From the Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095
Received for publication, February 1, 2001
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ABSTRACT |
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Plating suspended Swiss 3T3 cells onto
fibronectin-coated dishes promoted phosphorylation of endogenous focal
adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site,
and at Tyr-577, located in the activation loop, as revealed by
site-specific antibodies that recognize the phosphorylated form of
these residues. Treatment with the selective Src family kinase
inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the
phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin.
Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was
dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn
expression). Stimulation of Swiss 3T3 cells with bombesin also induced
a rapid increase in the phosphorylation of endogenous FAK at Tyr-397.
In contrast to the results obtained with fibronectin, PP-2 did not
prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a
concentration (10 µM) that suppressed
bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not
prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with
other G protein-coupled receptor agonists including vasopressin,
bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic
acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our
results identify, for first time, the existence of
Src-dependent and Src-independent pathways leading to
FAK autophosphorylation at Tyr-397 stimulated by
adhesion-dependent signals and G protein-coupled
receptor agonists in the same cell.
A rapid increase in the tyrosine phosphorylation of the
non-receptor tyrosine kinase
FAK1 (1, 2), which localizes
to focal adhesion plaques, has been identified as a prominent early
event in cells stimulated by diverse signaling molecules that regulate
cell proliferation, migration, and apoptosis (3-5). In particular, FAK
is activated and tyrosine-phosphorylated in response to integrin
clustering induced by cell adhesion or antibody cross-linking (1, 2, 6-9). In addition, FAK is rapidly tyrosine-phosphorylated in cells
stimulated by mitogenic neuropeptide agonists including bombesin
(10-16) and bioactive lipids including LPA (17-19) that act via
heptahelical GPCRs, polypeptide growth factors (20-24), bacterial
toxins (25, 26), and activated variants of pp60Src
(27, 28). The importance of FAK-mediated signal transduction is
underscored by experiments implicating this tyrosine kinase in
embryonic development (29) and in the control of cell migration (30-33), proliferation (30, 34), and apoptosis (35, 36). In addition,
there is increasing evidence linking overexpression of FAK to the
invasive properties of cancer cells (37, 38). It is increasingly
recognized that the tyrosine phosphorylation and activation of FAK is
an important point of convergence in the action of integrins, GPCR
agonists, growth factors, and oncogenes (5, 39, 40). However, the
molecular pathways leading to FAK tyrosine phosphorylation in response
to multiple extracellular factors remain incompletely understood.
Plating suspended cells onto fibronectin-coated dishes, a paradigm of
integrin receptor activation (41), induces
adhesion-dependent phosphorylation of FAK at multiple sites
including tyrosines 397, 576, 577, 861, and 925 (32, 42-45). Tyr-397,
the only apparent autophosphorylation site (46-51), has emerged as a
critical residue in FAK-mediated signaling (5). The autophosphorylation
of FAK at Tyr-397 creates a high affinity binding site for the SH2
domain of Src family kinases including Src, Yes, and Fyn and leads to the formation of a signaling complex between FAK and Src family kinases
(46, 47, 49-52). A model has recently been proposed that envisages
reciprocal catalytic activation of FAK and Src family kinases in
response to adhesion-dependent signals. Src family kinases
associated with FAK are thought to phosphorylate FAK at additional
sites including Tyr-576 and Tyr-577 that are located in the activation
loop of the kinase catalytic domain of FAK (32, 42, 53), thereby
promoting maximal FAK catalytic activation. Because phenylalanine
mutation of Tyr-576 and Tyr-577 reduced adhesion-mediated FAK
autophosphorylation (at Tyr-397), it has been proposed that activation
loop phosphorylation of FAK by Src stimulates intermolecular
phosphorylation at Tyr-397, thereby leading to signal amplification at
sites of integrin-mediated cell adhesion (32). Therefore, in this model
Src family kinases are thought to play a major role leading to
autophosphorylation of FAK at Tyr-397 as part of an
adhesion-dependent signaling response. Recently, we
demonstrated that GPCR agonists including bombesin and LPA also
induce rapid activation of Src family kinases (54) and promote the
formation of a FAK/Src signaling complex (55). However, it is not known
whether Src family kinases are also required for promoting FAK
phosphorylation at Tyr-397 in cells stimulated with GPCR agonists, as
would be predicted by the signal amplification model.
