From Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208
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ABSTRACT |
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The protein tyrosine phosphatase PTP-PEST is a
cytosolic enzyme that displays a remarkable degree of selectivity for
tyrosine-phosphorylated p130Cas as a substrate, both
in vitro and in intact cells. We have investigated the
physiological role of PTP-PEST using Rat1 fibroblast-derived stable
cell lines that we have engineered to overexpress PTP-PEST. These cell
lines exhibit normal levels of tyrosine phosphorylation of the majority
of proteins but have significantly lower levels of tyrosine
phosphorylation of p130Cas than control cells. Initial
cellular events occurring following integrin-mediated attachment to
fibronectin (cell attachment and spreading) are essentially unchanged
in cells overexpressing PTP-PEST; similarly, the extent and time course
of mitogen-activated protein kinase activation in response to integrin
engagement is unchanged. In contrast, the reduced phosphorylation state
of p130Cas is associated with a considerably reduced rate
of cell migration and a failure of cells overexpressing PTP-PEST to
accomplish the normally observed redistribution of p130Cas
to the leading edge of migrating cells. Furthermore, cells
overexpressing PTP-PEST demonstrate significantly reduced levels of
association of p130Cas with the Crk adaptor protein. Our
results suggest that one physiological role of PTP-PEST is to
dephosphorylate p130Cas, thereby controlling tyrosine
phosphorylation-dependent signaling events downstream of
p130Cas and regulating cell migration.
PTP-PEST is a ubiquitously expressed mammalian, cytosolic protein
tyrosine phosphatase (PTP)1
that was cloned in our laboratory (1) and by others (2-4). Several
signaling proteins have been shown to be capable of binding to
PTP-PEST, including Shc (5, 6), paxillin (7), Grb2 (8), and Csk (9),
but the physiological significance of these interactions and their
relevance to the function of PTP-PEST is unclear. In order to
investigate the physiological substrate specificity of members of the
PTP family, we developed a novel method involving expression of
substrate-trapping mutant forms of PTPs (10, 11). These mutants retain
a high affinity for substrates but are catalytically impaired.
Therefore, they form complexes with appropriate target substrates that
are amenable to isolation and characterization. We have used this
method to identify substrates of PTP-PEST and thus gain insight into
its physiological function. We have shown that the adaptor protein p130Cas is a specific substrate for PTP-PEST in
vitro and in intact cells (11). Similar results have also been
obtained in studies performed on fibroblasts derived from PTP-PEST p130Cas was initially identified as a major
tyrosine-phosphorylated protein in cells transformed by the oncogenes
v-crk (14, 15) and v-src (16, 17). In addition,
p130Cas is rapidly phosphorylated following mitogenic
stimulation by a wide variety of agonists (18-22), during B-cell
activation via antigen receptor ligation (23, 24), and following cell
attachment to fibronectin (25-29). Although these observations
implicate tyrosine phosphorylation of p130Cas in the
control of a variety of cellular processes, the precise role of
p130Cas remains largely obscure. The structure of
p130Cas suggests that it is involved in the formation of
multiprotein complexes via several types of protein-protein
interaction. Thus, p130Cas contains an SH3 domain, which
has been shown to interact with proline-rich sequences in several
proteins (13, 25, 30-32), a proline-rich segment, representing a
potential SH3 domain binding sequence, a central domain with multiple
copies of the motif YXXP which, when
tyrosine-phosphorylated, represent potential binding sites for a
variety of SH2 domains (33), and a C-terminal segment containing high
affinity binding sites for the SH2 and SH3 domains of Src (34).
In order to investigate further the cellular function of PTP-PEST, we
have generated Rat1 fibroblast lines that overexpress the wild-type
phosphatase protein. These cells display a specific defect in
p130Cas phosphorylation following cell attachment to
fibronectin and exhibit a considerably reduced rate of migration. The
data suggest that regulation of the tyrosine phosphorylation status of
p130Cas by PTP-PEST is a critical control element of cell motility.
Generation of Cell Lines--
cDNA encoding full-length
PTP-PEST was subcloned into the retroviral transfer vector pWZL(hygro),
from which gene expression is driven by the viral long terminal repeat
promoter, and the hygromycin resistance gene is expressed from the same
transcript via an internal ribosomal entry site. PTP-PEST-encoding
retrovirus (as well as a control retrovirus containing empty
pWZL(hygro) vector) was then generated by calcium phosphate-mediated
transfection of the recombinant vector into the Bosc23
replication-incompetent ecotropic virus-packaging cell line.
