(Received for publication, August 22, 1995; and in revised form, December 13, 1995)
From the
The role of phosphatidylinositol 3`-kinase (PI 3`-kinase)
activity in platelet-derived growth factor (PDGF)-stimulated tyrosine
phosphorylation of focal adhesion kinase (p125) and
paxillin has been examined. The tyrosine phosphorylation of
p125
and paxillin in response to PDGF was markedly
inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI
3`-kinase activity, membrane ruffle formation, and tyrosine
phosphorylation of p125
and paxillin were all inhibited
by the same low concentrations of wortmannin (>90% inhibition at 40
nM). In contrast, tyrosine phosphorylation of p125
and paxillin in response to bombesin, endothelin, and phorbol
12,13-dibutyrate was not inhibited by wortmannin in these cells.
Furthermore, LY294002, an inhibitor of PI 3`-kinase structurally
unrelated to wortmannin, also inhibited PDGF-stimulated p125
tyrosine phosphorylation. PDGF was shown to stimulate the
tyrosine phosphorylation of p125
in porcine aortic
endothelial (PAE) cells transfected with the wild type PDGF-
receptors, but not in PAE cells transfected with PDGF-
receptors
in which the PI 3`-kinase binding sites (Tyr-740/751) were replaced by
phenylalanine. PDGF-stimulated, PI 3`-kinase-dependent tyrosine
phosphorylation of p125
was not inhibited by rapamycin,
and thus it was dissociated from the activation of p70 S6 kinase,
previously identified as a molecular downstream target of PI 3`-kinase.
Thus, we have identified a PI 3`-kinase-dependent signal
transduction pathway in the action of PDGF, which leads to the
phosphorylation of p125 and paxillin.
Platelet-derived growth factor (PDGF) ()is a 30-kDa
polypeptide dimer that regulates cellular proliferation, chemotaxis,
and cytoskeletal reorganization(1, 2, 3) .
The binding of PDGF to individual receptor chains stimulates their
dimerization and subsequent
transphosphorylation(2, 4) . Cytoplasmic effector
proteins such as phospholipase
C
(5, 6, 7, 8) , SH2-containing
protein phosphotyrosine phosphatase(9) ,
GAP(10, 11) , the p85 subunit of PI
3`-kinase(12, 13) , GRB-2/sem-5(14) ,
and the Src family of protein tyrosine kinases (15) associate
with specific phosphorylated tyrosine residues on the receptor chains
via their SH2 domains(16, 17) . Once bound to the
receptor, many of these proteins are phosphorylated on tyrosine by the
intrinsic tyrosine kinase activity of the receptor. PDGF also
stimulates the tyrosine phosphorylation of the focal
adhesion-associated proteins, p125
and paxillin (18, 19) . p125
is a cytosolic tyrosine
kinase that lacks SH2 and SH3 domains but associates with other
proteins including v-Src and
paxillin(20, 21, 22, 23, 24) .
Paxillin contains multiple domains that can interact with vinculin,
p125
, and other cytoskeletal
proteins(24, 25, 26) . The signal
transduction pathways underlying PDGF-stimulated tyrosine
phosphorylation of p125
and paxillin have not yet been
elucidated.
PI 3`-kinase phosphorylates inositol phospholipids on
the D3 position. In vivo this enzyme is thought to
phosphorylate the head group of PtdIns(4, 5) P to yield PtdIns (3, 4, 5) P
, and this lipid has
been postulated to act as a second
messenger(27, 28, 29, 30, 31) .
p70
has been identified as one of the putative molecular
downstream targets of PI 3`-kinase activity (32, 33, 34, 35) (but see also (36) ). There is evidence, however, that PI 3`-kinase-regulated
signaling bifurcates upstream of p70
, implying that there
must be other molecular downstream targets of PI 3`-kinase
activity(32, 33) . PDGF stimulates the recruitment of
polymerized actin into membrane ruffles and this cytoskeletal response
is a PI 3`-kinase-dependent event (37, 38, 39) . Likewise, PDGF-stimulated
chemotaxis is dependent on PI 3`-kinase activity(37) . It is
not known, however, whether p70
or other molecular
downstream targets of PI 3`-kinase are involved in these
PDGF-stimulated processes.
