From the Department of Pathology, Yale University
School of Medicine, New Haven, Connecticut 06520-8023, the
§ Cancer Biology Laboratories, Department of Molecular
Medicine, College of Veterinary Medicine, Cornell University, Ithaca,
New York 14853, and the
Department of Pediatrics, Ohio State
University School of Medicine, Columbus, Ohio 43205
Received for publication, October 4, 2000, and in revised form, February 16, 2001
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ABSTRACT |
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Recent studies suggest that focal adhesion kinase
(FAK) is important for cell migration. We now suggest a mechanism by
which FAK activates the signal transducer and activator of
transcription (STAT) pathway, regulating cell adhesion and migration.
In particular, we observe that FAK is capable of activating Stat1, but
not Stat3. Co-immunoprecipitation and in vitro binding
assays demonstrate that Stat1 is transiently and directly associated
with FAK during cell adhesion, and Stat1 is activated in this process.
FAK with a C-terminal deletion (FAK Extracellular matrix
(ECM)1 proteins and integrins
play essential roles in the regulation of cell adhesion and migration
(1, 2). Integrins are heterodimeric transmembrane receptors (3-5). Similar to the signal transduction induced by cytokine-receptor binding, interaction of integrins with the ECM proteins can induce tyrosine phosphorylation of many intracellular proteins.
Integrin-induced tyrosine phosphorylation is critical for cell adhesion
and migration since cell spreading and migration are diminished by
tyrosine phosphorylation inhibitors (6). Focal adhesion kinase (FAK) becomes tyrosine-phosphorylated during integrin-mediated cell adhesion
and is believed to play important roles in integrin signal transduction
(7-9). Similar to receptor tyrosine kinases, FAK interacts with a pool
of intracellular signaling proteins, including c-Src,
phosphatidylinositol 3-kinase, Grb2, and p130CAS (10-14).
Recent studies have suggested that FAK is involved in cell survival
(15, 16). Furthermore, FAK-deficient mice demonstrate an early
embryonic lethal phenotype, suggesting FAK is essential for development
(17, 18). Interestingly, this developmental defect is believed to be
caused by the impairment of cell migration and the enhancement of cell
adhesion, as suggested by studies of FAK The signal transducer and activator of transcription (STAT) pathway is
a general route of signal transduction from cell surface receptors to
gene regulation (22-24). Many cytokines, such as interferons, activate
STAT proteins to induce gene (25-29) expression. In addition to JAK
family kinases, which mediate signals from cytokine receptors, many
kinds of protein-tyrosine kinases (PTKs) also activate STAT proteins
under a variety of physiological or pathological conditions. In
particular, EGF receptor kinase can directly activate STAT proteins
(30-34). Interestingly, STAT activation by EGF results in inhibition
of cell proliferation and apoptosis, which contrasts with the well
documented EGF function of stimulation in cell growth, suggesting the
STAT signaling pathway can negatively control cell growth (35, 36).
Furthermore, fibroblast growth factor receptor kinase, and Src
family kinases may also activate STAT proteins (37-41). Therefore,
these results suggest that STAT proteins are common substrates of a
number of PTKs.
Since cell adhesion and cell migration involve PTK activation, it is
reasonable to examine if STAT activity plays a role in these processes.
Here we demonstrate that Stat1 activity is induced by the integrin
signaling pathway. Intriguingly, FAK can interact with and activate
Stat1 during cell attachment. Furthermore, we show that Stat1
activation reduces cell adhesion and stimulates cell migration.
Cell Lines--
293T and A431 cells were cultured in DMEM with
10% or 5% fetal bovine serum (FBS, Life Technologies, Inc.). Stat1
Plasmids and Antibodies--
Expression vectors encoding
HA-tagged wild type Stat1 and Stat1-SH2RQ mutant were described
previously (30). Stat1-CYF has a single amino acid change on position
701 from Tyr to Phe. Plasmid pCX-Stat1 was constructed by inserting
non-tagged Stat1 into StuI site of pCX vector that has a
cytomegalovirus promoter. Expression plasmids encoding HA-tagged wild
type FAK, kinase-defective mutant FAK (KD), Y397F mutant, and
C-terminal 14-amino acid deletion mutant FAK
Rabbit polyclonal anti-Stat1 antibody (C-24) and monoclonal anti-Stat1
antibody (C-111) (Santa Cruz Biotechnology) were used for
immunoprecipitation, Western blotting, supershift assay, and immunofluorescence staining. A monoclonal anti-FAK antibody
(Transduction Laboratories and PharMingen) was used for
immunofluorescence staining. Another rabbit polyclonal anti-FAK
antibody (C-20) (Santa Cruz Biotechnology) was used for
immunoprecipitation, Western blotting, and immunofluorescence staining.
A rabbit polyclonal anti-phospho-Stat1 antibody (New England Biolabs)
was used for immunoblotting; mouse anti-HA antibody (12CA5) and a
rabbit polyclonal anti-FAK antibody (described previously (see Refs. 42
and 43)) were used for immunoprecipitation, Western blotting, and
immunofluorescence staining. Polyclonal anti-Stat2 (45) and
anti-NF- Cell Extracts and electrophoresis mobility shift assay
(EMSA)--
Tissue culture plates were coated overnight with 10 µg/ml human plasma fibronectin (Life Technologies, Inc.) in 1× PBS,
washed twice with PBS, and then incubated with 2 mg/ml heat-inactivated (1 h at 70 °C) BSA in 1× PBS for 2 h at 37 °C. Cells were
harvested by brief trypsinization and washed twice with PBS containing
0.5 mg/ml soybean trypsin inhibitor (Sigma). The cells were resuspended in DMEM without serum and added to coated plates (100 mm) at 8 × 106. After various times of incubation at 37 °C, cells
were washed twice with cold PBS and lysed in whole cell-extract (WCE)
buffer (15 mM Hepes, pH 7.9, 250 mM NaCl, 0.5%
Nonidet P-40, 10% glycerol, and 1 mM EDTA) containing a
mixture of protease and phosphatase inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml pepstatin, 1 mM vanadate, 10 mM NaF, and
1 mM dithiothreitol), left on ice for 45 min, and
centrifuged for 10 min at 4 °C. WCE containing the same amount of
total proteins were subjected to EMSA with 10 fmol of
32P-labeled high affinity SIE probe
(5'-AGCTTCATTTCCCGTAAATCCCTAAAGCT-3') as described previously (35).
X-Gal Analysis--
Forty-eight hours after transfection, cells
were fixed by 1% glutaraldehyde (in PBS) in 37 °C for 15 min. Cells
were stained with 0.2% X-gal (Amersham Pharmacia Biotech) (in 10 mM Na3PO4, pH 7.0, 150 mM NaCl, 1 mM MgCl2, 3.3 mM K4Fe(CN)6, 3.3 mM
K3Fe(CN)6) for 1 h, washed with 70%
ethanol, then covered with PBS. The number of blue-stained and
transfected cells was counted in three different fields under
microscopy (magnification, ×100). All experiments were repeated at
least three times.
