette tip, and cells were permitted to migrate into the wound
for 18 h. Cells were maintained in 0.5% serum with or without a
pharmacological inhibitor of Abl (STI 571) during the entire course of
the experiment. Cells were fixed and then lightly stained with crystal
violet, and phase contrast micrographs were taken at × 100.
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Fig. 7.
abl /
arg / show
decreased actin fibers and vinculin-positive focal adhesions compared
with abl+/+ arg+/+ cells.
abl+/+ arg+/+ or abl /
arg / cells were plated on human fibronectin (10 µg/ml)-coated coverslips overnight in serum-free media either with or
without the Abl kinase inhibitor, STI 571. Cells were stained with
antibody recognizing vinculin and secondary fluorescein
isothiocyanate-conjugated goat anti-mouse antibody (green)
to identify focal contacts, whereas rhodamine-phalloidin
(red) was used to visualize F-actin. Confocal images were
taken at × 630.
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abl
/
arg
/
Cells Contain Less Tyrosine-phosphorylated Crk
and More Crk-CAS Complexes Than Wild-type Cells--
Previous
experiments (Fig. 3) suggested that Abl may prevent cell migration by
phosphorylating Crk, thereby disrupting Crk-CAS association. To examine
endogenous Crk-CAS coupling in abl
/
arg
/
cells, lysates were prepared from cells in suspension or at various
times after attachment to fibronectin, which promotes migration of
these cells (shown above, Fig. 5A). Significantly more CAS
was associated with Crk in abl
/
arg
/
cells relative to wild-type cells, even though the levels of Crk and
CAS proteins were similar in these cells (Fig.
8). Furthermore, no apparent change in
CAS phosphorylation was detected between cell types following cell
attachment to fibronectin, indicating that Abl and Arg do not regulate
CAS tyrosine phosphorylation under these conditions. Instead, it
appears that Abl and Arg play a primary role in basal phosphorylation
and regulation of Crk function, independent of CAS activation. In fact,
the upper tyrosine-phosphorylated band recognized by anti-Crk antibody
could only be detected in cells expressing Abl and Arg proteins (Fig.
8B). Phosphorylation of Crk specifically at tyrosine 221 has
been associated with decreased mobility by SDS-PAGE and inhibition of
Crk function (20, 24). The phosphorylation of Crk at tyrosine 221 in
wild-type, but not abl
/
arg
/
cells was
confirmed using an antibody that recognizes only tyrosine
221-phosphorylated Crk (8, 24) (Fig. 8B). Importantly, reconstitution of abl
/
arg
/
cells with
Abl not only inhibited cell migration (shown above, Fig. 5C)
but resulted in phosphorylation of Crk at tyrosine 221 and a
subsequent decrease in Crk-CAS complexes (Fig. 8C). Thus, it
appears that Abl and Arg kinase activity are primarily responsible for
Crk tyrosine 221 phosphorylation in these cells. Together these
findings indicate that Abl and Arg promote tyrosine 221 phosphorylation
of Crk leading to disruption of Crk-CAS complexes and the prevention of
cell migration. Based on the above findings and the work of
others, a model can be proposed illustrating the regulation of cell
migration by Abl. Cells are stimulated to migrate following integrin
ligation and/or cytokine exposure through formation of Crk-CAS
complexes (22). However, integrin and cytokine binding also promote Abl
kinase activity leading to phosphorylation of Crk at the regulatory
tyrosine 221 and inhibition of Crk-CAS complexes (10, 12, 17, 18). This
may serve as a negative feedback mechanism to control the level of
Crk-CAS association and cell migration on the extracellular matrix
(Fig. 9 and "Discussion").

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Fig. 8.
The highly migratory
abl /
arg / cells show
increased Crk-CAS complexes and decreased Crk
tyrosine 221 phosphorylation. A,
abl+/+ arg+/+ or abl /
arg / cells were serum-starved overnight and maintained
in suspension for 1 h before being incubated in human fibronectin
(10 µg/ml)-coated plates for 30 or 60 min. Lysates were prepared and
immunoprecipitated (IP) with anti-Crk antibodies before
immunoblotting with an antibody specific for CAS. An aliquot of whole
cell lysate from these cells was immunoblotted using anti-CAS antibody,
then stripped and reprobed with anti-phosphotyrosine antibody
(PTyr). B, Crk immunoprecipitated from lysates
prepared as described in A were immunoblotted with an
antibody recognizing Crk. The blot was then stripped and reprobed with
antibody specifically recognizing Crk phosphorylated at position 221 (Crk 221). C, abl /
arg / cells were reconstituted with HA-tagged Abl
(Abl) protein. For controls, either abl /
arg / or abl+/+ arg+/+ cells were
transfected with empty expression vector. Lysates were
immunoprecipitated with anti-CAS antibody and then immunoblotted with
antibodies specific for CAS and Crk. Whole cell lysates were also
immunoblotted for total Crk protein.