In the present study, we report that stimulation with the mitogenic
GPCR agonists bombesin, bradykinin, endothelin, vasopressin, and LPA
induced rapid phosphorylation of endogenous FAK at Tyr-397 in intact
Swiss 3T3 cells, an effect comparable to that stimulated by
integrin-mediated cell adhesion. Treatment with the selective Src
family kinase inhibitor PP-2 inhibited the phosphorylation of Tyr-397
induced by fibronectin. Furthermore, integrin-mediated FAK
phosphorylation at Tyr-397 was dramatically reduced in SYF cells
(deficient in Src, Yes, and Fyn expression). In striking contrast,
PP-2, at a concentration that abolished activation loop phosphorylation, did not prevent FAK Tyr-397 phosphorylation in Swiss
3T3 cells stimulated by bombesin, LPA, or other GPCR agonists. In
addition, LPA also induced FAK phosphorylation at Tyr-397 in SYF cells.
Our results demonstrate, for the first time, that the signaling events
leading to the phosphorylation of FAK at Tyr-397 induced by GPCR
agonists are Src-independent and thus can be distinguished from those
stimulated by integrin receptors that require Src family kinase activity.
Cell Culture--
Stock cultures of Swiss 3T3 cells were
maintained in DMEM, supplemented with 10% fetal bovine serum in a
humidified atmosphere containing 10% CO2 and 90% air at
37 °C. For experimental purposes, Swiss 3T3 cells were plated in
100-mm dishes at 6 × 105 cells/dish in DMEM
containing 10% fetal bovine serum and used after 6-8 days when the
cells were confluent and quiescent.
SYF cells (deficient in Src, Yes, and Fyn expression) and YF cells
(deficient in Yes and Fyn expression) were obtained from ATCC (CRL-2459
and CRL-2497). Stock cultures of these cells were maintained in DMEM,
supplemented with 10% fetal bovine serum in a humidified atmosphere
containing 10% CO2 and 90% air at 37 °C. For
experimental purposes, SYF and YF cells were plated in 100-mm dishes at
7 × 105 cells/dish in DMEM containing 10% fetal
bovine serum and were used after 5 days when the cells were confluent.
Cell Stimulation with Bombesin and Other Agonists--
Confluent
and quiescent Swiss 3T3 cells were washed twice with DMEM, equilibrated
in the same medium at 37 °C for at least 30 min, and then treated
with bombesin or other agonists for the times indicated. The
stimulation was terminated by aspirating the medium and solubilizing
the cells in 1 ml of ice-cold RIPA buffer containing 50 mM
HEPES, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
150 mM NaCl, 10% glycerol, 1.5 mM
MgCl2, 1 mM EGTA, 1 mM sodium
orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride.
Confluent cultures of SYF and YF cells were incubated for 40 h in
DMEM containing 0.1% fetal bovine serum and then treated as described
above for Swiss 3T3 cells.
Cell Stimulation with Fibronectin or Adherence to
Poly-L-lysine--
Confluent and quiescent Swiss 3T3 cells
were harvested by limited trypsin/EDTA treatment (0.05% trypsin, 2 mM EDTA in PBS). The trypsin was inactivated by soybean
trypsin inhibitor (0.5 mg/ml) with 0.25% bovine serum albumin in DMEM.
Cells were collected by centrifugation, resuspended in DMEM containing
0.1% bovine serum albumin, and held in suspension for 1 h at
37 °C. Cell culture dishes (10 cm) were pre-coated with fibronectin
purified from bovine plasma (10 µg/ml) or poly-L-lysine
(100 µg/ml) in PBS overnight at 4 °C, rinsed with PBS, and warmed
to 37 °C for 1 h prior to plating. Suspended cells were
distributed onto ligand-coated dishes and incubated at 37 °C, and at
various times following plating, protein extracts were made in RIPA
buffer as described previously. Confluent cultures of SYF and YF cells
were incubated for 40 h in DMEM containing 0.1% fetal bovine
serum and then treated as described for Swiss 3T3 cells.
Immunoprecipitation--
Lysates were clarified by
centrifugation at 15,000 rpm for 10 min. Supernatants were transferred
to fresh tubes, and proteins were immunoprecipitated at 4 °C for
4 h with protein A-agarose linked to polyclonal anti-FAK (C-20)
antibody, as described previously (10, 11). Immunoprecipitates were
washed three times with RIPA buffer and extracted in 2× SDS-PAGE
sample buffer (200 mM Tris-HCl, pH 6.8, 1 mM
EDTA, 6% SDS, 4% 2-mercaptoethanol, 10% glycerol) by boiling 10 min
and resolved by SDS-PAGE.