Immunoprecipitation and Western Blot Analysis--
For
immunoprecipitation, cells plated on fibronectin were lysed with WCE
buffer. Four hundred micrograms of proteins were incubated with
anti-HA, anti-FAK (C-20), or anti-Stat1 (C-24) antibodies at 4 °C
overnight. Twenty-five microliters of protein G- or protein A-agarose
was added for 3 h of additional incubation at 4 °C. After
washing the precipitates three times with WCE buffer with protease and
phosphatase inhibitors, samples were electrophoresed in 6% or 8%
SDS-polyacrylamide gels. Following electrophoresis, proteins were
transferred to polyvinylidene difluoride membrane and blotted with
anti-HA, C-24 anti-Stat1, C-20 anti-FAK, or anti-phospho-Stat1 antibodies. For immunoblot, 10 µg of proteins from each sample were analyzed.
In Vitro Translation and Kinase Assay--
The cDNAs of
Stat1 (pSG-Stat1) and FAK (pBluescript-FAK) were in vitro
transcribed and translated using the TNT Coupled Reticulocyte Lysate
systems or TNT Coupled Wheat Germ Lysate systems (Promega) in the
presence of Redivue L-[35S]methionine
(>1,000 Ci/mmol at 10 mCi/ml; Amersham Pharmacia Biotech). Stat1
protein from in vitro translation reaction was mixed in a
kinase reaction buffer (10 mM PIPES, pH 7.0, 5 mM MnCl2, 1 mM NaCl, 0.1 mM dithiothreitol, and 10 µM ATP) (38) with
insect cell SF21 lysates with or without FAK expression. After a 20-min incubation period at 30 °C, the samples were immunoprecipitated with
an anti-Stat1 antibody (C-24) or non-related serum at 4 °C and
applied to SDS-polyacrylamide gel electrophoresis as described above.
Similar procedures were applied for other in vitro
translation reactions. Stat1 protein levels were detected by direct
autoradiography. Immunoprecipitation was performed in the buffer
containing 15 mM Hepes, pH 7.9, 400 mM NaCl,
0.5% Nonidet P-40, 10% glycerol, and 1 mM EDTA. C-24
anti-Stat1, C-20 anti-FAK, C-20 anti-NF- GST-Stat1 Construction, Purification, and in Vitro Binding
Assay--
The GST-Stat1 construct was generated by inserting a
full-length Stat1 cDNA fragment from pSG-Stat1 (released by
EcoRI) into pGEX-4T-3 (Amersham Pharmacia Biotech)
EcoRI site. The GST-Stat1 and GST proteins were produced and
purified according to the manufacturer's instructions. In
vitro translated FAK was pre-cleared by GST-conjugated glutathione-Sepharose 4B beads and then incubated with 10 µg of purified GST-Stat1, or GST-conjugated glutathione-Sepharose 4B beads in
the WCE buffer for 4 h at 4 °C. Following three washes with WCE
buffer, the precipitates were separated by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography.
Immunofluorescence Staining--
Cells were collected and
treated (as described under "Cell Extracts and EMSA") before
plating in six-well plates on fibronectin-coated coverslips. After
incubation for various periods of time, cells were washed with PBS
twice, and fixed with 4% paraformaldehyde for 30 min, permeabilized
with 0.5% Triton X-100 for 5 min, blocked with 3% BSA for 1 h,
and then processed for immunofluorescence by using different primary
antibodies. IFN- Cell Adhesion Assays--
Different concentrations of human
plasma fibronectin (FN) (Life Technologies, Inc.) were adsorbed onto
plastic 96-well tissue culture plates (100 µl/well). After using
0.5% BSA to block the plates in 37 °C, certain numbers of cells
depending the cell types (see figure legends), were plated and
incubated at 37 °C to indicating time points. The plates were washed
with PBS twice, and cells were fixed with 4% paraformaldehyde, pH 7.4, for 30 min in 4 °C. Cells were washed again, stained with 0.5%
crystal violet, and incubated overnight at room temperature. The extent
of cell adherence was determined by plate reader at
OD630.
Cell Migration Assays--
Migration assays in 24-well transwell
chambers (8-µm pore size, Costar) were carried out as described
previously (46). Briefly, 0.6 ml of serum-free medium with 10 µg/ml
fibronectin was added to the lower chamber, whereas cells were added
into the upper chamber in serum-free medium. After an indicated time of
incubation at 37 °C to allow cells to migrate, membranes were fixed
with 3% paraformaldehyde, pH 7.4, for 30 min in 4 °C and stained.
Cells that did not migrate were removed by wiping the upper side of membranes, and the migrated cells were counted under a microscope (magnification, ×100). Six different views were counted.
Expression of FAK Causes Activation of Stat1, but Not
Stat3--
We first determined whether FAK could induce Stat1
activation. In this experiment, 293T cells were transfected with
vectors expressing FAK and Stat1 separately, or in combination. Using a
gel EMSA, we observed Stat1 activation in cells transfected with FAK
(Fig. 1A, lane
4) but not in mock-transfected cells (Fig. 1A,
lane 2), suggesting that FAK activated endogenous
Stat1 in vivo in these cells. This result was confirmed by a
supershift assay (data not shown). Transfection of a HA-tagged Stat1
(30) also generated a weak Stat1 complex, which migrated slightly
slower than the endogenous Stat1 complex, possibly due to the added HA tag in the protein (Fig. 1A, lane 3).
More impressively, in cells co-transfected with FAK and Stat1, Stat1
was strongly activated (Fig. 1A, lane
5). This Stat1 complex was recognized by an anti-Stat1 antibody, forming a supershifted complex (SS) in the EMSA
(Fig. 1A, lane 6). To investigate
whether this Stat1 activation by FAK-cotransfection is specific for
Stat1, we further assessed possible activation of Stat3 by FAK under
the same conditions. In contrast to Stat1, Stat3 was weakly activated
when Stat3 and FAK were co-transfected (Fig. 1B). However,
Src could activate Stat1 as well as Stat3 at similar levels in
co-transfection studies. These results indicate that Stat1, not Stat3,
is a preferable target of FAK signaling. More importantly, endogenous
Stat1, not Stat3 or other STAT proteins, appeared to be activated in
cells transfected with FAK only, although there are several members of
endogenous STAT proteins in these cells (Fig. 1A,
lane 4). Jak1 kinase is required for tyrosine phosphorylation of Stat1 in response to many cytokines (34). However,
Jak1 was not necessary for the STAT activation by FAK, since Stat1 was
activated by FAK in a Jak1-deficient HeLa cell line, E2A4 (47),
similarly to results observed in 293T cells (Fig. 1B,
lane 10).
To visualize the effect of Stat1 activation on cell adhesion, these
cells were co-transfected with a vector that expressed Characterization of Functional Domains Involved in Stat1 Activation
by FAK--
We next determined the functional domains that were
involved in Stat1 activation by FAK. The C-terminal tyrosine
(Stat1-Y701) or the SH2 domain of Stat1 is essential for Stat1
activation in response to cytokines. We found that mutations of the
critical C-terminal tyrosine (Stat1-CYF) or of the SH2 domain
(Stat1-SH2RQ) also prevented Stat1 activation by FAK (Fig.
2A, compare lanes 6 and 8 with lane 4).
Almost equal levels of Stat1 protein in the various transfected cells
were verified by a Western blot analysis (Fig. 2A,
lower panel). Similarly, expression of wild type
Stat1 in these cells occasionally resulted in a low level of Stat1
activation (Fig. 2A, lane 2); however,
expression of either C-terminal tyrosine mutant (Stat1-CYF) or SH2
mutant (Stat1-SH2RQ) alone did not generate this Stat1 activation (Fig.