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Fig. 9.
Model depicting how Abl serves as a negative
regulator of migration through the disruption of Crk-CAS
complexes. Interaction of cells with cytokines and adhesive
proteins present in the extracellular matrix promote receptor tyrosine
kinase activation and integrin ligation leading to activation of
downstream cell signaling components. Focal adhesion kinase and/or Src
represent two tyrosine kinases that operate downstream of cytokine
receptors and integrins to phosphorylate the substrate domain of CAS.
Crk-CAS complex formation is then facilitated through the interaction
of the SH2 domain of Crk with tyrosine-phosphorylated CAS. The
polyproline region of Abl interacts with the SH3 domain of Crk leading
to tyrosine phosphorylation at tyrosine 221. Through intramolecular
folding, the SH2 domain of Crk binds the phosphorylated tyrosine 221, prohibiting further Crk-CAS interaction. In this way Abl regulation of
Crk-CAS coupling serves as a negative feedback mechanism on cell
migration.
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DISCUSSION |
Appropriate control of cell migration is achieved through a
complex balance of both positive and negative signaling mechanisms. An
inducible negative feedback mechanism is necessary to modulate the
process of movement as well as to prevent unwanted cell migration. Although loss of negative controls or checkpoints would certainly lead
to aberrant cell migration, little is known about these mechanisms. Our
findings that Abl and Arg negatively regulate cell migration indicate
an important biological role for this family of tyrosine kinases in the
control of cell migration. We provide several lines of evidence that
Abl family kinases serve as negative regulators of cell migration
through their ability to control the molecular coupling of Crk and CAS
proteins. First, cells transiently expressing mutationally inactivated
KD Abl showed significantly increased cell migration and increased
Crk-CAS complexes. In contrast, expression of mutationally activated
Abl in cells prevented migration and formation of endogenous Crk-CAS
complexes. Second, induction of cell migration by exogenous expression
of Crk and CAS was specifically blocked by the coexpression of
activated Abl. Third, pharmacological inhibition of Abl kinase activity
with STI 571 was sufficient to induce cell migration. Finally,
embryonic fibroblast cells isolated from animals genetically deficient
for Abl family kinases showed significantly enhanced cell migration and
increased Crk-CAS complexes. Together these findings illustrate a role
of Abl kinases in the control of Crk-CAS complexes and cell migration.
An important question that remains is how Abl kinase activity itself is
regulated in migratory and stationary cells. Abl kinase activity is
induced both by cytokine stimulation and integrin ligation. Following
cell exposure to the growth factors, platelet-derived growth factor and
epidermal growth factor, Abl is phosphorylated by Src kinase, leading
to a more activated form of Abl (12, 24). Abl is also activated via
phosphorylation following plating on fibronectin, which leads to
transient localization of Abl to actin structures (10, 11). However,
activation of cytokine receptors and integrin ligation also facilitates
enhanced Crk-CAS complex formation leading to increased cell migration
(22, 23, 38). Our findings suggest that the concomitant activation of Abl kinase upon cell adhesion or exposure to cytokines could serve as a
negative feedback mechanism to control Crk-CAS complexes and cell
migration. That Crk, CAS, and Abl localize to focal contacts and
actin-rich membrane ruffles suggests that the localization of Abl
likely promotes its interaction with Crk-CAS complexes. Based on our
work and the work of others, a model can be proposed showing how
Abl kinases regulate CAS-Crk complexes and the migration machinery of
cells (Fig. 9). Initial integrin ligation and cytokine stimulation
promote focal adhesion kinase and Src activation leading to CAS
phosphorylation (23, 38). Crk then binds to tyrosine-phosphorylated CAS
inducing Rac activity and cell migration (22, 35, 39). Abl kinases are
also activated in response to integrins and cytokines providing a
negative feedback to modulate the level of Crk-CAS complexes and
downstream signals that regulate the actin cytoskeleton. An important
component of this model is the ability of Abl to associate with Crk and
phosphorylate it on tyrosine 221. Our findings indicate that Abl
expression induces tyrosine 221 phosphorylation of Crk and
disruption of Crk-CAS complexes. Abl and Arg have been reported to bind
to Crk and phosphorylate tyrosine 221 in vitro (17, 18).
Once phosphorylated, tyrosine 221 of Crk forms an intramolecular bridge
with its own SH2 domain, thereby preventing interaction with its
effector proteins and downstream signals (8, 9, 18). However, our
findings do not exclude the possibility that Crk tyrosine 221 phosphorylation may be indirect in response to Abl activation through
changes in an unknown kinase or phosphatase that specifically regulates
tyrosine 221 phosphorylation or dephosphorylation. Nevertheless,
significant evidence exists that favors a direct association of Abl
kinases with Crk, and a similar mechanism has been proposed to control
Crk-mediated neurite outgrowth in response to nerve growth factor (8).