PI 3`-kinase activity, membrane ruffle
formation, and tyrosine phosphorylation of p125 and
paxillin are stimulated by the same low concentrations of PDGF in Swiss
3T3 cells(18) . Furthermore, the tyrosine phosphorylation of
p125
and paxillin by PDGF is critically dependent on the
integrity of the actin cytoskeleton(18) . Cytochalasin D, an
agent that prevents actin polymerization, inhibits PDGF-stimulated
tyrosine phosphorylation of these cytoskeletal-associated
proteins(18) . We reasoned, therefore, that PI 3`-kinase may
lie upstream in a common signal transduction pathway stimulating the
formation of membrane ruffles and the tyrosine phosphorylation of
p125
and paxillin. To test this hypothesis, we utilized
two different experimental approaches. First, PI 3`-kinase activity was
directly inhibited by pretreatment of Swiss 3T3 cells with two
structurally unrelated inhibitors, wortmannin and LY294002. Second, PAE
cells were transfected with either wild type PDGF-
receptors or
PDGF
receptors in which the PI 3`-kinase binding sites
(Tyr-740/751) were replaced by phenylalanine. Using these two
complementary approaches, we demonstrate here, for the first time, that
the inhibition of PDGF-stimulated PI 3`-kinase activity, prevents the
tyrosine phosphorylation of p125
and paxillin in response
to PDGF in a selective manner.
Figure 1: Effect of wortmannin on PDGF-stimulated PI 3`-kinase activity and the tyrosine phosphorylation of proteins in response to PDGF and bombesin. A, quiescent Swiss 3T3 cells were preincubated for 10 min at 37 °C with 0, 20, or 40 nM wortmannin. Cells were subsequently incubated with 3 ng/ml PDGF for 10 min and then lysed. The lysates were immunoprecipitated with anti-Tyr(P) mAb, and the PI 3`-kinase activity in the immunoprecipitates was assayed. The autoradiogram obtained was scanned with an LKB Ultrascan XL densitometer to quantify phospholipids in terms of peak area. Values correspond to the phosphorylation of PI expressed as a percentage of the maximal response. The results shown are representative of at least three experiments. B, cells were preincubated in the presence (+) or absence(-) of 30 nM wortmannin for 10 min, subsequently incubated for 10 min with either 3 or 30 ng/ml PDGF or 10 nM bombesin and then lysed. The lysates were immunoprecipitated with the anti-Tyr(P) mAb PY72 and then analyzed by immunoblotting with either anti-Tyr(P) mAbs (PY) (a mixture of PY20 and 4G10) or a GAP antiserum (GAP) as indicated. In this and subsequent figures, representative autoradiograms are shown of experiments repeated at least three times.
To determine the effect of wortmannin on the tyrosine
phosphorylation of proteins in response to PDGF and bombesin, quiescent
Swiss 3T3 cells were preincubated with 30 nM wortmannin for 10
min and then incubated for another 10 min in the presence of either 3
or 30 ng/ml PDGF or 10 nM bombesin. The cell lysates were
immunoprecipitated with the anti-Tyr(P) mAb PY72 and then analyzed by
SDS-PAGE, followed by immunoblotting with a mixture of the anti-Tyr(P)
mAb PY20 and 4G10. In agreement with our previous results(18) ,
PDGF stimulated the tyrosine phosphorylation of distinct substrates at
different concentrations (Fig. 1B). Bands with M 110,000-130,000 and 70,000-75,000
were phosphorylated on tyrosine in response 3 ng/ml PDGF, while their
intensity was markedly reduced at 30 ng/ml PDGF, a concentration of
PDGF that stimulated the phosphorylation of numerous other proteins (Fig. 1B). Preincubation of cells with 30 nM wortmannin caused a marked decrease in the tyrosine
phosphorylation of the M
110,000-130,000 and
70,000-75,000 bands stimulated by 3 ng/ml PDGF. Several lines of
evidence indicate that this effect of wortmannin was selective. (a) At both 3 and 30 ng/ml PDGF, the most prominent band
phosphorylated on tyrosine has a M
170,000-190,000 and corresponds to the autophosphorylated
and
PDGF receptor chains(2, 3) .