Specifically, 20 µg of vector DNA was added to a 50% confluent 10-cm
diameter tissue culture plate of Bosc23 cells growing at 37 °C in 10 ml of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal
bovine serum; 10 ml of fresh medium was then added 16 h after
transfection. Virus-containing culture supernatants were harvested
after a further 32 h of incubation, passed through a 0.45-µm
syringe-loaded filter (Millipore), then added to 30% confluent
cultures of Rat1 fibroblasts (5 ml of virus supernatant per 10-cm
diameter plate of cells) in the presence of 4 µg/ml Polybrene
(Sigma). An identical second round of infection was performed using
fresh virus supernatant obtained by replenishing the medium of the
transfected Bosc23 cells and incubation for a further 5 h at
37 °C. Fresh medium (DMEM with 5% fetal bovine serum) was added to
the cells 16 h after the second round of infection, and the cells
were grown for a further 24 h. Drug selection was initiated by
splitting the infected population of cells into growth medium
containing 100 µg/ml hygromycin B (Life Technologies, Inc.); the
medium was changed every 2 days, and drug-resistant colonies were
picked 10 days after the initial addition of hygromycin. All cell lines
were routinely grown in DMEM containing 5% fetal bovine serum and 100 µg/ml hygromycin B. Expression of PTP-PEST was found to be stable
over at least 40 passages.
Antibodies and Reagents--
Rabbit polyclonal (CSH8) and mouse
monoclonal (AG10) antibodies to human PTP-PEST have been described
previously (13). Antibodies to p130Cas (rabbit polyclonal
B+F, mouse monoclonal 8G4 (35)) were provided by Dr. Amy Bouton
(University of Virginia). Mouse monoclonal anti-phosphotyrosine antibody G104 was generated as described previously (11). Antibodies to
paxillin (mouse monoclonal, clone 349), p130Cas (mouse
monoclonal, clone 21), Crk (mouse monoclonal, clone 22), and Shc
(rabbit polyclonal) were from Transduction Laboratories; antibody to
p125FAK (rabbit polyclonal sc-558) was from Santa Cruz
Biotechnology; mouse monoclonal antibody to vinculin (VIN-11-5) was
from Sigma, polyclonal rabbit antibody specific for activated ERK1 and
ERK2 MAP kinases was from Promega. Human plasma fibronectin was from Life Technologies, Inc., and polylysine and soybean trypsin inhibitor were from Sigma.
Cell Adhesion to Fibronectin--
Cells were grown to Cell Lysis, Immunoprecipitation, and Immunoblotting--
Cells
were lysed for 30 min at 4 °C in 50 mM Tris-HCl (pH
7.4), 100 mM NaCl, 1 mM benzamidine, 1 µg/ml
leupeptin, 1 µg/ml aprotinin, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 5 mM sodium
pyrophosphate, 10 mM NaF, and insoluble material was removed by centrifugation at 4 °C for 10 min at 14,000 × g. Immunoprecipitation was performed on a rocking platform
for 2 h at 4 °C using antibodies that were pre-coupled to 10 µl of protein A-Sepharose beads (Amersham Pharmacia Biotech). The
beads were collected by a brief centrifugation (10 s at 5000 × g), washed three times with 1 ml of 20 mM
Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM
benzamidine, 1 µg/ml leupeptin, 1 µg/ml of aprotinin, 10%
glycerol, 0.5% Triton X-100, resuspended in SDS-PAGE sample buffer,
and heated at 95 °C for 5 min. Immunoprecipitated proteins were then
analyzed by SDS-PAGE and visualized by immunoblotting using horseradish
peroxidase-coupled secondary antibodies and enhanced chemiluminescence
reagents from Amersham Pharmacia Biotech.
Indirect Immunofluorescence--
Cells attached to glass
coverslips were rinsed in PBS and fixed at room temperature for 15 min
in 2% paraformaldehyde in PBS. The cells were then washed twice in PBS
and solubilized on ice for 5 min in 0.2% Triton X-100, 3% goat serum
in PBS. After washing three times at room temperature with PBS
containing 3% goat serum, primary antibody solutions were added (in
PBS containing 3% goat serum), and the samples were incubated for
1 h at room temperature. Excess antibody was removed by washing a
further four times as above. Appropriate secondary antibodies
conjugated to either fluorescein isothiocyanate or Texas Red (obtained
from Cappel) were then added, and the samples were incubated for a
further 30 min at room temperature. Finally, the samples were rinsed
four times with PBS containing 3% goat serum and once with PBS, and
the stained coverslips were mounted onto microscope slides using
ProLong antifade reagent from Molecular Probes Inc. Samples were
analyzed using a Zeiss Axiophot epifluorescence microscope; images were
collected digitally using a CCD camera and image processing software
from Oncor Image.