2A, lanes 5 and 7). A weak
Stat1 activity was observed in cells co-expressing either of these two
mutant Stat1 proteins with FAK. This might be attributed to endogenous
STAT activation by FAK, as was also observed in cells transfected by
FAK alone (Fig. 2A, lane 3). These
results indicate that the C-terminal tyrosine and the SH2 domain are
essential for Stat1 activation by FAK.
To verify whether the kinase activity of FAK is required for the STAT
activation, we used kinase-defective FAK with a K454R mutation at the
ATP binding site of the catalytic domain (48). This mutation of FAK
(KD) dramatically reduced Stat1 activation compared with that by the
wild type FAK (Fig. 2B, compare lanes 5 and 6 with lanes 3 and
4). The observed Stat1 activity (lane 6) was at the same level as that of Stat1 alone
(lane 2), demonstrating that this FAK mutant was
defective in Stat1 activation. Tyrosine 397 of FAK is a major
autophosphorylation site of the protein and is required for the binding
of Src family kinases. The Src-FAK association appeared to increase the
tyrosine phosphorylation of FAK and other substrates (13, 49). We found
that this Src association site of Tyr-397 was also involved in the STAT
activation, because a point mutation that replaced tyrosine 397 with
phenylalanine (Y397F) significantly decreased Stat1 activation (Fig.
2B, lanes 7 and 8). These
results suggest that the FAK is essential for Stat1 activation, and the
Src binding may also be involved in further activation of Stat1. In the
above experiments, the mutant FAK proteins were expressed at a level
comparable to that of wild type FAK, whereas endogenous FAK expression
in these cells was relatively low (see lower panel).
Stat1 Is Associated with FAK and Is Tyrosine-phosphorylated during
Cell Adhesion--
After cytokine stimulation, STAT proteins can bind
directly to phosphorylated receptor-tyrosine kinase complexes. Since
the SH2 domain of Stat1 is required for Stat1 activation by FAK, we examined possible interaction between Stat1 and FAK. To avoid potential
artifacts arising from protein overexpression, we performed the
experiment using untransfected cells. An antibody specific to FAK was
used to perform immunoprecipitation in untransfected 293T cells,
followed by an examination of the immunoprecipitated complexes using an
anti-Stat1 antibody (Fig. 3A).
In this assay, Stat1 was clearly co-immunoprecipitated with an anti-FAK
antibody (lanes 1-4, upper
panel). However, the migration of co-immunoprecipitated Stat1 was slower than that of the major Stat1 band (indicated as
Stat1), immunoprecipitated by an anti-Stat1 antibody
(lane 5). We suspected that these slower
migrating Stat1 bands were resulted from protein phosphorylation after
Stat1 protein had interacted with FAK. This notion was confirmed by a
protein blot with another antibody that specifically recognizes
tyrosine-phosphorylated, but not unphosphorylated, Stat1. Only these
slower migrating bands were recognized by this anti-phosphotyrosine
Stat1 (Stat1p) antibody (Fig. 3A, middle
panel), whereas the major unphosphorylated Stat1 band (shown
in lane 5, upper panel) was
not recognized by this antibody (lane 5,
middle panel). Intriguingly, it appeared that only tyrosine-phosphorylated Stat1 was co-immunoprecipitated with FAK,
and this FAK-Stat1 association transiently reached the maximal level
when cells were attached to fibronectin for a brief period (at 0.5-h
time point). With the progression of cell attachment, the amount of
Stat1 associated with FAK was significantly reduced. The levels of
precipitated FAK protein were almost the same (Fig. 3A,
lower panel). These results suggest that Stat1
can associate transiently with FAK at early time points of cell
adhesion when FAK is activated (7-9). A similar observation was also
made in A431 cells (Fig. 3B) and U3A-Stat1 cells (data not
shown) in which tyrosine phosphorylation of Stat1 was
co-immunoprecipitated with FAK.
Consistent with the transient association of Stat1 with FAK and Stat1
tyrosine phosphorylation, a specific Stat1 DNA binding activity was
observed only in an early time of the cell attachment and this Stat1
activity was reduced gradually as the cell attachment proceeded (Fig.
3C). The Stat1 activation induced by interferon-
To examine whether Stat1 and FAK are capable to interact directly or
indirectly through certain adapters, Stat1 and FAK were in
vitro transcribed and translated in reticulocyte lysate in the
presence of [35S]methionine. Stat1 was also expressed as
a GST fusion protein and FAK was expressed in an insect cell line,
SF21, using a baculovirus vector (see "Experimental Procedures" for
detail). As shown in Fig. 3D, in vitro translated
Stat1 was incubated with FAK from the SF21 cell lysates
(upper panel, lanes 1 and
3) or with cell lysate alone without FAK (lanes
2 and 4). Assuming that only phosphorylated Stat1
can bind to FAK, we used the buffer conditions that support kinase
activities (see "Experimental Procedures" for detail). Complexes
immunoprecipitated with an anti-Stat1 antibody (lanes 3 and 4) or non-related serum (lanes
1 and 2) were blotted with antibodies against FAK
and phospho-Stat1 (Fig. 3D, upper
panel, upper and middle
rows). FAK protein was clearly detected in the Stat1
immunoprecipitates (lane 3), but not in the
control samples without FAK (lane 4), or in the
complexes immunoprecipitated by non-related serum (lanes
1 and 2). Stat1 was tyrosine-phosphorylated as a
result of interacting with FAK (Fig. 3D, upper
panel, middle row, lane
3). The interaction between FAK and Stat1 was also confirmed in an experiment in which GST-Stat1 could precipitate FAK protein from
in vitro translation system (Fig. 3D,
middle panel, lane 2) or
FAK from SF21 lysate (data not shown). To further exclude the
possibility that there were additional adapters existing in the
reticulocyte lysate, Stat1 and FAK were translated in wheat germ lysate
and analyzed similarly (Fig. 3D, bottom
panel). In this system, Stat1 and FAK were also
co-immunoprecipitated (lanes 4 and 5),
and GST-Stat1 but not GST alone could pull down FAK (lanes
1 and 2). Taken together, these results strongly
indicate that FAK can directly interact with and phosphorylate Stat1.
Importantly, kinase activity of FAK was required for this direct
interaction because no FAK could be precipitated with Stat1 without
pre-incubation these two proteins in the kinase buffer (data not
shown). Equal Stat1 protein levels were shown by autoradiography of
35S (upper panel, lower
row). We further examined whether FAK-Stat1 interaction is
specific by co-translating FAK and STATs using reticulocyte lysate
(middle panel) and wheat germ lysate
(bottom panel). We found that no FAK and Stat2
could be co-immunoprecipitated by either an anti-Stat2 or an anti-FAK
antibody, respectively (middle panel
(lanes 8 and 9) or lower
panel (lanes 7 and 8), only
one band was detected, representing either Stat2 or FAK; these proteins
were visualized by autoradiography of 35S), although FAK
and Stat2 were translated together. In contrast, Stat1 and FAK were
co-immunoprecipitated under the same conditions (middle
panel (lanes 5 and 6) or
bottom panel (lanes 4 and
5), in which both Stat1 and FAK bands were detected in the
same lane). We further found that, as another negative control,
translated FAK and NF- A FAK Mutant with the C-terminal Deletion Diminishes Stat1
Activation and Blocks FAK-Stat1 Interaction--
The C-terminal domain
of the FAK protein is required for FAK localization at focal contacts,
and a
To examine this possibility, 293T cells were transfected with Stat1 and
FAK or Stat1 Phosphorylation Induced by Fibronectin Is Greatly Reduced in
FAK Increase of Cell Adhesion and Reduction of Cell Motility in
Stat1-deficient Cells--
Recently, it has been shown that
FAK-deficient cells have defects in cell migration. Thus, it is logical
to examine whether or not Stat1 can also affect integrin-mediated cell
adhesion and cell migration and if there is any functional connection
between FAK and Stat1.