Western Blotting--
After SDS-PAGE, proteins were transferred
to Immobilon membranes. After transfer, membranes were blocked using
5% nonfat dried milk in PBS, pH 7.2, and incubated overnight at
4 °C with the anti-FAK-Tyr(P)-397 Ab (0.1 µg/ml) or
anti-FAK-Tyr(P)-577 Ab (0.1 µg/ml), as indicated. The membranes were
washed three times with PBS, 0.1% Tween 20 and then incubated with
secondary antibodies (horseradish peroxidase-conjugated donkey
antibodies to rabbit, NA 934) (1:5000) for 1 h at 22 °C. After
washing three times with PBS, 0.1% Tween 20, the immunoreactive bands
were visualized using ECL detection reagents. Autoradiograms were
scanned using the GS-710 Calibrated Imaging Densitometer (Bio-Rad), and
the labeled bands were quantified using the Quantity One software
program (Bio-Rad).
Materials--
Bombesin, endothelin, bradykinin, vasopressin,
LPA, bovine fibronectin, and poly-L-lysine were obtained
from Sigma. Horseradish peroxidase-conjugated donkey antibodies to
rabbit, NA 934, and ECL reagents were from Amersham Pharmacia Biotech.
PP-2 and PP-3 were obtained from Calbiochem-Novabiochem. FAK polyclonal
Ab C-20 was from Santa Cruz Biotechnology (Santa Cruz, CA). The
phosphospecific polyclonal Abs to Tyr-397 and Tyr-577 of FAK were
obtained from BIOSOURCE International (Camarillo,
CA). All other reagents used were of the purest grade available.
The Role of Src Family Kinase Activity in FAK Phosphorylation at
Tyr-397 in Response to Fibronectin--
In order to examine the role
of Src family kinases in integrin-mediated phosphorylation of FAK at
Tyr-397, we determined the effect of the pyrazolopyrimidine PP-2, a
selective inhibitor of Src family kinase members (56, 57), on the
phosphorylation FAK at Tyr-397 induced by fibronectin. Previously, we
demonstrated that this compound, at concentrations that completely
inhibited the catalytic activity of Src family kinases, did not
interfere with FAK autophosphorylation activity (54, 57). Cultures of Swiss 3T3 cells placed in suspension were treated in the absence or in
the presence of increasing concentrations of PP-2 (0.1-10 µM) and then plated onto dishes coated with either
fibronectin or poly-L-lysine for 30 min. Cell lysates were
immunoprecipitated with anti-FAK Ab, and the immune complexes were
analyzed by SDS-PAGE followed by Western blotting with a
phosphospecific antibody directed against the autophosphorylation site
of FAK (anti-FAK-Tyr(P)-397 Ab). As shown in Fig.
1A, treatment of cells with
PP-2 strikingly inhibited the phosphorylation of FAK at Tyr-397 in a
concentration-dependent fashion. Maximal inhibition was
obtained at 10 µM. In contrast, treatment with 10 µM PP-3, a structurally related but inactive analog of
PP-2, did not interfere with integrin-mediated FAK phosphorylation at
Tyr-397 (Fig. 1B).
FAK autophosphorylation at Tyr-397 creates a high affinity binding site
for the SH2 domain of Src, and Src associated with FAK is thought to
phosphorylate FAK at additional sites including Tyr-576 and Tyr-577,
which are located in the kinase catalytic domain of FAK. Treatment with
10 µM PP-2 completely blocked the phosphorylation of FAK
Tyr-577 induced by fibronectin (inset, Fig. 1B,
right). These results suggest that Src family kinases are required
for fibronectin-induced FAK phosphorylation at both activation loop and
autophosphorylation sites.
FAK Phosphorylation at Tyr-397 in Response to Fibronectin Is
Greatly Diminished in Cells Deficient in Src, Yes, and Fyn (SYF
Cells)--
Recently, Klinghoffer et al. (58) reported that
integrin-induced tyrosine phosphorylation of FAK was markedly reduced
in SYF cells, but the phosphorylation of specific sites was not
analyzed. In order to substantiate that Src family kinases are required for FAK Tyr-397 phosphorylation induced by fibronectin, as suggested by
the pharmacological results shown in Fig. 1A, we examined
integrin-induced FAK Tyr-397 phosphorylation in SYF cells and in cells
deficient in Yes and Fyn but not in Src (YF cells). As illustrated in
Fig. 2, plating YF cells (which express
c-Src) onto fibronectin induced a marked increase in FAK Tyr-397
phosphorylation as compared with either parallel cultures plated on
poly-L-lysine or to SYF cells plated on fibronectin. The
increase in FAK Tyr-397 phosphorylation induced by fibronectin in YF
cells was virtually abrogated by treatment with 10 µM
PP-2. In contrast, plating SYF cells on fibronectin induced only a
small (but measurable) increase in FAK Tyr-397 phosphorylation, as
compared with SYF cells plated on poly-lysine. Interestingly, the small
increase in fibronectin-induced FAK Tyr-397 phosphorylation in the
absence of Src, Yes, and Fyn was not affected by treatment with 10 µM PP-2. Taken together, the results presented in Figs. 1
and 2 indicate that Src family kinases are required for maximal
integrin-induced FAK autophosphorylation and substantiate the
specificity of PP-2 as an Src family inhibitor since this agent did not
exert any inhibitory effect on FAK Tyr-397 phosphorylation in cells
lacking Src, Yes, and Fyn.