Cell Migration Assays--
Cell migration was assessed using a
monolayer wound healing assay. Cells were first grown to confluence in
plastic tissue culture dishes, and a wound was made in the cell
monolayer using a sterile 1-ml pipette tip. Cell movement into the
cleared area was then assessed by visual inspection of the cells during
growth at 37 °C in DMEM containing 5% fetal bovine serum and 100 µg/ml hygromycin. Cells growing on coverslips were also subjected to wounding and allowed to recover for 16 h at 37 °C, at which
time they were fixed and analyzed by indirect immunofluorescence as described above.
PTP-PEST Expression in v-Crk-transformed Cells--
3Y1 rat
fibroblast cells that stably express the v-Crk oncoprotein were grown
in DMEM containing 5% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin. Human PTP-PEST cDNA was introduced into
3Y1v-Crk cells on glass coverslips by calcium phosphate-mediated
transfection. The vector (PMT2) expresses PTP-PEST under the control of
the adenovirus major late promoter. Transfected cells were allowed to
grow for 2 days before fixation and immunofluorescence analysis.
Fibroblasts Overexpressing PTP-PEST Exhibit a Specific Reduction in
Phosphotyrosine Content of p130Cas--
We have previously
demonstrated that PTP-PEST displays selectivity for
tyrosine-phosphorylated p130Cas as a substrate both
in vitro and in intact cells (11, 13), suggesting that a
major function of PTP-PEST in vivo is to regulate the
tyrosine phosphorylation state, and thus the function, of p130Cas. In order to identify the cellular processes that
are controlled through regulation of the phosphorylation status of
p130Cas by PTP-PEST, we generated Rat1 fibroblast lines
that stably express human PTP-PEST protein and compared their
properties with control cell lines into which an empty vector construct
was introduced. Immunoblot analysis of cell lysates derived from these
clones, using a monoclonal antibody (AG10) that recognizes a segment
within PTP-PEST which is identical in the rat and human PTP-PEST
proteins (residues 216-305), demonstrated that the level of PTP-PEST
expression was increased by 5-10-fold in the PTP-PEST-overexpressing
clones, compared with the endogenous levels in the vector control
clones (Fig. 1, lower panels).
Immunoblot analysis of these cell lysates with an anti-phosphotyrosine
antibody indicated that the phosphorylation state of the majority of
tyrosine-phosphorylated proteins present in Rat1 fibroblasts under
normal growth conditions was not significantly affected by this level
of overexpression of PTP-PEST (Fig. 1, upper panels).
However, the phosphotyrosine content of a protein of
Mr
Since PTP-PEST recognizes p130Cas as a substrate with a
high degree of selectivity (11, 13), it appeared likely that the protein of Mr 130,000 in which we observed
Tyr(P) content was reduced in PTP-PEST-overexpressing cells was
p130Cas. This was confirmed by direct assessment of the
Tyr(P) content of p130Cas following immunoprecipitation
(Fig. 2). In contrast, neither the focal adhesion kinase
p125FAK nor paxillin, which have been reported to associate
in stable complexes with PTP-PEST (7), exhibited detectable alteration in Tyr(P) content (Fig. 2). Similarly, no differences were observed in
the levels of phosphorylation of Shc (which has also been reported to
bind to PTP-PEST under certain conditions (5, 6)) or of Src (data not
shown). Similar observations were made in five different
PTP-PEST-overexpressing cell lines. These results reinforce the
previously described high degree of specificity of PTP-PEST for
p130Cas and demonstrate, for the first time, that this
specificity for p130Cas is maintained at physiological
levels of tyrosine phosphorylation within the cell.
Analysis of Fibronectin-induced Signaling Events in
PTP-PEST-expressing Cells--
Integrin-mediated cell adhesion to the
extracellular matrix protein fibronectin rapidly induces a high level
of tyrosine phosphorylation of p130Cas (25-28, 36, 37)
suggesting a role for p130Cas in integrin signaling.
Therefore, we compared various fibronectin-induced signaling processes
in control and PTP-PEST overexpressing Rat1 fibroblasts. We observed
that during attachment and initial spreading on fibronectin-coated
plates PTP-PEST-overexpressing lines adhere to fibronectin at similar
rates (cells attached within 10 min of replating) and displayed similar
initial rates of spreading on fibronectin when compared with control
cells (data not shown). In addition, the morphology of the spread cells
was similar in both cell types, and immunofluorescence analysis
demonstrated that there was no significant effect of PTP-PEST
expression on the rapid accumulation of phosphotyrosine and of focal
adhesion proteins, such as paxillin, into focal adhesion structures
following cell attachment to fibronectin (Fig.
3). Under these conditions, very little
accumulation of p130Cas in focal adhesion structures was
observed. The majority of the p130Cas protein was localized
to the cytoplasm, particularly in the perinuclear region, and there was
no discernible difference between PTP-PEST-overexpressing and control
cells (data not shown).
Anti-Tyr(P) immunoblots of lysates prepared from control and
PTP-PEST-overexpressing fibroblasts during their attachment to fibronectin revealed patterns of Tyr(P)-containing proteins distinct from that observed in lysates of randomly growing cells (compare Fig.