Pretreatment of cells with wortmannin had no effect on the degree of
tyrosine phosphorylation of the PDGF receptor chains stimulated by
either 3 or 30 ng/ml PDGF, implying that wortmannin did not interfere
with PDGF receptor autophosphorylation in Swiss 3T3 cells (Fig. 1B). (b) Wortmannin had no apparent
effect on the tyrosine phosphorylation of multiple proteins stimulated
by 30 ng/ml PDGF. (c) Accordingly, wortmannin pretreatment of
cells had no effect on PDGF-stimulated tyrosine phosphorylation of GAP (Fig. 1B) or the mobilization of intracellular
Ca
that occurs as a result of PDGF activation of
phospholipase C
(results not shown). These results suggest that
the inhibitory effect of wortmannin on PDGF-stimulated tyrosine
phosphorylation is selective for a subset of proteins.
The
neuropeptide, bombesin, stimulates the tyrosine phosphorylation of
multiple proteins including the broad bands of M 110,000-130,000 and 70,000-80,000(45) . In
contrast to PDGF, the effects of neuropeptides are mediated through
G-protein-coupled seven transmembrane receptors (53) that do
not stimulate PI 3`-kinase activity in Swiss 3T3
cells(45, 54, 55) . Hence,
bombesin-stimulated tyrosine phosphorylation of proteins should not be
inhibited by wortmannin. As shown in Fig. 1B,
preincubation of cells with 30 nM wortmannin had no effect on
the bombesin-stimulated tyrosine phosphorylation of the broad bands of M
110,000-130,000 and 70,000-80,000.
Indeed wortmannin up to 100 nM had no effect on
bombesin-stimulated tyrosine phosphorylation of proteins (results not
shown). In addition, bombesin-stimulated formation of actin stress
fibers is not affected by wortmannin pretreatment of cells (results not
shown). Thus wortmannin distinguishes between PDGF and
bombesin-stimulated tyrosine phosphorylation of specific proteins and
cytoskeletal changes.
Cells were preincubated with wortmannin (0-40 nM) and
then stimulated with either 3 ng/ml PDGF or 10 nM bombesin.
The cell lysates were immunoprecipitated with an anti-Tyr(P) mAb and
the immunoprecipitates analyzed by immunoblotting with an
anti-p125 mAb. The results shown in Fig. 2(A and B) indicate that wortmannin induced a dramatic
dose-dependent inhibition of the tyrosine phosphorylation of
p125
in response to 3 ng/ml PDGF. In five independent
experiments, pretreatment of cells with 30 nM wortmannin for
10 min produced an 85 ± 6% inhibition of the tyrosine
phosphorylation of p125
in response to subsequent
stimulation with PDGF (3 ng/ml). In contrast the tyrosine
phosphorylation of p125
stimulated with 10 nM bombesin was not inhibited by preincubation of the cells with
wortmannin up to 40 nM.
Figure 2:
Effect of wortmannin on PDGF-, EGF-,
bombesin-, endothelin- and PDB-stimulated tyrosine phosphorylation of
p125. A, quiescent Swiss 3T3 cells were
preincubated for 10 min at 37 °C with wortmannin (0-40
nM). Cells were subsequently incubated with either 10 nM bombesin or 3 ng/ml PDGF for 10 min, lysed, and then
immunoprecipitated with the anti-Tyr(P) mAb PY72. Immunoprecipitates
were analyzed by immunoblotting with anti-p125
mAb. B, the autoradiogram obtained in A was scanned with
an LKB Ultrascan XL densitometer to quantify phosphoproteins in terms
of peak area. Values correspond to the phosphorylation of p125
expressed as a percentage of the maximal response in response to
10 nM bombesin (open circles) or 3 ng/ml PDGF (closed circles). C, quiescent Swiss 3T3 cells were
preincubated for 10 min at 37 °C with or without wortmannin (30
nM). Cells were subsequently incubated with either 3 ng/ml
PDGF, 10 ng/ml EGF, 10 nM bombesin, 10 nM endothelin (End), or 100 nM PDB for 10 min and lysed. The cell
lysates were then immunoprecipitated with the anti-Tyr(P) mAb PY72.
Immunoprecipitates were analyzed by immunoblotting with
anti-p125
mAb.
To extend the results presented
above, we examined the wortmannin sensitivity of p125 tyrosine phosphorylation in response to EGF, endothelin, and PDB
in Swiss 3T3 cells. Here we demonstrate, for the first time, that EGF
stimulated an increase in the tyrosine phosphorylation of p125
(3.6 ± 1-fold, n = 4) in Swiss 3T3 cells.