To further investigate potential functions of STAT proteins in cell
adhesion and migration, embryonic fibroblasts isolated from Stat1 null
(
To confirm whether this Stat1-mediated negative effect on cell adhesion
was through the interaction with integrin, these fibroblasts were
plated on poly-L-lysine. All these fibroblasts, with or
without Stat1, showed no difference in cell adhesion on
poly-L-lysine (data not shown), indicating the difference
in cell adhesion caused by the Stat1 protein may be mediated through
integrin receptors.
To exclude the possibility that the above observation was limited to
embryonic fibroblasts, we next examined Stat1-deficient U3A and
U3A-Stat1 cells, in which Stat1 was stably transfected (35, 53). In
contrast to the parental Stat1-deficient U3A-pSG5 cells, the U3A-Stat1
clone showed significantly decreased cell adhesion at the coating
concentrations of 1.0 and 2.5 µg/ml FN (Fig. 6B). Longer
incubation time resulted in greater differences between control
U3A-pSG5 and U3A-Stat1. However, no significant difference was observed
when these cells were plated on poly-L-lysine (data not
shown). Three different U3A-Stat1 clones were examined, and similar
results were obtained (data not shown), indicating that the difference
of adhesion was not due to clonal variations. These results further
suggest that Stat1 has a role in inhibiting cell adhesion. No
significant differences of integrin receptor expression were found
between Stat1
Since cell adhesion is intrinsically linked to cell migration, we
further examined cell migration using a modified Boyden chamber assay.
Stat1 FAK and Stat1 Are Partially Co-localized in the Focal Adhesion
Sites--
The above biochemical analyses suggest that FAK and Stat1
interact with each other during cell adhesion. In intact cells, a
fraction of FAK protein is found in the focal contacts during cell
adhesion. Therefore, if FAK-Stat1 interaction is preserved in the cells
in vivo, it is possible that a fraction of Stat1 may be
co-localized with FAK in the focal adhesion sites.
We next examined cultured A431 cells for this possibility. Cells were
plated on the fibronectin-coated coverslips for 30 and 60 min. Stat1
and FAK were then visualized by immunofluorescence microscopy using
fluorescein isothiocyanate- or Texas Red-conjugated secondary
antibodies (Fig. 8A; see
"Experimental Procedures" for detail). Focal contacts were revealed
in both time points using anti-FAK (green) (a and
d, arrows). The majority of Stat1
(red) was found in the cytoplasm and partly in the nucleus
during cell adhesion (b and e). Confocal images
showed that, at the 30-min time point, many cells were not spread very
well on fibronectin, and FAK and Stat1 co-localization was observed on
the focal contacts (c, yellow color,
arrowheads). Interestingly, at the 60-min time point, the
co-localization was not observed in well spread cells, but still
remained in some cells that were less well spread (f, arrows and arrowheads). These results indicate
that Stat1 and FAK may interact with each other mainly during the early
stage of cell adhesion at the focal contacts, which was consistent with the co-immunoprecipitation results (Fig. 3, A and
B). We also observed the co-localization of FAK and Stat1 in
other cell lines (data not shown).
We then examined IFN- Integrin-mediated cell adhesion and migration play essential roles
in cell growth and development, and FAK is believed to play a critical
role in ECM-integrin initiated signaling processes (5, 54, 55).
Recently, it has been shown that cells deficient in FAK exhibit
enhanced formation of focal contacts and a defect in cell migration
(19-21), suggesting that FAK has an important role in
integrin-mediated cell migration. However, the molecular mechanism by
which FAK exerts its effect on cell migration is not completely understood.
In this report we demonstrate that Stat1-deficient cells, like
FAK-deficient cells, have enhanced cell adhesion (Fig. 6) and form
stronger focal contacts when attached to ECM (Fig. 8 and data not
shown). Moreover, Stat1 deficiency also results in slower cell
migration (Fig. 7). These observations raise the possibility that there
is a molecular link between integrin, FAK and Stat1 function. Our
biochemical studies have shown that integrin engagement can indeed
activate Stat1 (Fig. 3C). More intriguingly, we have demonstrated that Stat1 directly interacts with FAK in vivo
and in vitro, and the FAK-Stat1 interaction leads to
transient Stat1 tyrosine phosphorylation during cell adhesion (Fig. 3).
Functional FAK-Stat1 interaction is further supported by the
observations that a small portion of Stat1 and FAK are co-localized at
the focal adhesions (Fig. 8), consistent with the observation that FAK-Stat1 co-localization and interaction are dependent on C-terminal domain of the FAK that is required for FAK localization to focal contacts (Fig. 4). The evidence indicating that FAK-Stat1 activation may have a significant physiological role in cell migration was obtained using FAK-deficient cells. We have shown that Stat1 activation during cell adhesion is diminished in FAK-deficient cells (Fig. 5),
which is correlated with decreased integrin-mediated migration of these
cells. On the basis of these results we suggested a model that
integrin-FAK signaling can activate Stat1, which plays an important
role in reducing cell adhesion and enhancing cell migration.
One critical question is whether FAK can directly activate Stat1. We
have presented evidence indicating that STAT and FAK interact directly
and Stat1 is a substrate of FAK tyrosine kinase in a number of in
vivo and in vitro systems (Fig. 3). We also have shown
that the FAK-Stat1 interaction was specific, since Stat3 is not
activated by FAK under the same in vivo conditions that
Stat1 is activated (Fig. 1B). Additionally, in a number of in vitro experiments, FAK was found to directly interact
with and phosphorylate Stat1, but not Stat2 and NF- Although our data indicate that FAK can directly activate Stat1, we do
not suggest that FAK is the only kinase that is involved in Stat1
activation during the cell adhesion. We have shown the mutation Y397F,
which abolishes Src interaction with FAK, could also partially reduce
Stat1 activation (Fig. 2), indicating the possible role of Src in
enhancing the FAK function. Src activates Stat1 in an overexpression
system (Fig. 1B), and Src has been shown to interact with
Stat3 (37, 38). In this case, either Src can be recruited to further
phosphorylate Stat1 or Src can enhance FAK activity, and indirectly
increase Stat1 activation. However, Src was observed to be incapable to
act on Stat1 alone, since Src and Stat1 can not be
co-immunoprecipitated in vitro, whereas FAK-Stat1 are
co-immunoprecipitated in a number of untransfected cells. Furthermore,
in contrast to the partial role of Src, FAK is essential under the same
conditions; if FAK is defective due to the mutation in the kinase
domain, there is little Stat1 phosphorylation (Fig. 2B).