4 upper panel with Fig. 1).
This is because the fibronectin replating experiment was performed
using serum-starved cells in the absence of soluble growth factors;
these conditions result in a reduction in the level of tyrosine
phosphorylation of many proteins within these cells in comparison to
that observed in samples derived from cells grown in the presence of
serum. Detachment of serum-starved fibroblasts and incubation in
suspension resulted in extensive dephosphorylation of the majority of
tyrosine-phosphorylated proteins present in the initial attached
population of cells, regardless of the expression level of PTP-PEST
within the cells. Replating these cells onto fibronectin-coated dishes
rapidly (within 20 min) induced the tyrosine phosphorylation of a
similar array of proteins in both cell types, with the exception of a
prominent tyrosine-phosphorylated protein of Mr
Since paxillin and p125FAK constitute two of the major
Tyr(P)-containing proteins present in fibroblasts following adhesion to fibronectin, the phosphorylation state of these proteins was also analyzed directly by anti-Tyr(P) immunoblotting of the
immunoprecipitated proteins (Fig. 5). The extent of
fibronectin-stimulated tyrosine phosphorylation of both paxillin and
p125FAK was unchanged in cells expressing PTP-PEST compared
with the control cells (Fig. 5), further demonstrating the high degree of specificity of PTP-PEST for p130Cas.
Many previous studies have suggested that the adaptor protein Crk is a
major downstream effector of tyrosine-phosphorylated p130Cas following fibronectin stimulation. The sequence of
p130Cas contains 15 copies of the motif YXXP
(38) which, when tyrosine-phosphorylated, corresponds to the consensus
site for binding of the Crk SH2 domain (33). Furthermore, cell
attachment to fibronectin has been shown to result in the formation of
stable complexes between tyrosine-phosphorylated p130Cas
and the SH2 domain of Crk (24, 27, 36). Therefore, we investigated the
formation of Crk-p130Cas complexes in
fibronectin-stimulated control and PTP-PEST overexpressing cells by
immunoprecipitation of the Crk protein, followed by immunoblotting the
precipitates with a p130Cas antibody. Lysates prepared from
fibronectin-stimulated control (vector-expressing) cells contained
significantly higher levels of stable complexes between Crk and
p130Cas than lysates prepared from PTP-PEST-overexpressing
cells; no Crk-p130Cas complexes were detected in lysates
prepared from cells held in suspension (Fig. 5B). These
results indicate that PTP-PEST is capable of regulating
Crk-dependent, fibronectin-induced signaling events
downstream of p130Cas, via reducing the level of tyrosine
phosphorylation of Crk SH2 domain binding sites on
p130Cas.
In addition to the rapid induction of protein tyrosine phosphorylation
that occurs in cells during their attachment to fibronectin, ERK1 and
ERK2 MAP kinases are concomitantly activated, rapidly and transiently,
under these conditions (26, 39-41). The function of activated MAP
kinases in integrin-induced cellular processes is currently unclear, as
is the mechanism whereby integrin receptor ligation leads to their
activation. One potential mechanism is via binding of the adaptor
protein Grb2 to Tyr(P)-925 on p125FAK, recruitment of
Grb2-associated SOS (a potent guanine nucleotide exchange factor for
Ras) to focal adhesion complexes containing tyrosine-phosphorylated
p125FAK, and subsequent activation of Ras (39, 42, 43).
However, several reports now suggest alternative pathways for
activation of ERK1/ERK2 MAP kinases in many cell types (44-46). These
include the formation of signaling complexes based on
tyrosine-phosphorylated p130Cas, via the binding of Crk or
Nck adaptor proteins and recruitment of SOS (36, 37, 47). Therefore, we
compared fibronectin-induced activation of MAP kinase in control and
PTP-PEST-expressing fibroblasts to investigate the possible involvement
of p130Cas tyrosine phosphorylation. Both cell types
responded to fibronectin attachment by rapidly (within 10-20 min) and
transiently activating ERK1/ERK2 (Fig. 4, lower panels); the
level of activation was found to be similar to that observed following
serum stimulation (data not shown). The rate and extent of MAP kinase
activation were similar in both cell types indicating that, at least in
Rat1 fibroblasts, MAP kinase activation in response to fibronectin attachment is largely independent of p130Cas tyrosine
phosphorylation. Furthermore, this observation demonstrates that
overexpression of PTP-PEST in fibroblasts affects only a limited subset
of fibronectin-induced signaling pathways.