EGF-stimulated phosphorylation of p125
was inhibited by
85 ± 12% when the cells were pretreated with 30 nM wortmannin (Fig. 2C), consistent with an
inhibition of EGF-stimulated PI 3`-kinase activity by wortmannin (data
not shown). In accord with the data obtained with PDGF, EGF stimulated
the rapid formation of membrane ruffles in Swiss 3T3 cells, a response
that was markedly inhibited by pretreatment of the cells with 30 nM wortmannin for 10 min (data not shown).
Endothelin, a
neuropeptide that acts through a different G-protein-coupled receptor
to bombesin also stimulates tyrosine phosphorylation of p125 and formation of actin stress fibers in Swiss 3T3
cells(44, 57) . In contrast to the results obtained
with PDGF and EGF, endothelin-stimulated p125
tyrosine
phosphorylation and actin reorganization were not affected by
pretreatment of the cells with 30 nM wortmannin for 10 min (Fig. 2C and results not shown). In addition, the
tyrosine phosphorylation of p125
in response to direct
activation of protein kinase C by PDB (56) was not affected by
pretreatment of the cells with 30 nM wortmannin (Fig. 2C).
Figure 3:
Effect of LY294002 on the tyrosine
phosphorylation of p125 in response to PDGF and bombesin.
Quiescent Swiss 3T3 cells were washed and preincubated for 1 h at 37
°C in DMEM with or without LY294002 (1-15 µM).
Cells were subsequently incubated with either 10 nM bombesin
or 3 ng/ml PDGF for 10 min, lysed, and then immunoprecipitated with the
anti-Tyr(P) mAb PY72. Immunoprecipitates were analyzed by
immunoblotting with anti-p125
mAb.
The
results presented in Fig. 4demonstrate that PDGF stimulates the
tyrosine phosphorylation of p125 in PAE cells expressing
wild type PDGF-
receptors. In contrast, PDGF failed to stimulate
an increase in the tyrosine phosphorylation of p125
in
PAE cells expressing Y740F/Y751F PDGF-
receptors (Fig. 4).
This result suggests that PDGF-stimulated tyrosine phosphorylation of
p125
is dependent on the interaction of the p85
regulatory subunit of PI 3`-kinase with the PDGF receptor chains. It is
interesting to note that PAE cells expressing the Y740F/Y751F mutated
PDGF-
receptors exhibit a higher basal level of p125
tyrosine phosphorylation and that these cells have previously
been demonstrated to contain higher levels of PI 3`-lipids (59) . This result provides an independent line of evidence
supporting the conclusion that PDGF-stimulated tyrosine phosphorylation
of p125
is dependent on the activation of PI 3`-kinase.
Figure 4:
PDGF-stimulated tyrosine phosphorylation
of p125 in PAE cell lines expressing wild type or mutant
PDGFR-
receptors. PAE cells expressing wild type PDGFR-
or
PDGFR-
Y740F/Y751F mutant receptors molecules were incubated with
PDGF (0-30 ng/ml) for 10 min at 37 °C. The cells were lysed,
and the lysates were immunoprecipitated with the anti-Tyr(P) mAb PY72
and then analyzed by immunoblotting with anti-p125
mAb.
Figure 5: Effect of wortmannin on PDGF- and bombesin-stimulated tyrosine phosphorylation of paxillin. Upper, quiescent Swiss 3T3 cells were preincubated for 10 min at 37 °C with 0, 20 or 30 nM wortmannin and subsequently with 3 ng/ml PDGF or with 10 nM bombesin (Bom), as indicated. Lower, other cell cultures were preincubated for 10 min at 37 °C with (+) or without(-) 30 nM wortmannin, and subsequently incubated with PDGF (0-10 ng/ml) for 10 min. Cell lysates were immunoprecipitated with the anti-paxillin mAb 165 and then analyzed by immunoblotting with a mixture of anti-Tyr(P) mAbs.
The
immunosuppressant rapamycin is a selective inhibitor of p70 activation in many cell types, including Swiss 3T3
cells(47) . We therefore examined the effect of rapamycin on
the PDGF-stimulated tyrosine phosphorylation of p125
.