Bombesin Stimulates FAK Phosphorylation at Tyr-397 through an
Src-independent Pathway--
Although an increase in the
phosphorylation of FAK at Tyr-397 in response to an integrin stimulus
has been well documented (45, 59), the effect of GPCR agonists on the
phosphorylation of this residue has not been demonstrated. In order to
determine whether bombesin induces FAK phosphorylation at Tyr-397 in
Swiss 3T3 cells, quiescent cultures of these cells were treated with 10 nM bombesin for various times and lysed. The extracts were immunoprecipitated with a polyclonal anti-FAK Ab, which recognizes the
C-terminal sequence of FAK, and the immune complexes were analyzed by
SDS-PAGE followed by Western blotting using a site-specific antibody
(anti-FAK-Tyr(P)-397) that recognizes the phosphorylated state of FAK
at Tyr-397.
As shown in Fig. 3A
(upper panel), bombesin stimulation of Swiss 3T3 cells
induced a rapid and marked increase in the phosphorylation of FAK at
Tyr-397. Densitometric scanning showed that the phosphorylation of this
residue reached a maximum 2 min after the addition of bombesin to
intact cells. Immunoblotting with anti-FAK antibody of FAK
immunoprecipitates verified that similar amounts of FAK were recovered
after different times of bombesin treatment (Fig. 3A, lower
panel). These results demonstrate that bombesin induces FAK
phosphorylation at Tyr-397, the major autophosphorylation site of FAK
that plays a critical role in FAK signaling.
To determine whether Src family kinase activity is required for FAK
phosphorylation at Tyr-397 induced by bombesin, cultures of Swiss 3T3
cells were treated in the absence or in the presence of increasing
concentrations of PP-2 and then stimulated with this agonist. As shown
in Fig. 3B, exposure to PP-2 did not reduce the level of
phosphorylation at Tyr-397 induced by bombesin stimulation, even at a
concentration (10 µM) that prevented integrin-induced phosphorylation of FAK at Tyr-397 and Tyr-577 (Fig. 1). In fact, the
level of bombesin-induced FAK Tyr-397 phosphorylation in cells treated
with PP-2 was indistinguishable from that obtained in control
(untreated) cultures or in cultures treated with the inactive analog
PP-3 (Fig. 3B, inset). We verified that similar amounts of
FAK were recovered after treatment with or without bombesin and with or
without PP-2 and PP-3 (Fig. 3B, lower panels).
Western blotting of FAK immunoprecipitates with the site-specific
antibody anti-FAK-Tyr(P)-577 revealed that bombesin stimulation also
induced phosphorylation of FAK at Tyr-577 in Swiss 3T3 cells (Fig.
3C). Treatment of Swiss 3T3 cells with increasing
concentrations of PP-2 prevented phosphorylation of FAK at Tyr-577
induced by bombesin in a concentration-dependent manner.
Half-maximal and maximal inhibitory effects were achieved at 2 and 10 µM, respectively. We verified that similar amounts of FAK
were recovered after treatment with increasing concentrations of PP-2
(Fig. 3C). Thus, PP-2, at a concentration that completely
blocked FAK activation loop phosphorylation (Fig. 3C), did
not prevent FAK autophosphorylation at Tyr-397 stimulated by bombesin
(Fig. 3B).
Differential Contribution of Src Family Kinase Activity to
FAK Phosphorylation at Tyr-397 in Response to Bombesin and
Fibronectin--
The results presented in Figs. 1-3 indicate that Src
family kinases are required for fibronectin but not for bombesin
receptor stimulation of FAK phosphorylation at Tyr-397. In order to
substantiate further the existence of Src-dependent and
-independent pathways leading to phosphorylation of FAK at Tyr-397, we
examined the effect of PP-2 on the phosphorylation of FAK at Tyr-397
in Swiss 3T3 cells stimulated with either bombesin or fibronectin
for various lengths of time.