Effect of PTP-PEST Expression on Cell Motility--
As outlined
above, ectopic expression of PTP-PEST in Rat1 fibroblasts had no
apparent effect on the majority of the initial cellular events induced
by fibronectin, even though p130Cas exhibited a markedly
reduced level of tyrosine phosphorylation. However, many important
integrin receptor-mediated cellular processes, including the movement
of cells across the extracellular matrix, occur over an extended time
course; such processes are also potentially influenced by
fibronectin-induced tyrosine phosphorylation events and might therefore
be significantly affected by PTP-PEST overexpression. We assessed the
motility of the PTP-PEST expressing Rat1 fibroblasts by visual
inspection of wound closure following wounding of a cell monolayer.
Cells overexpressing PTP-PEST were found to migrate into the wound much
less rapidly than control, vector-expressing cells (Fig.
6A). Thus, control cells
migrated into the center of the cleared area to close the wound within
24 h, whereas PTP-PEST-overexpressing cells required 3-4 days to
migrate a similar distance. This difference in cell motility was
observed in all clones examined, i.e. three vector control
clones and three PTP-PEST overexpressing clones (Fig. 6B),
and in five independent experiments.
The potential involvement of p130Cas in controlling cell
motility was investigated further by indirect immunofluorescence
analysis of cells during their migration into a wound. Staining of
vector control cells with appropriate antibodies revealed that
p130Cas, as well as the focal adhesion proteins paxillin,
vinculin, and p125FAK, was localized to the leading edge of
migrating cells in lamellar structures (Fig. 6C). This
subcellular location is clearly consistent with the idea that
p130Cas plays a role in controlling cell movement across
the extracellular matrix. In contrast, p130Cas was found
throughout PTP-PEST expressing cells, with prominent staining in the
perinuclear region and very little of the protein discernible at the
cell periphery. Furthermore, in these cells vinculin and paxillin were
localized in focal adhesion structures, with no significant
accumulation at the leading edge of the cell (Fig. 6C).
These observations suggest that the ability of PTP-PEST to interfere
with cell motility may result from its ability to prevent the
redistribution of p130Cas from a cytoplasmic location to a
specific cytoskeletal location, namely the leading edge of the cell, in
cells that are migrating across the matrix. This inhibition of
p130Cas redistribution by PTP-PEST presumably results from
the reduced level of tyrosine phosphorylation of p130Cas
observed in PTP-PEST-overexpressing cells, since several studies have
highlighted the involvement of p130Cas tyrosine
phosphorylation in the redistribution of p130Cas from
soluble to insoluble fractions of cell lysates (38, 48, 49). Thus, by
dephosphorylating p130Cas, PTP-PEST can prevent its
association with appropriate Tyr(P)-dependent binding
partners, including the Crk adaptor protein (Fig. 5B), and
these Tyr(P)-dependent interactions appear to be essential for the efficient recruitment of p130Cas to the leading
edge of the migrating cell.
PTP-PEST Affects the Subcellular Location of p130Cas in
v-Crk-transformed Fibroblasts--
The ability of PTP-PEST to
influence the function of p130Cas by affecting its
subcellular location was analyzed further by transient transfection of
PTP-PEST into v-Crk-transformed rat fibroblasts. In these transformed
cells p130Cas is highly tyrosine-phosphorylated and is
predominantly localized to prominent focal adhesion structures (Fig.
7, upper panels). In contrast,
in cells overexpressing PTP-PEST, p130Cas displays a
diffuse cytoplasmic staining pattern, with little detectable staining
of focal adhesions (Fig. 7, lower panels). This effect of
PTP-PEST appears to be selective for the p130Cas protein
since the location of paxillin within the cell was unaffected by
PTP-PEST overexpression. Thus, paxillin was predominantly observed in
focal adhesion structures regardless of the level of PTP-PEST expression within the cell (Fig. 7). These results demonstrate that
PTP-PEST is capable of overcoming the potent effects of the v-Crk
oncoprotein on p130Cas and support the idea that PTP-PEST
controls specifically the phosphorylation state, and thereby the
subcellular location, of the p130Cas protein.
The results described in this paper suggest that PTP-PEST is a
regulator of integrin-mediated movement of fibroblasts across the
extracellular matrix via its regulation of the tyrosine phosphorylation state of p130Cas. Although previous reports have suggested
a potential involvement of p130Cas in cell motility
(50-52), our data are the first to reveal a requirement for tyrosine
phosphorylation of p130Cas in this process. The precise
role of p130Cas in controlling cell movement is not clear.
However, data recently obtained from mouse embryos lacking the
p130Cas protein suggest that it is involved in organization
of the actin cytoskeleton in fibroblasts and cardiocytes (53).