Moreover, we have shown that the deletion of the C terminus of FAK
abolishes FAK-Stat1 interaction and Stat1 activation (Fig. 4), although
this C-terminal deletion should have no effect on Src interaction with
FAK (44). Thus, we conclude that FAK is the major kinase, Src may play
an accessory role in Stat1 activation, and the location of FAK in the
focal contacts is critical for FAK-Stat1 interaction and Stat1
phosphorylation during cell adhesion.
In cytokine signaling, the JAK family kinases are activators of STAT
proteins. It was reported recently that integrin-mediated endothelial
cell adhesion induces JAK2 and Stat5 activation (56). However, in our
studies, we observe direct activation of Stat1 through FAK, and members
of JAK family may not be necessary. It is common that the multiple
kinases are involved in STAT activation. For example, EGF receptor
kinase and JAK kinases may both be involved in phosphorylation of STAT
proteins in response to EGF, although EGF receptor kinase itself is
sufficient to activate STAT proteins without JAK (30, 33, 34). It is
believed that PTKs other than JAKs activate STATs during early
evolution, and the JAK-STAT pathway was evolved from certain original
PTK-STAT pathways (57).
Similar to the situation after cytokine treatment, Stat1 activation
during cell adhesion is transiently induced. Thus, cells reach their
full attachment state only when Stat1 activity is diminished. For
example, in A431 cells, Stat1 activity diminished after being plated on
fibronectin for 4 h (Fig. 3), and, correlatively, cells were fully
attached. To avoid possible artifacts generated by protein
overexpression, most of our assays were performed in non-transfected
cells. In particular, we have used Stat1-defective cells such as Stat1
( We have demonstrated that FAK and Stat1 are associated in
vivo (Fig. 3). This association cannot be attributed to the
overexpression of these proteins in transfected cells, since, in
untransfected A431 and 293T cells, Stat1 was shown to associate with
FAK. This association was enhanced transiently at the early stages of
cell attachment; therefore, the peak of this association may correspond to FAK phosphorylation and activation during focal adhesion formation. Moreover, Stat1 that associates with FAK is tyrosine-phosphorylated, as
detected by a specific anti-phosphotyrosine Stat1 antibody (Fig. 3,
A and B). Using confocal microscopy, we further
demonstrated that, in non-transfected A431 cells, a fraction of Stat1
protein was found to be co-localized with FAK at the focal adhesion
sites, and the co-localization occurred during cell adhesion (data not shown). In addition, we examined whether the C-terminal domain is
required for FAK-Stat1 interaction. It has been shown that this region
of FAK is essential for FAK to be localized at the focal adhesion
sites. Thus, if the C-terminal domain of FAK is required for FAK-Stat1
interaction, it would suggest that Stat1 has a role in affecting focal
adhesion dynamics. Indeed, the Finally, although our results suggest that Stat1 is downstream of FAK
in an adhesion (integrin)-initiated signaling cascade, the downstream
genes regulated by the STAT pathway are not defined. Stat1-mediated
gene transcription may be involved in this process, since both the
Stat1 tyrosine phosphorylation site and the SH2 domain mutants that
have no DNA binding activity after stimulation exhibit no effect on
cell adhesion after being co-transfected with FAK (data not shown). It
would be interesting to investigate which specific integrin family
members are involved in the activation of Stat1 by FAK, and whether
other adhesion molecules in addition to the fibronectin-integrin
interaction can also mediate the STAT activation.
C14) completely abolishes this
interaction, indicating this association is dependent on the C-terminal
domain of FAK, which is required for FAK localization at focal
contacts. Moreover, Stat1 activation during cell adhesion is diminished in FAK-deficient cells, correlating with decreased migration in these
cells. Finally, we show that depletion of Stat1 results in an
enhancement of cell adhesion and a decrease in cell migration. Thus,
our results have demonstrated, for the first time, a critical signaling
pathway from integrin/FAK to Stat1 that reduces cell adhesion and
promotes cell migration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
fibroblasts (19-21).
However, mechanisms by which FAK regulates cell adhesion and migration
are still not fully understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and wild type fibroblast cells were cultured in RPMI 1640 with
10% FBS. U3A was cultured in DMEM with 10% FBS and 250 µg/ml
hygromycin B. U3A-pSG5 and U3A-Stat1 cells were cultured in DMEM with
10% FBS, 250 µg/ml hygromycin B (Roche Molecular Biochemicals) and 500 µg/ml Geneticin (Life Technologies, Inc.). FAK wild type and
/
fibroblasts were cultured in DMEM (high glucose) with 10% FBS, 1 mM sodium pyruvate (Life Technologies, Inc.), 100 µM non-essential amino acids (Life Technologies, Inc.),
and 100 µM 2-mercaptoethanol (Sigma).
C14, had been described
previously (42-44).
B/p65 (C-20) (Santa Cruz Biotechnology, Inc.) antibodies
were used for in vitro assays. A monoclonal anti-vinculin
antibody was used for immunofluorescence staining (Upstate
Biotechnology Inc.).
B/p65 (all from Santa Cruz
Biotechnology, Inc.), and anti-Stat2 antibodies (45) were used.
Non-related serum was used as a control.
-treated cells were also cultured on coverslips (no
fibronectin) overnight before treatment. Thirty minutes after
treatment, cells were fixed as above. The primary antibodies were
diluted as follows:
-HA, 1:100; monoclonal
-FAK and
-Stat1
(C-111), 1:100; polyclonal
-FAK and
-Stat1 (C-24), 1:300. The
secondary antibodies were fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (AlexaTM 488, Molecular Probes, 1:200) or Texas
Red-conjugated goat anti-mouse IgG (Jackson Immunoresearch
Laboratories, Inc., 1:200).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of FAK activates Stat1, but not
Stat3. A, exogenous FAK-induced endogenous
(lane 3) and exogenous (lane
4) Stat1 activation in 293T cells. Extracts from 293T cells
were analyzed by EMSA using a high affinity probe, M67-SIE, 48 h
after transfection with Stat1, FAK, or in combination. Lane 1, control
showing IFN- -induced Stat1 activity; lane 2,
vector; lane 3, Stat1 (HA-tagged);
lane 4, FAK; lane 5, FAK
and Stat1; lane 6, Stat1 supershift by an
anti-Stat1 antibody. B, FAK failed to activate Stat3 in
co-transfected 293T cells and Jak1 was not necessary for Stat1
activation by FAK. Although Stat1 activation was strongly induced by
FAK (lane 5), Stat3 activation was not induced by
the similar FAK expression (lane 1).
Co-transfection of Src with Stat1 or Stat3 induced both Stat1 and Stat3
activation at comparable levels (lanes 3 and
7). The nature of STAT-containing complexes was confirmed by
supershift complexes recognized by specific Stat3 or Stat1 antibodies
(lanes 2 and 4 and lanes
6 and 8, respectively). Stat1 DNA binding
activity was also observed in Jak1-deficient E2A4 cells co-transfected
with Stat1 and FAK (lane 10), and it was seen in
control transfected with vector alone (lane 9).
C, expression of FAK and Stat1 caused changes in cell
morphology. Cells transfected with mock plasmid (vector only)
(a), non-tagged Stat1 (b), Stat3 (c),
or FAK with Stat3 (f) showed no morphological change. Cells
transfected with FAK alone (d) exhibited a modest change in
cell morphology, whereas FAK- and Stat1-co-transfected cells
(e) exhibited a dramatic change in cell shape. Transfected
cells were indicated by blue staining (see "Experimental
Procedures"). Arrows indicate weakly transfected cells
that are light blue. The scale
bar represents 10 µm.