As shown in Fig. 4A, exposure
to PP-2 markedly attenuated the phosphorylation of FAK at Tyr-397 in
cells plated onto fibronectin-coated dishes at all the times examined,
supporting the participation of Src in FAK autophosphorylation induced
by adhesion-dependent signals. In contrast, treatment with
PP-2 did not affect the phosphorylation of FAK at Tyr-397 in response
to bombesin stimulation for various times (Fig. 4B). We
verified that similar amounts of FAK were recovered after different
times of exposure to fibronectin, bombesin, or PP-2 (Fig. 4,
lower panel). These results substantiate further the notion
that Src family kinases are required for fibronectin but not for
bombesin receptor stimulation of FAK phosphorylation at Tyr-397.
Role of Src in FAK Phosphorylation at Tyr-397 and Tyr-577 in
Response to Vasopressin, Bradykinin, LPA, and Endothelin--
The
preceding results with bombesin prompted us to determine the role of
Src family kinase activity in the phosphorylation of FAK at Tyr-397 and
Tyr-577 induced by activation of other endogenously expressed GPCRs in
Swiss 3T3 cells. Cultures of these cells were treated in the absence or
in the presence of 10 µM PP-2 and then stimulated with 20 nM vasopressin, 20 nM bradykinin, 2 µM LPA, or 20 nM endothelin for 10 min and
lysed. The lysates were immunoprecipitated with anti-FAK Ab, and the
immune complexes were analyzed by SDS-PAGE followed by Western blotting
with either anti-FAK-Tyr(P)-397 or anti-FAK-Tyr(P)-577 Abs. As shown in
Fig. 5, cell stimulation with these
agonists induced a marked increased in the phosphorylation of FAK at
both Tyr-397 and Tyr-577. Treatment with 10 µM PP-2 completely prevented the increase in the phosphorylation of FAK at
Tyr-577 (Fig. 5A) but did not affect the phosphorylation of FAK at Tyr-397 in response to vasopressin, bradykinin, LPA, or endothelin (Fig. 5B). We verified that similar amounts of
FAK were recovered after treatment with these GPCR agonists with or without PP-2.
Since previous results indicated that SYF and YF cells express
receptors for LPA (58), we also examined whether this agonist induces
FAK Tyr-397 phosphorylation in these cells. As illustrated in Fig.
6, FAK Tyr-397 phosphorylation was
enhanced by LPA stimulation in both SYF cells and YF cells to the same
degree. In addition, treatment with PP-2 did not exert any detectable
inhibitory effect on LPA-induced FAK Tyr-397 phosphorylation in either
SYF or YF cells. These results indicate that activation loop
phosphorylation by Src family kinases is not required for the tyrosine
phosphorylation of FAK at Tyr-397 triggered by a variety of GPCR
agonists.
Concluding Remarks--
A rapid increase in the overall tyrosine
phosphorylation of the non-receptor tyrosine kinase FAK has been
extensively documented as an early event in the action of multiple
extracellular stimuli that modulates cell growth, motility,
differentiation, and apoptosis in a variety of cell types. It is
increasingly recognized that the function of FAK in signal transduction
depends on the phosphorylation of specific residues in this enzyme. In
particular, autophosphorylation of FAK at Tyr-397 has emerged as a
crucial event in FAK-mediated signal transduction. The phosphorylation
of FAK at Tyr-397 triggers the formation of molecular complexes with
other signaling proteins including Src family kinases (5), the p85
regulatory subunit phosphatidylinositol 3-kinase (60), phospholipase
C
The best characterized function of FAK autophosphorylation at Tyr-397
is the creation of a high affinity binding site for the SH2 domain of
Src family members. Given that competition for the SH2 and/or SH3
domains of Src by high affinity allosteric ligands promotes enzymatic
activation of this kinase (64, 65), the association of Src with FAK
should lead to the formation of a molecular complex in which Src
kinases are activated. A model has recently been proposed that
envisages reciprocal catalytic activation of FAK and Src family kinases
in response to adhesion-dependent signals (32). In the
framework of this model, Src activated by binding to FAK Tyr-397
phosphorylates FAK at Tyr-576 and -577 which are located in the
activation loop of the kinase catalytic domain of FAK (42, 53) and
thereby promotes maximal FAK catalytic activity. Because FAK mutants in
which Tyr-576 and Tyr-577 were substituted by phenylalanines show
substantially reduced FAK Tyr-397 phosphorylation, relative to that of
FAK, it has been proposed that activation loop phosphorylation FAK by
Src family kinases stimulates intermolecular phosphorylation of FAK at
Tyr-397, thereby leading to signal amplification at sites of
integrin-mediated cell adhesion (32, 45). The results presented here
demonstrate that treatment with the selective Src family kinase
inhibitor PP-2, at concentrations that suppress activation loop
phosphorylation, markedly attenuates FAK Tyr-397 phosphorylation in
Swiss 3T3 cells plated onto fibronectin-coated dishes. Moreover, FAK
phosphorylation at Tyr-397 in response to integrin engagement is
drastically reduced in triple mutant cells lacking Src, Yes, and Fyn.