Integrin-mediated cell movement across the extracellular matrix is a
highly dynamic process requiring continual assembly and disassembly of
focal contacts, together with extensive remodeling of the actin
cytoskeleton (54-57). Many of the proteins found in focal contacts,
including p130Cas, are highly tyrosine-phosphorylated; in
fact, focal contacts represent one of the major sites of tyrosine
phosphorylation within the cell, as assessed by immunofluorescence
analysis using anti-Tyr(P) antibodies (58) (see Fig. 3). Furthermore,
at least two protein tyrosine kinases, p125FAK and Src, are
localized to focal contacts (59, 60), and both kinases have been
implicated in the control of cell movement (52, 61-63). These
observations have led to the suggestion that tyrosine phosphorylation
plays a significant role in controlling the assembly and disassembly of
focal adhesion complexes, thereby regulating cell motility. However,
the control of cell movement is a reversible, dynamic process
suggesting that its regulation by tyrosine phosphorylation must involve
both kinases and phosphatases. Our results suggest that the protein
tyrosine phosphatase PTP-PEST regulates cell motility by modulating
specifically the extent of tyrosine phosphorylation of
p130Cas.
The observation that p130Cas is localized at the leading
edge of migrating fibroblasts suggests a model for the observed effects of PTP-PEST expression on cell movement. By limiting the accumulation of tyrosine-phosphorylated p130Cas, the ability of
p130Cas to interact with appropriate SH2 domain-containing
binding partners is impaired in cells overexpressing PTP-PEST. It
appears likely that some of these interactions are essential for
targeting tyrosine-phosphorylated p130Cas to the correct
cytoskeletal location. Thus, it has been shown that the SH2 domain of
v-Crk is required for the v-Crk-induced translocation of
tyrosine-phosphorylated p130Cas to a detergent-insoluble
fraction (48). Also, in p130Cas-overexpressing COS cells,
fibronectin-induced relocation of p130Cas to focal adhesion
complexes requires both the SH3 domain of p130Cas (which
can bind directly to the focal adhesion kinase p125FAK (25,
30, 49)) and a C-terminal segment which contains binding sites for the
SH2 and SH3 domains of Src (34, 64). Our observation that cells
overexpressing PTP-PEST display a defect in the formation of complexes
between p130Cas and Crk following fibronectin stimulation
(Fig. 5B) suggests that such complexes are involved in
regulating cell movement. Indeed, recent evidence obtained using COS
cells transfected with mutant forms of Crk and p130Cas
suggested an involvement of p130Cas-Crk complexes in COS
cell migration on vitronectin-coated membranes (50).
Recently, p130Cas The results in this paper represent the first description of the
physiological role of PTP-PEST. The data suggest that PTP-PEST is
involved in controlling the rate of cell movement via the
dephosphorylation of p130Cas. Our results also provide new
insight into the function of p130Cas in integrin-mediated
processes. Thus, it appears that tyrosine phosphorylation of
p130Cas is largely dispensable for many of the cellular
responses to fibronectin, including cell attachment, cell spreading,
and MAP kinase activation. However, tyrosine phosphorylation of
p130Cas is required for the efficient movement of cells
across the extracellular matrix and therefore is likely to represent an
important mechanism for regulating the dynamic process of cell
migration in vivo.
INTRODUCTION
Top
Abstract
Introduction
References
/
mice (12). This high degree of selectivity of PTP-PEST for
p130Cas is derived from two distinct high affinity
interactions. The catalytic domain of PTP-PEST itself displays
intrinsic specificity for tyrosine-phosphorylated p130Cas
(11), but this specificity is greatly enhanced by an interaction between the SH3 domain of p130Cas and a proline-rich
sequence surrounding Pro-337 in the phosphatase (13). These data
strongly suggest that a major role of PTP-PEST is to regulate the level
of tyrosine phosphorylation of p130Cas within the cell.
EXPERIMENTAL PROCEDURES
80%
confluence and then serum-starved for 16 h in DMEM with 0.5%
fetal bovine serum in the absence of hygromycin. Removal of hygromycin
from the medium was necessary to maintain cell viability during serum
starvation and had no effect on the level of expression of PTP-PEST.
The cells were removed from the plates by the addition of 0.05%
trypsin, 0.53 mM EDTA and were subsequently suspended in
serum-free DMEM containing 125 µg/ml soybean trypsin inhibitor (5 ml
of medium per 10-cm diameter tissue culture dish). The cells were
recovered by centrifugation, washed three times with serum-free DMEM,
suspended in this medium at a concentration of 106
cells/ml, placed into tissue culture dishes on a slowly rotating platform (the dishes were pre-coated with 1% bovine serum albumin to
prevent cell attachment at this point), and allowed to recover for 45 min at 37 °C. The resultant cell suspension was added to fibronectin-coated tissue culture dishes (5 × 106
cells per 10-cm diameter dish) or fibronectin-coated glass coverslips. Coating with fibronectin was achieved by incubation for 16 h at 4 °C in PBS containing 10 µg/ml fibronectin; excess matrix protein was then removed by rinsing with PBS. The coated surfaces were blocked
with 1% bovine serum albumin in PBS for 1 h at 37 °C and washed twice in DMEM at 37 °C prior to addition of cells.