-galactosidase. Thus, transfectants could be specifically recognized by the blue color after X-gal staining. We observed dramatic
morphological changes in transfected cells that seemed to parallel
Stat1 activation by FAK. The FAK- and Stat1-co-transfected cells
clearly lost their cell spreading ability and were detached from the
plate (Fig. 1C, e; arrows indicate
light blue cells, which were transfected at a
lower level.). For the cells transfected with FAK alone, a portion of
transfected cells also underwent similar morphological alterations that
might result from the endogenous Stat1 activity induced by FAK (Fig.
1C, d). The cells that were mock-transfected or
Stat1 only transfected showed no effect (Fig. 1C,
a and b). In contrast to cells co-transfected
with FAK and Stat1, those co-transfected with FAK and Stat3 showed no
significant change on morphology (Fig. 1C, f).
Therefore, these data further demonstrate that Stat1 activation by FAK,
but not Stat3, can negatively affect cell adhesion.
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Fig. 2.
Functional Stat1 and FAK are necessary for
activation. A, Stat1 activity in Stat1 wild type and
mutant transfected 293T cells (Stat1-CYF indicates Y701F mutant, and
Stat1-SH2RQ indicates SH2 domain mutant) revealed that
Stat1-CYF and Stat1-SH2RQ diminished Stat1 activation induced by FAK
(lanes 6 and 8). Similar levels of
Stat1 or Stat1 mutant proteins (lower panel) in
transfected cells were detected by Western blot with an anti-Stat1
antibody. Lane 1, vector; lane
2, Stat1; lane 3, FAK; lane
4, Stat1 and FAK; lane 5, Stat1-CYF;
lane 6, Stat1-CYF and FAK; lane
7, Stat1-SH2RQ; lane 8, Stat1-SH2RQ
and FAK. B, FAK (KD) cannot activate Stat1 in co-transfected
293T cells (compare lane 6 to lane
4) and Stat1 activity was reduced in cells co-transfected
with FAK (Y397F) and Stat1 (lane 8). Exogenous
FAK protein levels were similar (lower panel).
Lane 1, vector; lane 2,
Stat1; lane 3, FAK; lane 4,
Stat1 and FAK; lane 5, FAK (KD); lane
6, Stat1 and FAK (KD); lane 7, FAK
(Y397F); lane 8, Stat1 and FAK (Y397F).
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Fig. 3.
FAK and Stat1 were transiently associated
following cell adhesion. A, 293T cells were plated on
fibronectin for indicated time points (0, 0.5, 1.5, and 4 h). The
lysates from these cells were used for immunoprecipitation with
anti-FAK antibodies, blotted with anti-Stat1 (upper
panel, lanes 1-4), or, after
stripping, blotted with anti-phospho-Stat1 antibody (middle
panel). A 91-kDa band co-immunoprecipitated with FAK was
recognized by an anti-Stat1 antibody (upper
panel). Lane 5 is the control showing
the positions of phosphorylated Stat1 (Stat1p) and unphosphorylated
Stat1, which were immunoprecipitated using an anti-Stat1 antibody. The
higher bands (Stat1p) co-immunoprecipitated with FAK, were also
recognized by a specific anti-phosphotyrosine Stat1 antibody
(middle panel), but the lower band (Stat1,
indicated in lane 5, upper
panel) was not recognized by this anti-phosphotyrosine Stat1
antibody (lane 5, middle
panel). The immunoprecipitated FAK protein was at the equal
level (lower panel). B, A431 cells
were plated on FN for the indicated time points (0, 0.5, 1.5, and
4 h) and a transient association of tyrosine phosphorylated Stat1
with FAK was noted. The slower migrating Stat1 band was recognized by
an anti-phospho-Stat1 antibody (Stat1p). C, using an EMSA,
transient Stat1 activities were observed at the early time points
during A431 cell adhesion. Lane 1, IFN- (10 ng/ml) treatment; lane 2, Stat1 complex was
supershifted (SS) by the anti-Stat1 antibody. Similarly,
extracts from A431 cells that were plated on FN at the different time
points indicated (lanes 3-7) were analyzed by
EMSA. The same anti-Stat1 antibody supershifted the inducible complex
generated at the 30-min time point (lanes 8 and
9). D, direct interaction of FAK and Stat1
in vitro. Upper panel, in
vitro translated Stat1 using reticulocyte lysates was incubated
with FAK protein from SF21 cell lysate (lanes 1 and 3) or from SF21 cell lysate control (lanes
2 and 4). Non-related serum (lanes
1 and 2) and an anti-Stat1 antibody
(lanes 3 and 4) were used for
immunoprecipitation. A co-immunoprecipitated FAK protein was observed
using anti-FAK C-20 antibody (upper row,
lane 3). Phosphorylated Stat1 was revealed by an
anti-phospho-Stat1 antibody (middle row,
lane 3). Stat1 protein levels were similar after
immunoprecipitation (lower row, lanes
3 and 4). Middle panel,
left, in vitro translated FAK using reticulocyte
lysates was pulled down by GST-Stat1. FAK translated in
vitro was shown in lane 3. Although FAK was
pulled down by GST-Stat1 (lane 2), GST alone did
not pull down FAK (lane 1). Right,
Stat1 and FAK (lanes 4-6) and Stat2 and FAK
(lanes 7-9) were in vitro translated
together. Immunoprecipitation using indicated antibodies revealed no
interactions between Stat2 and FAK (lanes 8 and
9), but Stat1 and FAK were precipitated together
(lanes 5 and 6). Non-related serum was
used as controls (lanes 4 and 7). The
proteins were detected by autoradiography of 35S. Some
partial translated products could be also observed below the Stat1
band. Bottom panel, similar experiments were
conducted in wheat germ lysates. Note that bands represent FAK and
Stat2 have small but distinct migration differences.
treatment in A431 cells was used as a control (lane
1; in lane 2, Stat1 complex was
supershifted by an anti-Stat1 antibody). Apparently, IFN-
is a much
stronger inducer of Stat1 activation than fibronectin. However,
activation of Stat1 after plating on fibronectin was clearly observed
at the 0.5-h time point (lane 4). The nature of
Stat1 in this complex was confirmed when this induced complex was
recognized by a Stat1-specific antibody, generating a supershifted
complex (SS, lane 9). These data
further indicate that Stat1 is transiently activated, directly or
indirectly, by FAK during cell adhesion.
B/p65 did not co-precipitate (data not shown).
These results demonstrate that FAK and Stat1 interaction is specific.
C14 mutant FAK, which lacked C-terminal 14 amino acids, had a
diffuse cytoplasmic distribution (44). Since FAK and Stat1 were
co-precipitated, it is possible that the C-terminal domain of FAK may
also be necessary for FAK-Stat1 interaction.
C14 mutant FAK. All these exogenously introduced proteins
were HA epitope-tagged (30, 44). After immunoprecipitation by an
anti-Stat1 antibody, the immunocomplexes were blotted with anti-HA
antibody that could reveal all exogenous proteins (Fig. 4A, upper
panel). Blotting with an anti-Stat1 antibody showed that
Stat1 protein was expressed at similar levels (Fig. 4A,
middle panel). As expected, wild type FAK was
precipitated with either endogenous or exogenous Stat1 (Fig.