Taken together, these findings are consistent with a model in which
integrin-induced FAK autophosphorylation at Tyr-397 requires Src family
kinase function.
Despite its importance, little is known about the effect of GPCR
agonists on the phosphorylation of FAK at specific tyrosines, especially Tyr-397, the major site of autophosphorylation. Our results
demonstrate that stimulation of intact Swiss 3T3 cells with bombesin
induces a rapid increase in FAK phosphorylation at Tyr-397. Stimulation
with other GPCR agonists including endothelin, vasopressin, bradykinin,
and LPA also stimulates the phosphorylation of FAK at specific
residues, the critical Tyr-397 as well as Tyr-577, as revealed using
antibodies that specifically recognized these residues in their
phosphorylated form. Our findings show that activation of GPCRs induces
rapid multisite tyrosine phosphorylation of endogenously expressed FAK
in attached Swiss 3T3 cells, i.e. without overexpressing FAK
or Src and without subjecting the cells to detachment and subsequent replating.
In view of the results obtained with integrin-stimulated cells, we also
examined whether Src family kinases are required for FAK
phosphorylation at Tyr-397 in response to bombesin or other GPCR
agonists. Our results demonstrate that treatment with PP-2, a selective
inhibitor of Src family kinases, completely prevents the increase in
FAK Tyr-577 phosphorylation induced by bombesin, in line with the
notion that Src family kinases mediate phosphorylation of the
activation loop of FAK. However, in striking contrast to the results
obtained with integrin-stimulated cells, treatment with PP-2, at a
concentration that abolished bombesin-induced FAK phosphorylation at
Tyr-577, did not affect the phosphorylation of Tyr-397 in response to
this agonist. Similarly, we found that PP-2 abrogated Tyr-577
phosphorylation in response to bradykinin, endothelin, vasopressin, and
LPA but did not prevent the increase in FAK phosphorylation at Tyr-397
induced by these GPCR agonists. In agreement with these results, FAK
Tyr-397 phosphorylation was enhanced by LPA stimulation in both SYF
cells and YF cells. Thus, GPCR-mediated FAK phosphorylation at Tyr-397,
unlike that promoted by integrin binding to fibronectin, is independent
of Src-mediated activation loop phosphorylation.
In conclusion, the results presented here indicate that phosphorylation
of FAK at Tyr-397 induced by bombesin and other GPCRs is
Src-independent, whereas the phosphorylation of this residue promoted
via integrin-mediated cell adhesion requires Src family kinase
function. Our results identify, for first time, the existence of
distinct pathways leading to FAK phosphorylation at Tyr-397 stimulated
by GPCR agonists and adhesion-dependent signals in the same cell.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Role of Src activity in FAK phosphorylation
at Tyr-397 in response to fibronectin. A, Swiss 3T3
cells held in suspension were treated for 15 min in the absence
(0) or in the presence of increasing concentrations of PP-2,
as indicated, and then plated on either
poly-L-lysine-coated dishes (P-LYS) or
fibronectin-coated dishes (FIB) for 30 min and subsequently
lysed. FAK-Tyr-397 phosphorylation was analyzed by immunoprecipitation
(IP) using anti-FAK Ab C-20 followed by Western blotting
with anti-FAK-Tyr(P)-397. B, Swiss 3T3 cells held in
suspension were treated for 15 min in the absence ( ) or in the
presence (+) of 10 µM PP-2 or 10 µM PP3, as
indicated. Cells were then plated on poly-L-lysine-coated
dishes (P-LYS) or fibronectin-coated dishes (FIB)
for 30 min, as indicated. The cells were then lysed, and the extracts
were incubated with anti-FAK antibody C-20. The immunoprecipitates were
analyzed by Western blotting with anti-FAK-Tyr(P)-397 Ab.
Inset, Swiss 3T3 cells held in suspension were treated as
described in B. The cells were lysed, and the extracts were
incubated with anti-FAK Ab C-20, and the immunoprecipitates were
analyzed by Western blotting with anti-FAK-Tyr(P)-577 Ab. In all cases,
the membranes were analyzed further by Western blotting with anti-FAK
Ab. The positions of FAK-Tyr(P)-397, FAK-Tyr(P)-577, and FAK are
indicated by arrows. The autoradiograms shown re
representative of at least three independent experiments.