RESULTS
130,000 was significantly reduced in
lysates of PTP-PEST overexpressing cells. There was some variability in
the Tyr(P) content of protein(s) of Mr
65,000-70,000. Thus, although two of the vector control clones (65 and
73) exhibited slightly higher Tyr(P) content at 65,000-70,000 when
compared with the PTP-PEST-overexpressing cell lines, this difference
was not observed in control clone 67 (Fig. 1, upper panels).
In addition, this difference was not observed consistently in different
lysates prepared from the same cell lines. The identity of this
65,000-70,000 protein is not known, but it appears not to be paxillin
since direct analysis of the Tyr(P) content of paxillin demonstrated
that its tyrosine phosphorylation was not altered following
overexpression of PTP-PEST (Fig. 2).
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Fig. 1.
Effect of PTP-PEST overexpression on
phosphotyrosine levels in Rat1 fibroblast cells. Aliquots (30 µg
of protein) of lysates prepared from Rat1 fibroblast cells stably
expressing PTP-PEST (right panels) or empty vector
(left panels) were analyzed by SDS-PAGE and immunoblotting
with antibodies to phosphotyrosine (monoclonal antibody G104,
upper panels) or PTP-PEST (monoclonal antibody AG10,
lower panels). Three clonal cell lines of each type were
analyzed, as indicated by the numbers above the upper
panels. The position of the arrow indicates the
approximate molecular weight of tyrosine-phosphorylated
p130Cas.
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Fig. 2.
Specific reduction in phosphotyrosine content
of p130Cas in cells overexpressing PTP-PEST. Proteins
were immunoprecipitated from aliquots (0.3 mg of protein) of lysates
prepared from Rat1 fibroblast cells stably expressing PTP-PEST
(right panels) or empty vector (left panels). The
antibodies used for immunoprecipitation were as follows: rabbit
polyclonal serum B+F (p130Cas), affinity purified rabbit
polyclonal sc-558 (FAK), and mouse monoclonal antibody 349 (paxillin).
In each case, the immunoprecipitated (IP) protein was
divided into two equal aliquots, which were analyzed by SDS-PAGE and
immunoblotting with either anti-phosphotyrosine antibody G104
(pY) or with the antibodies used for immunoprecipitation of
the protein, as indicated.
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Fig. 3.
Fibronectin-induced formation of focal
adhesion complexes in cells overexpressing PTP-PEST. Cells
overexpressing PTP-PEST (clone 50, lower panels) or empty
vector (clone 73, upper panels) were serum-starved and
attached to fibronectin-coated glass coverslips as described under
"Experimental Procedures." Following incubation at 37 °C for
1 h the cells were fixed and stained with the appropriate
antibodies for immunofluorescence analysis. The primary antibodies used
were as follows: affinity purified rabbit polyclonal CSH8 (PTP-PEST),
mouse monoclonal G104 (phosphotyrosine, pY), mouse
monoclonal 349 (paxillin).
130,000 which was significantly reduced in intensity in lysates of
cells overexpressing PTP-PEST (Fig. 4, upper panels). We
identified this 130-kDa protein as p130Cas by
immunoprecipitation with a p130Cas-specific antibody,
followed by anti-Tyr(P) immunoblotting (Fig. 5). Since the initial response of cells
to fibronectin was largely unchanged in cells overexpressing PTP-PEST,
these observations suggest that even though p130Cas is one
of the major Tyr(P)-containing proteins induced by cell attachment to
fibronectin, tyrosine phosphorylation of p130Cas may be
dispensable for the majority of initial fibronectin-induced cellular
events, including cell attachment and spreading.
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Fig. 4.
Fibronectin-induced protein tyrosine
phosphorylation and MAP kinase activation in cells overexpressing
PTP-PEST. Cells overexpressing PTP-PEST (clone 50, right
panels) or empty vector (clone 73, left panels) were
serum-starved (lanes A), suspended (lanes
S), seeded onto fibronectin-coated tissue culture plates, and
incubated at 37 °C for the indicated times (in min) as described
under "Experimental Procedures." Lysates prepared from these cells
were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine
antibody (pY) G104 (upper panels) or with a
polyclonal antibody specific for the phosphorylated, active forms of
p44 and p42 MAP kinase (ERK 1 and 2) (lower panels). The
position of the arrow (upper panels) indicates
the approximate molecular weight of tyrosine-phosphorylated
p130Cas.
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Fig. 5.