4A, upper panel, lanes
3 and 4), indicating endogenous Stat1 and
exogenous FAK associated efficiently. Interestingly,
C14 mutant FAK
completely lost its ability to interact with Stat1 (Fig. 4A,
lane 6), indicating that the C-terminal domain is
also essential for interaction with Stat1. Consistent with this
observation, cells co-expressing
C14 mutant FAK and Stat1 did not
generate Stat1 DNA binding activity (Fig. 4B,
lane 6) and did not exhibit morphological changes
(Fig. 4C). Furthermore,
C14 and Stat1 were not observed
to be co-localized in focal adhesion sites (data not shown). These
results strongly argue that, in addition to autophosphorylation ability
and kinase activity, which are intact in the
C14 mutant FAK, the
specific location of FAK at the focal contacts was also essential for
its activation with Stat1 in vivo; the results also indicate
that FAK-Stat1 must co-localize to focal contacts to have an effect on
cell adhesion.
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Fig. 4.
A mutant FAK with C-terminal deletion
( C14) failed to interact with Stat1.
A, wild type FAK, but not FAK
C14, interacted with Stat1
in transfected 293T cells. Control plasmid (lane
1), HA-Stat1 (lane 2), HA-wild type
FAK (lane 3), and HA-FAK
C14 (lane
4) were transfected into 293T cells separately or in
combination as indicated. Cell lysates were prepared 48 h after
transfection. Immunoprecipitation was performed with an anti-Stat1
antibody, blotted with either anti-HA antibody (upper
panel) or anti-Stat1 antibody (middle
panel). Wild type FAK, but not FAK
C14, was precipitated
with Stat1 (lanes 3 and 4 were
compared with lanes 5 and 6,
upper panel). Exogenous Stat1 proteins were
expressed at comparable levels (lanes 2,
4, and 6, middle panel).
Similarly, exogenously expressed FAK protein was revealed by
immunoprecipitation and blotting with anti-HA antibody
(lanes 3-6, lower panel).
A nonspecific band that migrated slower than FAK protein, was
identified in lanes 1 and 2 (lower panel). B,
C14 mutant FAK
does not activate Stat1 in transfected 293T cells. Cell lysates from
different transfected cells were used for EMSA. No Stat1 (non-HA Stat1)
activity was detected in cells transfected with
C14 mutant FAK
(lane 5) and co-transfected with Stat1 and
C14
mutant FAK (lane 6), whereas strong Stat1
activation was observed in cells co-transfected with Stat1 and wild
type FAK (lane 4) and weak endogenous Stat1
activation was generated by transfection of FAK alone (lane
3 and Fig. 2a). C, expression of the
C14 mutant FAK alone or with Stat1 did not cause a change in cell
morphology. Transfected cells were stained blue. The
scale bar represents 10 µm.
/
Fibroblasts--
If FAK is one of the tyrosine kinases that
activates Stat1 during cell adhesion, Stat1 phosphorylation should be
decreased in FAK-deficient cells. To verify this possibility, Stat1 was precipitated from cell lysates of FAK-deficient and wild type fibroblasts (19) after plating on fibronectin. Induced Stat1 phosphorylation was greatly reduced in FAK
/
cells (Fig.
5, upper panel,
lane 2) compared with that in FAK wild type cells (upper panel, lane 5).
However, there was a certain level of Stat1 phosphorylation in FAK
/
(Fig. 5, upper panel, lanes
2 and 3), suggesting that other tyrosine kinases
besides FAK may also be able to activate Stat1 during the adhesion
process. Stat1 phosphorylation was eventually reduced in both cells
(upper panel, lanes 3 and 6). Stat1 was re-blotted showing equal protein level
(lower panel). The Stat1 activation reached its
highest level at 90 min, which was slower than that in 293T and A431
cells, probably due to intrinsic cell line differences in the rates of
cell adhesion. These results further support the model that FAK is
involved in Stat1 phosphorylation and activation during cell
adhesion.
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Fig. 5.
Stat1 activity was diminished in FAK
/
fibroblasts during cell
adhesion to fibronectin. Cell lysates from FAK
/
and FAK wild
type fibroblasts that had been plated on FN for indicated time points
(0, 90, and 240 min) were precipitated by anti-Stat1 antibody and
blotted with anti-phospho-Stat1 antibody (upper
panel), or, after stripping, blotted with anti-Stat1
antibody (lower panel). Although Stat1
phosphorylation was induced in both cell types by fibronectin, the
degree of phosphorylation was much stronger in wild type cells
(upper panel, lane 5)
compared with FAK
/
cells (lane 2).
/
) and wild type mice (50) were used. These cells were
comparatively examined for cell adhesion (see "Experimental Procedures" for detail), in which cell attachment was measured over a
time course at variable concentrations of FN (Fig.
6A and data not shown). Cells
did not attach well at the low coating concentrations of FN (data not
shown). It appeared that a minimal coating concentration of 2.5 µg/ml
FN (bound concentration of 0.013 ng/mm2 as shown previously
(51, 52) was necessary for sufficient cell adhesion of both Stat1 null
and wild type fibroblasts. An increase of FN coating concentrations to
5 and 10 µg/ml (bound concentrations of 0.11 and 0.3 ng/mm2, respectively) did not affect cell attachment
further (Fig. 6A, lower panel). We
found that Stat1 null fibroblasts had statistically higher levels of
adhesiveness than wild type cells (Fig. 6A; 30 min,
p = 0.0003; 60 min, p = 0.005; 120 min,
p = 0.002). The difference in cell adhesion between
Stat1 null and wild type cells did not change when higher
concentrations of FN (Fig. 6A, lower
panel; 5 µg/ml, p = 0.009; 10 µg/ml,
p = 0.007) were used, indicating that this difference
is not a function of the amounts of cell matrix protein bound to the
plate. Furthermore, the difference of cell adhesion between Stat1 null
and wild type cells was observed as early as 30 min after plating;
longer plating times up to 180 min did not diminish the difference
(Fig. 6A). Thus, these results indicated that there is an
intrinsic difference in cell attachment between Stat1 null and wild
type cells, suggesting that the presence of Stat1 may negatively affect
cell adhesion.
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Fig. 6.
Stat1-deficient cells exhibit increased cell
adhesion. A, Stat1 /
fibroblasts (white
boxes) exhibited greater adhesion than wild type fibroblasts
(black boxes). 2 × 104
cells/well were seeded on 96-well plates coated with different
concentrations of FN (1.0 and 2.5 µg/ml) and incubated for 30, 60, and 120 min (upper panel). Cells were also plated
on higher concentration FN (2.5, 5.0, and 10 µg/ml) for 180 min in a
separate experiment (lower panel; the higher
scale was due to increased fully attached cells with longer incubation
time). Stat1
/
fibroblasts exhibited 20-40% higher cell adhesion
than wild type fibroblasts during shorter or longer incubation times
(from 30 to 180 min). B, the presence of Stat1 resulted in
decreased cell adhesion. Stat1-deficient U3A cells (U3A-pSG5,
white boxes) were stably transfected by an empty
vector pSG5. U3A-Stat1 cells were stably transfected by Stat1
expressing vector pSG-Stat1 (U3A-Stat1, black
boxes). Stat1 protein levels were essentially equal in the
different clones that were used in the experiments and comparable to
the parental cell line, 2fTGH. 5 × 104/well cells
were plated onto FN coated 96-well plates as described above and
similar experiments were performed as shown in A. Results
are the averages from three different experiments. The
asterisk (*) indicates statistical significance
(p < 0.05). The vertical lines
denote standard deviations.