Quantification of FAK phosphorylation at Tyr-397 and Tyr-577 was
performed by densitometric scanning of the bands. Values shown are the
mean ± S.E. of at least three independent experiments and are
expressed as the percentage of the maximal increase in FAK tyrosine
phosphorylation value above control (unstimulated) values.
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Fig. 2.
FAK phosphorylation at Tyr-397 in response to
fibronectin is greatly diminished in cells deficient in SYF cells.
SYF and YF cells held in suspension were treated for 15 min in the
absence ( ) or in the presence (+) of 10 µM PP2. Cells
were then plated on either poly-L-lysine- or
fibronectin-coated dishes for 30 min, as indicated. The cells were then
lysed, and the extracts were incubated with anti-FAK Ab C-20 followed
by Western blotting with anti-FAK-Tyr(P)-397 Ab. The membranes were
analyzed further by Western blotting with anti-FAK Ab. The positions of
FAK-Tyr(P)-397 and FAK are indicated by arrows. The
autoradiograms shown are representative of at least three
independent experiments. Quantification of FAK phosphorylation at
Tyr-397 was performed by densitometric scanning of the bands. Values
shown are the mean ± S.E. of at least three independent
experiments and are expressed as the percentage of the maximal increase
in FAK tyrosine phosphorylation value above control (unstimulated)
values.
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Fig. 3.
Bombesin stimulates FAK phosphorylation at
Tyr-397 through an Src-independent pathway in Swiss 3T3 cells.
A, confluent and quiescent Swiss 3T3 cells were stimulated
with 10 nM bombesin for various times at 37 °C, as
indicated and subsequently lysed. FAK phosphorylation at Tyr-397 was
analyzed by immunoprecipitation using anti-FAK antibody C-20 followed
by Western blotting with anti-FAK-Tyr(P)-397 Ab. B,
confluent and quiescent cells were treated for 15 min in the absence
(0) or in the presence of increasing concentrations of PP-2,
as indicated, and then stimulated without or with 10 nM
bombesin (BOM, closed symbols) for a further 10 min and subsequently lysed. FAK phosphorylation at Tyr-397 was analyzed
by immunoprecipitation using anti-FAK antibody C-20 followed by Western
blotting with anti-FAK-Tyr(P)-397. Inset, confluent and
quiescent Swiss 3T3 cells were treated for 15 min either in the absence
( ) or in the presence (+) of 10 µM of PP-2 or 10 µM of PP3. Cells were then incubated for a further 10 min
either in the absence (
) or in the presence (+) of 10 nM
of bombesin (BOM), as indicated. The cells were then lysed
and the extracts were incubated with anti-FAK antibody C-20, followed
by Western blotting with anti-FAK-Tyr(P)-397 Ab. C,
confluent and quiescent Swiss 3T3 cells were treated for 15 min in the
absence or in the presence of increasing concentrations of PP-2 as
indicated, and then stimulated without or with 10 nM of
bombesin for a further 10 min and subsequently lysed. FAK
phosphorylation at Tyr-577 was analyzed by immunoprecipitation using
anti-FAK antibody C-20 followed by Western blotting with
anti-FAK-Tyr(P)-577 Ab. In all cases, the membranes were analyzed
further by Western blotting using anti-FAK Ab. The positions of
FAK-Tyr(P)-397, FAK-Tyr(P)-577, and FAK are indicated by
arrows. The autoradiograms shown are
representative of at least three independent experiments.
Quantification of FAK phosphorylation at Tyr-397 and Tyr-577 was
performed by densitometric scanning of the bands. Values shown are the
mean ± S.E. of at least three independent experiments and are
expressed as the percentage of the maximal increase in FAK tyrosine
phosphorylation value above control (unstimulated) values.
View larger version (39K):
[in a new window]
Fig. 4.
Kinetics of FAK phosphorylation at Tyr-397 in
response to bombesin or fibronectin in Swiss 3T3 cells.