Effect of PTP-PEST overexpression on
fibronectin-stimulated tyrosine phosphorylation of
p130Cas. Cells overexpressing PTP-PEST (clone 50) or
empty vector (clone 73) were serum-starved, suspended in serum-free
medium (lanes S), and then seeded onto fibronectin-coated
tissue culture plates and incubated at 37 °C for 60 min (lanes
FN). A, proteins were immunoprecipitated from aliquots
(0.5 mg of protein) of lysates prepared from these cells, using the
same antibodies as in Fig. 2. In each case, the immunoprecipitated
protein (IP) was divided into two equal aliquots, which were
analyzed by SDS-PAGE and immunoblotting with either
anti-phosphotyrosine antibody G104 (pY, left
panels) or with the antibodies used for immunoprecipitation of the
protein (right panels) as indicated. B, aliquots
of the same lysates (0.8 mg of protein) were also analyzed by
immunoprecipitation using a monoclonal Crk antibody (clone 22)
(upper panels). The immunoprecipitated protein was divided
into two equal aliquots, which were analyzed by SDS-PAGE and
immunoblotting with antibodies to p130Cas (monoclonal 8G4,
left panels) or with the Crk antibody (right
panels). Aliquots of the cell lysates were also immunoblotted with
the Crk and p130Cas antibodies to ensure that all lysates
contained similar concentrations of the two proteins (lower
panels).
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Fig. 6.
Effect of PTP-PEST overexpression on cell
motility. Cells overexpressing PTP-PEST (clone 50, right
panels) or empty vector (clone 73, left panels) were
grown to confluence in tissue culture dishes (A), and an
area (approximately 2 mm wide) was cleared of cells by scraping with a
sterile pipette tip. The cells were then photographed immediately
(upper panels) or following incubation in growth medium for
24 h at 37 °C (lower panels). B, results
of cell motility assays were quantitated from the resultant photographs
by counting the number of cells present after 24 h within areas
corresponding to cell migration distances of 0.2-0.6 mm and 0.6-1.0
mm. Results shown represent the average number of cells per
mm2 counted at the indicated distances from the edge of the
initial wound; quadruplicate cell counts were performed at each
distance per clone, and error bars represent standard
deviations. Results are representative of 5 independent experiments.
Shaded bars represent vector control clones; striped
bars represent PTP-PEST-overexpressing clones. C,
wounds were made in cell monolayers growing on glass coverslips, and
the cells were incubated in growth medium for 15 h at 37 °C,
fixed, and stained for immunofluorescence using antibodies to
p130Cas (rabbit polyclonal B+F, upper panels)
and vinculin (monoclonal VIN 11-5, lower panels).
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Fig. 7.
Effect of PTP-PEST overexpression on the
subcellular location of p130Cas in v-Crk-transformed
fibroblasts. cDNA encoding PTP-PEST was transfected into
3Y1v-Crk rat fibroblasts growing on glass coverslips, and the cells
were allowed to grow for 48 h before fixation followed by
immunofluorescence analysis using antibodies to PTP-PEST (affinity
purified rabbit polyclonal antibody CSH8), p130Cas (mouse
monoclonal, clone 21), and paxillin (mouse monoclonal, clone 349).
Cells shown are representative either of transfected cells that
overexpress PTP-PEST (lower panels) or of cells from the
same coverslip that were not transfected (upper
panels).
DISCUSSION
/
fibroblasts have been reported to
display a defect in their response to an activated, transforming
variant of the Src tyrosine kinase; they fail to form colonies in soft agar, a defect that can be rescued by re-expression of
p130Cas (53). These results suggest that the involvement of
p130Cas in regulating the organization of actin
cytoskeleton structures may also influence the response of cells to
transforming oncogenes, a suggestion that is supported by several
earlier observations implicating a direct regulatory role for
p130Cas in cellular transformation (65-67). These data
further suggest that PTP-PEST may play a key role in suppressing
cellular transformation through its ability to dephosphorylate
p130Cas. Indeed, such a role is supported by our
observation that overexpression of PTP-PEST dramatically influences the
location of p130Cas within v-Crk-transformed cells,
suggesting that PTP-PEST potentially influences the response of cells
to a variety of transforming oncogenes.
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ACKNOWLEDGEMENTS |
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We thank Kim Pennino for assistance in characterizing the G104 monoclonal anti-phosphotyrosine antibody used in this work; Amy Bouton (University of Virginia) for p130Cas antibodies, Rat1 fibroblast and 3Y1v-Crk fibroblast cells; and Scott Lowe (Cold Spring Harbor Laboratory) for providing the pWZL(hygro) vector and Bosc23 cell line.
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FOOTNOTES |
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* This work was funded by Grant CA 53840 from the National Institutes of Health.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.
Current address: OSI Pharmaceuticals Inc., 106 Charles Lindbergh
Blvd., Uniondale, NY 11553-3649.
§ To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724-2208. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail:tonks{at}cshl.org.
The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.
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REFERENCES |
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