From the
The interaction of cells with extracellular matrix proteins
plays a critical role in a variety of biological processes. Recent
studies suggest that cell-matrix interactions mediated by integrins can
transduce biochemical signals to the cell interior that regulate cell
proliferation and differentiation. These studies have placed the focal
adhesion kinase (FAK), an intracellular protein tyrosine kinase, in a
central position in integrin-initiated signal transduction pathways
(Zachary, I., and Rozengurt, E.(1992) Cell 71, 891-894;
[Medline]
Schaller, M., and Parsons, J. T. (1993) Trends Cell Biol. 3,
258-262). Here, we report data suggesting a possible association
of FAK with the cytoskeletal protein talin in NIH 3T3 cells. We have
identified a 48-amino acid sequence in the carboxyl-terminal domain of
FAK necessary for talin binding in vitro. Furthermore, we have
correlated the ability of integrin to induce FAK phosphorylation with
its ability to bind talin using a mutant integrin lacking the
carboxyl-terminal 13 amino acids. These studies suggest talin may be a
mediator for FAK activation in signaling initiated by integrins and may
provide an explanation for the dependence on the integrity of
actin-cytoskeleton of multiple intracellular signaling pathways
converging to FAK activation and autophosphorylation.
Focal adhesion kinase (FAK)
The mechanism of FAK activation in
response to various stimuli is poorly understood(2) .
Inactivation of protein kinase C by a number of methods does not block
increase in FAK phosphorylation stimulated by neuropeptides, although
treatment of cells with protein kinase C activators lead to increased
tyrosine phosphorylation of FAK(22) . In contrast, inactivation
of p21
Studies using
cytochalasin D, which selectively disrupts the networks of actin
filaments, show that the integrity of the actin cytoskeleton is
required for increased phosphorylation of FAK in response to a variety
of extracellular stimuli(6, 22) . Furthermore, integrins
have been shown to interact with the actin cytoskeleton via their
direct binding to two cytoskeletal proteins talin and
In some experiments, SDS was added
to the cell lysates (final concentration, 2%) prepared in 1% Nonidet
P-40 lysis buffer. The lysates with SDS were then boiled for 5 min to
disrupt potential associations of talin with other proteins. The
samples were cooled and diluted (10-fold) in 1% Nonidet P-40 lysis
buffer and used for immunoprecipitation with anti-talin serum or normal
rabbit serum to prepare for immobilized talin in binding assays.
For
talin-integrin binding assays, about 5 µg of bacterially expressed
GST fusion proteins were immobolized on glutathione beads and then
incubated with 1 mg of NIH 3T3 cell lysates in 1% Nonidet P-40 lysis
buffer overnight at 4 °C. The bound complexes were eluted in SDS
sample buffer and analyzed by Western blotting with anti-talin
antibody. The membranes were then stained with Ponceau S to show that
similar amounts of fusion proteins were present in the binding assays.
To study the role of FAK in integrin signaling, we searched
for FAK-interacting proteins that may mediate FAK activation in
response to integrin aggregation in cell adhesion. Because both
integrins and FAK are localized in focal contacts, we were particularly
interested in potential interactions of FAK with other cytoskeletal
proteins localized in focal contacts. To examine the potential
association of FAK with talin, coimmunoprecipitation of these two
proteins was performed using lysates prepared from NIH 3T3 cells. As
shown in Fig. 1, talin is present in the anti-FAK
immunoprecipitates (lane3), which comigrates with
the intact talin as precipitated by anti-talin serum (lane4). Densitometry of the fluorograms showed that about
3-5% of cellular talin was associated with FAK. Another band that
migrates slightly faster than the intact talin was also detected in
anti-talin precipitates, which probably represents the large
proteolytic fragment of talin (lane4)(30) .
Talin was not detected in immunoprecipitates of preimmune anti-FAK
serum (lane1) or antiserum against PDGF receptor (lane2), showing the specificity of talin
association with FAK. Consistent with previous reports on their
interaction in vitro(31) , talin was also detected in
anti-vinculin immnoprecipitates (lane5). Taken
together, these data suggested the possible association of FAK with
talin in vivo.