A, Swiss 3T3 cells held in suspension were treated for 15 min in the absence ( ) or in the presence (+) of 10 µM
PP-2 and then plated on poly-L-lysine-coated dishes
(P-LYS) for 40 min or on fibronectin-coated dishes
(FIB) for various times, as indicated, and subsequently
lysed. FAK phosphorylation at Tyr-397 was analyzed by
immunoprecipitation (IP) using anti-FAK antibody C-20
followed by Western blotting with anti-FAK-Tyr(P)-397. B,
confluent and quiescent Swiss 3T3 cells were treated for 15 min in the
absence (
) or in the presence (+) of 10 µM PP-2 and
then stimulated with 10 nM bombesin (BOM) for
various times as indicated and subsequently lysed. FAK phosphorylation
at Tyr-397 was analyzed by immunoprecipitation using anti-FAK antibody
C-20 followed by Western blotting with anti-FAK-Tyr(P)-397. In all
cases, the membranes were analyzed further by Western blotting using
anti-FAK Ab. The positions of FAK-Tyr(P)-397 and FAK are indicated by
arrows. The autoradiograms shown are
representative of at least three independent experiments.
Quantification of FAK-Tyr(P)-397 was performed by scanning densitometry
of the bands. Values shown are the mean ± S.E. of at least three
independent experiments and are expressed as the percentage of the
maximal increase in FAK-Tyr(P)-397 value above control (unstimulated or
plated on poly-L-lysine) values.
View larger version (37K):
[in a new window]
Fig. 5.
Differential role of Src family kinase
activity in FAK phosphorylation at Tyr-397 and Tyr-577 induced by GPCR
agonists in Swiss 3T3 cells. A, confluent and quiescent
Swiss 3T3 cells were treated for 15 min either in the absence ( ) or
in the presence (+) of 10 µM PP-2. Cells were then
incubated for a further 10 min in the absence or in the presence of 20 nM vasopressin (VAS), 20 nM
bradykinin (BK), 2 µM LPA, or 20 nM endothelin (END), as indicated. The cells
were then lysed, and the extracts were incubated with anti-FAK antibody
C-20, followed by Western blotting with anti-FAK-Tyr(P)-577 Ab.
B, confluent and quiescent Swiss 3T3 cells were treated as
described in A. The cells were then lysed, and the extracts
were incubated with anti-FAK antibody C-20, followed by Western
blotting with anti-FAK-Tyr(P)-397 Ab. In all cases, the membranes were
analyzed further by Western blotting with anti-FAK Ab. The positions of
FAK-Tyr(P)-397, FAK-Tyr(P)-577, and FAK are indicated by
arrows. The autoradiograms shown are
representative of at least three independent experiments.
Quantification of FAK-Tyr(P)-397 and FAK-Tyr(P)-577 was performed by
scanning densitometry of the bands. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the
percentage of the maximal increase in FAK-Tyr(P)-397 or FAK-Tyr(P)-577
value above control (unstimulated or plated on
poly-L-lysine) values.
View larger version (42K):
[in a new window]
Fig. 6.
LPA induces FAK phosphorylation at Tyr-397 in
SYF and YF cells. SYF and YF cells were treated for 15 min in the
absence ( ) or in the presence (+) of 10 µM PP2. Cells
were then incubated for a further 10 min in the absence or in the
presence of 20 µM LPA, as indicated. The cells were then
lysed, and the extracts were incubated with anti-FAK Ab C-20 followed
by Western blotting with anti-FAK-Tyr(P)-397 Ab. The membranes were
analyzed further by Western blotting with anti-FAK Ab. The positions of
FAK-Tyr(P)-397 and FAK are indicated by arrows. The
autoradiograms shown are representative of at least three
independent experiments. Quantification of FAK phosphorylation at
Tyr-397 was carried out by densitometric scanning of the bands. Values
shown are the mean ± S.E. of at least three independent
experiments and are expressed as the percentage of the maximal increase
in FAK tyrosine phosphorylation value above control (unstimulated)
values.
-1 (61), the adapter proteins Grb-7 (62) and Shc (53), and the
tumor suppressor PTEN (63). These findings suggest that FAK
autophosphorylation promotes the activation of multiple effector pathways.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK 56930, DK 55003, and DK 17294.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.
Ronald S. Hirshberg Professor of Translational Pancreatic Cancer
Research. To whom correspondence should be addressed: Dept. of
Medicine, UCLA School of Medicine, Warren Hall Rm. 11-124, 900 Veteran
Ave., Los Angeles, CA 90095-178622. Tel.: 310-794-6610; Fax:
310-267-2399; E-mail: erozengurt@mednet.ucla.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100984200
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ABBREVIATIONS |
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The abbreviations used are: FAK, focal adhesion kinase; DMEM, Dulbecco's modified Eagle's medium; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PP-2, pyrazolopyrimidine 2; PP-3, pyrazolopyrimidine 3; SH2 and SH3, Src homology domain 2 and 3, respectively.
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