In either case, our findings that CAS phosphorylation is not impacted
by Abl kinases suggests that Crk is the specific target of regulation
by Abl kinase rather than upstream components that facilitate CAS phosphorylation.
Our findings that Abl kinases are endogenous regulators of Crk function
could be important for other signaling and biological processes. For
example, it would be interesting to determine the influence of Abl
kinase on Crk/paxillin association and cell migration. The SH2 domain
of Crk binds to tyrosine-phosphorylated paxillin, and this also
influences cell morphology and migration (8, 29, 39). Abl kinase was
also recently shown to regulate apoptosis via p53- or p73-mediated
pathways (40-42). Interestingly, the formation of Crk-CAS complexes
has been shown to protect invasive cells from apoptosis (31). In fact,
inhibition of Crk-CAS coupling through the expression of dominant
negative forms of these proteins induces cell apoptosis (31).
Therefore, Abl regulation of Crk-CAS complexes may influence both cell
migration as well as survival. During the course of our studies no
significant change in cell death was observed in Abl-expressing cells.
In fact, Abl-expressing cells readily attached and spread on ECM
proteins, indicative of healthy, viable cells. Therefore, the changes
in migration observed in our study are not the result of changes in
viability of Abl-expressing cells. Lack of cell death in the work
presented here is likely due to the short duration of Abl expression
and activation in transient transfection experiments. Nevertheless, our
data do not preclude the possibility that Abl regulates cell apoptosis
through a Crk-dependent mechanism under certain
circumstances such as DNA damage- or irradiation-induced death.
The data presented here suggests a high degree of similarity in Abl and
Arg function in the regulation of cell migration. Migration and Crk-CAS
complexes were not significantly altered in abl
/
cells
relative to abl+/+ cells. abl
/
cells have
previously been shown to have increased Arg expression, suggesting that
Arg may compensate for Abl in these cells (18). In fact, in our study
cells deficient of both Abl and Arg were needed to illustrate the
negative regulation of cell movement. This suggests redundant functions
of these enzymes in the regulation of cell migration. However, it will
be necessary to reconstitute abl
/
arg
/
cells independently with Abl or Arg to access the precise role of these proteins in the regulation of Crk-CAS coupling and migration. The
C-terminal regions of Abl and Arg are highly divergent, and the
localization patterns of the two enzymes are distinct, suggesting that
these enzymes could play unique roles in the regulation of cell
migration (9, 16).
It is interesting that the Bcr-Abl fusion protein has been reported to
enhance cell survival and migration. Therefore, Bcr-Abl appears to
function very differently than Abl, likely resulting from differences
in Bcr-Abl activity and localization. In contrast to the tightly
regulated activity of c-Abl, Bcr-Abl is a constitutively activated
kinase (43, 44) localized almost solely to the actin cytoskeleton (45).
Moreover, Bcr-Abl protein is not regulated in response to integrin
ligation and cell adhesion to the ECM (46), whereas c-Abl activity and
localization are tightly regulated under these conditions (47, 48).
Input from integrin receptors may serve to target Abl to distinct
regions of the migratory cell such as focal contacts or membrane
ruffles where it specifically regulates Crk-CAS complexes. In contrast,
Bcr-Abl may not respond to such localization signals preventing Bcr-Abl
from negatively regulating Crk-CAS coupling. In fact, the inappropriate
localization of Bcr-Abl could lead to a depletion of Abl activity from
specific regions of a cell. The loss of temporal and spatial regulation of Crk phosphorylation could lead to increased Crk-CAS coupling and
enhanced cell migration. Alternatively, Bcr-Abl may activate additional
signals that induce cell migration independent of Crk-CAS coupling. In
any case, these findings point to an important role for Abl family
kinases in controlling the actin cytoskeleton of migrating cells.
The Drosophila homologue of mammalian Abl, D-Abl, is
also responsible for regulating actin dynamics and cell adhesion of
neurons (5, 49, 50). This is consistent with observations that abl
/
arg
/
mice show lethal defects in
neurulation and changes in their actin cytoskeleton (3). In both
Drosophila and Caenorhabditis elegans, Rac, which
is a downstream component of the Crk-CAS pathway, is necessary for
correct axon guidance (49, 52-54). Regulation of Rac activity in
C. elegans has recently been attributed to Crk interaction
with DOCK 180, which is induced in mammalian cells by Crk association
with CAS (52). cas
/
mice demonstrate lethal heart
and vascular defects, and primary fibroblasts isolated from cas
/
embryos illustrate cytoskeletal
abnormalities (51). Together these data indicate an important role for
signaling mechanisms that control cytoskeletal components and regulate
cell migration in development. Deregulation of these conserved
signaling cascades in adult organisms may contribute to aberrant cell
migration associated with tumor cell metastasis and inflammation. Our
findings that Abl is a negative regulator of cell migration through its
ability to regulate Crk-CAS complexes help provide an understanding of how molecular signaling mechanisms are controlled in migrating cells.