Quiescent cultures of Swiss 3T3 cells were pretreated with either
rapamycin (20 nM for 20 min) or wortmannin (30 nM for
10 min) and then stimulated with PDGF (3 ng/ml) or bombesin (10
nM) for 10 min. Cell lysates were analyzed by immunoblotting
with an anti-p70
polyclonal Ab and the phosphorylated
form of p70
(pp70
) was identified by its
retarded mobility. The results shown in Fig. 6demonstrate that
pretreatment of cells with rapamycin completely inhibited both the
PDGF- and bombesin-stimulated phoshorylation of p70
. Cell
lysates derived from parallel cultures of Swiss 3T3 cells were
immunoprecipitated with an anti-Tyr(P) mAb and the immunoprecipitates
analyzed by immunoblotting with an anti-p125
mAb. As
shown in Fig. 6, rapamycin had no effect on either PDGF- or
bombesin-stimulated tyrosine phosphorylation of p125
.
Furthermore, pretreatment of Swiss 3T3 cells with rapamycin (20
nM) had no effect on PDGF-stimulated formation of membrane
ruffles (data not shown). In contrast, wortmannin, which only slightly
inhibited the PDGF-stimulated phoshorylation of p70
at 30
nM, dramatically inhibited the PDGF-stimulated tyrosine
phosphorylation of p125
at this concentration (Fig. 6). Wortmannin had little effect on either the
bombesin-stimulated phosphorylation of p70
or tyrosine
phosphorylation of p125
. These results therefore
dissociate p70
activation from both PDGF- and
bombesin-mediated p125
tyrosine phosphorylation.
Figure 6:
Effect of rapamycin and wortmannin on
PDGF- and bombesin-stimulated phosphorylation of p70 and
the tyrosine phosphorylation of p125
. Two parallel sets
of cells were preincubated in the presence (+) or absence(-)
of either 20 nM rapamycin (R) for 20 min or 30 nM wortmannin (W) for 10 min, the cells were subsequently
incubated for 10 min with either 3 ng/ml PDGF or 10 nM bombesin and then lysed. The whole cell lysates from one set of
cells were analyzed directly by immunoblotting with the
anti-p70
rabbit polyclonal Ab. The lysates from the other
set of cells were immunoprecipitated with anti-Tyr(P) mAb and then
analyzed by immunoblotting with anti-p125
mAb.
The signal transduction pathways implicated in PDGF-induced
tyrosine phosphorylation of the focal adhesion proteins p125 and paxillin had not been elucidated. The results presented here
demonstrate that wortmannin at nanomolar concentrations dramatically
inhibits PDGF-stimulated tyrosine phosphorylation of a subset of
proteins, including p125
and paxillin in Swiss 3T3 cells.
We verified that at these concentrations wortmannin inhibits
PDGF-stimulated PI 3`-kinase activity and the reorganization of actin
into membrane ruffles in Swiss 3T3 cells. In contrast, wortmannin at
nanomolar concentrations does not affect p125
tyrosine
phosphorylation induced by bombesin, which does not stimulate PI
3`-kinase in Swiss 3T3 cells(45, 54, 55) .
Furthermore, the PI 3`-kinase inhibitor LY294002, which is structurally
unrelated to wortmannin, also inhibited PDGF-stimulated p125
tyrosine phosphorylation in a selective manner. Utilizing PAE
cells transfected with wild type and mutant PDGF
receptors, we
have shown that PDGF stimulates an increase in the tyrosine
phosphorylation of p125
in PAE cells transfected with
wild type PDGF
receptors, but not in PAE cells that were
transfected with PDGF
receptors lacking the PI 3`-kinase binding
sites. Therefore, employing different experimental approaches, we were
able to demonstrate that the activation of PI 3`-kinase is necessary
for PDGF-stimulated tyrosine phosphorylation of p125
.
These results suggest that PI 3`-kinase lies upstream in the signal
transduction pathway linking the PDGF receptor to tyrosine
phosphorylation of p125
and paxillin.