/
and wild type fibroblasts (data not shown).
/
fibroblasts exhibited a dramatically decreased level of
migration on FN compared with Stat1 wild type fibroblasts in both short
term and long term cultures (3 and 9 h, respectively) (Fig.
7A, p < 0.05). Similarly, in contrast to Stat1-deficient U3A-pSG5 cells,
U3A-Stat1 cells showed a significant increase of migration on FN at
different time points (Fig. 7B, p < 0.05).
These results suggest that Stat1 plays important roles in both cell
adhesion and cell migration mediated by integrins.
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Fig. 7.
Stat1-deficient cells exhibited decreased
cell migration. A, migration of Stat1 /
cells
(white boxes) was significantly reduced compared
with wild type cells (black boxes) on fibronectin
substratum. Migration of Stat1
/
and wild type cells was assessed
in Transwell plates at 3 and 9 h after plating (see
"Experimental Procedures" for detail). B, cells
expressing U3A-Stat1 (black boxes) exhibited a
greater migration rate compared with U3A Stat1-negative cells
(U3A-pSG5, white boxes) at 3 and 9 h after
plating. Results represent the averages of six different experiments.
The asterisk (*) indicates statistical significance
(p < 0.05). The vertical lines
denote standard deviations.
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Fig. 8.
Localization of Stat1 and FAK in A431 cells
during adhesion. Fluorescein isothiocyanate- and Texas
Red-conjugated secondary antibodies were used for immunofluorescence
staining. After brief trypsinization and washing, A431 cells were
plated on coverslips that were coated with 5 µg/ml fibronectin.
A, at indicated time points (30 min, a-c; 60 min, d-f), cells were processed for immunofluorescence by
using FAK polyclonal and Stat1 monoclonal antibodies. Focal contacts
were shown by anti-FAK antibody (a and d,
arrows). Stat1 had cytoplasmic distribution (b
and e). Confocal merged images revealed that Stat1 and FAK
co-localized in the focal contacts and cell periphery at a point of 30 min after plating (c, arrowhead,
yellow). At the 60-min time point, the co-localization was
not observed in well spread cells, but still in some cells that were
less well spread (f, arrows and
arrowheads). Two enlarged pictures at right show
details of FAK-Stat1 co-localization, as indicated by
arrowheads. The arrows point to FAK alone at the
focal contacts. Similar results were observed using different FAK and
Stat1 antibodies. The lower panel showed that the
control primary antibody, mIgG, instead of mouse anti-Stat1, was used.
These experiments were repeated six times. B, IFN-
induced Stat1 and FAK co-localization. a-c, cells treated
with IFN-
(10 ng/ml) for 30 min; d-i, untreated cells or
control. Cells were processed as described under "Experimental
Procedures." FAK monoclonal antibody (a and d),
Stat1 (b and e) polyclonal antibody, or 3% BSA
control (g, h, and i) were used in
double staining. Focal contacts (a, arrows) and
co-localization of FAK with Stat1 (c, arrowheads)
were indicated in IFN-
treated cells. The scale
bar represents 10 µm. Two enlarged pictures at
right show details of FAK-Stat1 colocalization.
-treated A431 cells to determine if
co-localization would be increased after phosphorylation of Stat1. Stat1 was shown in the nucleus after IFN-
treatment, but some Stat1
was still detected in the cytoplasm (Fig. 8B, b).
In untreated cells, Stat1 was mainly in cytoplasm (e).
Intriguingly, in comparison with untreated control cells, Stat1
appeared to increase its presence in the focal contacts and
co-localized with FAK after IFN treatment, further suggesting a role of
Stat1 in cell adhesion (Fig. 8B, comparing f with
c, arrows). The negative control with the
secondary antibodies alone revealed some background staining
(g-i). We also observed the co-localization of FAK and
Stat1 in other cell lines (data not shown). These results suggest that
FAK-Stat1 interaction has a physiological role during cell adhesion to
matrix proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B p65 (Fig.
3D, and data not shown). Furthermore, in FAK-deficient
cells, Stat1 tyrosine phosphorylation and activation during cell
adhesion are significantly reduced, suggesting that FAK is important
for Stat1 activation.
/
) fibroblasts and the U3A cell lines, in comparison with the
control wild type fibroblasts or Stat1 re-introduced U3A-Stat1 cells.
The presence of Stat1 in wild type fibroblasts or in U3A-Stat1 cells
results in a lower cell adhesion and a higher rate of cell migration
than that of Stat1-defective cells (Fig. 6 and 7). However, it should
be pointed out that the Stat1 activation during cell adhesion is
significantly weaker than the Stat1 activation induced by cytokines
such as interferons. Since Stat1 activation is weak and transient, the
negative effect of Stat1 may be overcome by other signaling pathways
that increase the cell adhesion, and cells eventually become attached.
Intriguingly, the initial reports of Stat1 null mice suggested that
Stat1 is required for functions of interferons (50, 58). On the basis of on our current studies, we expect that these mice may also have
certain defects related to cell adhesion and migration.
C14 mutant FAK completely lost its
ability to interact with and to activate Stat1. Consistent with this
observation, cells expressing
C14 with Stat1 did not exhibit
morphological changes (Fig. 4). Furthermore, in contrast to FAK-Stat1
interactions,
C14-Stat1 was not observed to be co-localized in focal
adhesion sites (data not shown). These results strongly support our
suggestion that FAK and Stat1 interact and are co-localized during cell
adhesion. Thus, FAK-Stat1 association may be responsible, at least in
part, for the observed Stat1 activation during cell adhesion.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. T. Hunter, D. Ilic, L. Languino, D. Stern, and D. DiMaio for their critical reading of the
manuscript and their helpful discussions and advice; Dr. D. Levy of NYU
for Stat1 wild type and null (/
) fibroblasts; Drs. I. Kerr and G. Stark for U3A cells; Dr. D. Ilic for FAK wild type and deficient
fibroblasts; Dr. S. Ghosh for NF-
B p65 construct; J. Pober for
advice; Drs. L. Ji, H. Qin, D. Graesser, and S. Mahooti for their help;
and N. Bennett for administrative assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R01 44906 and AI34522 (both to X.-Y. F.) and GM48050 and GM52890 (both to J.-L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Dept. of Pathology, Chiba University School of Medicine, Chiba, Japan.
** Recipient of a career development award from the National Institutes of Health. To whom correspondence should be addressed. Fax: 203-785-7303; E-mail: xin-yuan.fu@yale.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M009063200
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ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
GST, glutathione
S-transferase;
SH, Src homology;
EGF, epidermal growth
factor;
FAK, focal adhesion kinase;
STAT, signal transducer and
activator of transcription;
JAK, Janus kinase;
PIPES, 1,4-piperazinediethanesulfonic acid;
HA, hemagglutinin;
KD, kinase-defective;
WCE, whole cell extract;
PBS, phosphate-buffered
saline;
FN, fibronectin;
IFN, interferon;
BSA, bovine serum albumin;
PTK, protein-tyrosine kinase;
DMEM, Dulbecco's modified Eagle's
medium;
FBS, fetal bovine serum;
EMSA, electrophoretic mobility shift
assay.
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REFERENCES |
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