The results
presented in this study should be distinguished from those recently
published by Chen and Guan(19) . These authors reported that a
small fraction (5-6%) of total cellular PI 3`-kinase activity can
be recovered from p125 immunoprecipitates derived from
lysates of NIH 3T3 cells treated with PDGF (19) . (
)This association was maximal when the cells were
stimulated with 25 ng/ml PDGF, a concentration of PDGF that did not
stimulate the tyrosine phosphorylation of p125
in these
cells. Furthermore, cytochalasin D, a potent inhibitor of
PDGF-stimulated p125
tyrosine phosphorylation, did not
affect the association(19) . Thus, the association between
p125
and PI 3`-kinase seen in NIH 3T3 cells treated with
high concentrations of PDGF
cannot account for the results
presented in this study in which PI 3`-kinase was identified as an
upstream element in a signal transduction pathway leading to
p125
and paxillin tyrosine phosphorylation in cells
treated with low concentrations of PDGF.
p70 has been
identified as a molecular downstream target of PI
3`-kinase(32, 33, 34, 35) . It was
important, therefore to determine whether p70
lies along
a linear signaling pathway leading to p125
tyrosine
phosphorylation in PDGF-treated cells. Here we have demonstrated that
inhibition of the phosphorylation and activation p70
,
with the immunosuppressant rapamycin (47) , did not inhibit the
PDGF-stimulated tyrosine phosphorylation of p125
. In
addition we found that rapamycin does not affect PDGF-stimulated
membrane ruffling. We can therefore conclude that p70
activation and p125
tyrosine phosphorylation
constitute two independent molecular downstream targets of PI 3`-kinase
in PDGF-treated cells.
PDGF is a potent chemotactic agent for
fibroblasts and other cell types (1, 37) . It has been
shown that PI 3`-kinase activation is required for the formation of
membrane ruffles and the stimulation of chemotaxis induced by growth
factors(37, 38, 55) . Recently it has been
shown that the small G protein Rac lies downstream of PI 3`-kinase, and
there is evidence that PI 3`-lipids may promote Rac-GTP
formation(59) . Furthermore, it has been demonstrated that
there is a GTP-dependent and PDGF-stimulated association of Rac with PI
3`-kinase in Swiss 3T3 cells(63) . Activated Rac has been
demonstrated to direct the formation of membrane ruffles and the
assembly of focal adhesions(64) . In a previous study we
demonstrated that the PDGF-stimulated tyrosine phosphorylation of
p125 and paxillin is dependent on the integrity of the
actin cytoskeleton(18) . The results presented here,
demonstrating that PI 3`-kinase activation is required for
PDGF-stimulated tyrosine phosphorylation of p125
and
paxillin and the formation of membrane ruffles, establish another link
between the reorganization of the actin cytoskeleton and the tyrosine
phosphorylation of these focal adhesion-associated proteins. Taken
together, all these findings suggest that there is a linear signal
transduction pathway whereby ligation of the PDGF receptor activates PI
3`-kinase and thereby stimulates Rac-GTP formation. Activated Rac
induces the formation of focal contacts, reorganization of the actin
cytoskeleton, and the tyrosine phosphophorylation of p125
and paxillin. This signal transduction pathway could function in
the regulation of chemotaxis. The recent demonstration that
p125
-deficient cells do not display polar migratory shape
and exhibit a striking reduction in motility (65) is in
agreement with this interpretation.
Activation of PI 3`-kinase in
response to PDGF is thought to occur as a result of the association of
the p85 regulatory subunit of PI 3`-kinase with the
tyrosine-phosphorylated PDGF receptor chains(12, 13) .
The recent demonstration that Ras may interact with and activate PI
3`-kinase suggests another mechanism for PI 3`-kinase activation in
response to either PDGF or EGF in at least some cell
types(66, 67) . The relative contribution of these two
pathways leading to PI 3`-kinase activation and hence p125 tyrosine phosphorylation in response to PDGF or EGF warrants
further experimental work.
The tyrosine phosphorylation of
p125 and paxillin induced by bombesin and endothelin is
dependent on the integrity of the actin cytoskeleton (56, 60) and the activity of the small GTP-binding
protein Rho(57) . Here we demonstrate that the tyrosine
phosphorylation of p125
in response to either bombesin or
endothelin is not prevented by wortmannin at concentrations that
virtually abolished the tyrosine phosphorylation of p125
and paxillin in response to PDGF and EGF. Similar results were
obtained when LY294002 was used instead of wortmannin. An important
implication of these results, therefore, is that there is a PI
3`-kinase-dependent and PI 3`-kinase-independent signal transduction
pathway stimulating the tyrosine phosphorylation of p125
and paxillin in the same cells.