Binding of recombinant FAK to
talin-coated beads could be due to its direct binding to talin or
mediated by other proteins that are associated with talin. For example,
vinculin has been shown to be associated with talin (31) and is
present in anti-talin immunoprecipitates under our experimental
conditions (see Fig. 4). To distinguish these two possibilities,
cell lysates were boiled in SDS to disrupt any protein-protein
associations before immunoprecipitations by anti-talin serum (see
``Experimental Procedures''). As shown in Fig. 4,
vinculin could no longer be detected in talin-coated beads prepared
from the treated lysates (compare lanes1 and 2). Other potential talin-binding proteins such as
The results presented here suggest a possible association of
FAK with a cytoskeletal protein talin. This association was observed in vivo by coimmunoprecipitations and in vitro by
binding of recombinant FAK to immobilized cellular talin. Evidence for
direct association of talin to FAK was obtained using talin from
SDS-denatured NIH 3T3 lysates in in vitro binding assays (Fig. 4). These results suggest a possible mechanism for FAK
activation and tyrosine phosphorylation by integrins because the
cytoplasmic domain of integrin
The potential
association of FAK with talin observed here may also help to explain
previous observations that disruption of actin-cytoskeleton with
cytochalasin D could block FAK phosphorylation in several
situations(6, 22) . FAK activation and tyrosine
phosphorylation have been implicated as a point of convergence in the
actions of integrins, oncoproteins encoding tyrosine kinases, and
neuropeptides with receptors coupled to
G-proteins(1, 2) . The mechanisms of cross-talk among
these diverse pathways are still unclear at present. However, the
integrity of actin-cytoskeleton seems to play a role in FAK responses
to all of these stimuli, and the actin-disrupting drug cytochalasin D
could block FAK phosphorylation in all of these
situations(6, 22) . Therefore, the actin cytoskeleton
may be involved in integrating these diverse signals, which is then
relayed to FAK by talin.
Using a series of chicken integrin
Tapley et al.(35) have
identified that amino acids 780-789 of the cytoplasmic domain of
We have identified amino acids 965-1012 in the
carboxyl-terminal domain of FAK (deleted in
We are grateful to Dr. R. O. Hynes for antisera
against talin and vinculin, Dr. Q. Yu for monoclonal antibody 12CA5,
and Mike Dolenga for critical reading of the manuscript and helpful
comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is a
cytoplasmic tyrosine kinase localized in focal
contacts(1, 2, 3) . Consistent with its
subcellular localization, FAK has been implicated in signal
transduction pathways initiated by integrin-mediated cell adhesion to
extracellular matrix proteins. FAK becomes rapidly phosphorylated in
response to cell attachment to fibronectin-coated surfaces or integrin
clustering by
antibodies(4, 5, 6, 7, 8, 9, 10) .
Integrin clustering on the cell surface also stimulates the tyrosine
kinase activity of FAK in both fibroblasts and
platelets(7, 11) . In addition, transformation of
fibroblasts by v-Src results in an elevated tyrosine phosphorylation of
FAK, associated with increased kinase activity(7, 12) .
FAK phosphorylation is also stimulated by several neuropeptides,
including bombesin, vasopressin, and
endothelin(13, 14) , which bind to cell surface
receptors coupled to
G-proteins(13, 15, 16, 17, 18) .
Finally, tyrosine phosphorylation of FAK was recently shown to be
modulated by platelet-derived growth factor (PDGF) (19) and
lysophosphatidic acid, a platelet-derived phospholipid that elicits a
wide variety of cellular responses in diverse cell types by acting on
its receptor coupled to G-proteins(20, 21) . Therefore,
FAK activation and phosphorylation are likely to be a point of
convergence of multiple intracellular signaling
pathways(1, 2) .
by ADP-ribosylation with botulinum C3
exoenzyme blocks the tyrosine phosphorylation of FAK induced by
lysophosphatidic acid (23) or bombesin and
endothelin(24) . It is unclear, however, if p21
is involved in FAK activation by integrins.
-actinin(25, 26) . Therefore, we hypothesized that
some of the cytoskeletal proteins colocalized with FAK and integrins in
the focal contacts may be involved in mediating FAK activation by
integrins. Here, we report data suggesting a possible association of
FAK with talin in NIH 3T3 cells and the identification of a 48-amino
acid sequence in the carboxyl-terminal domain of FAK necessary for
talin binding in vitro. Furthermore, we have correlated the
ability of integrin to induce FAK phosphorylation with its ability to
bind talin using a mutant integrin lacking the carboxyl-terminal 13
amino acids.
Materials
Protein A-Sepharose 4B,
glutathione-agarose beads, Ponceau S, and monoclonal antibody against
talin were purchased from Sigma. Rabbit antiserum against PDGF receptor
was from UBI. Antisera against talin and vinculin were kind gifts of
Dr. R. O. Hynes (Massachusetts Institute of Technology), and monoclonal
antibody (12CA5) against an epitope of the hemagglutinin (HA1) protein
of the influenza virus was a kind gift of Dr. Q. Yu (Boston University
Medical School). Rabbit anti-FAK serum was prepared as previously
described(27) .
Cell Culture, Immunoprecipitation, and Western
Blot
NIH 3T3 cells or insect Spodopterafrugiperda (Sf21) cells were maintained as previously
described(27) . Cell lysates were prepared in 1% Nonidet P-40
lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1%
Nonidet P-40, 10% glycerol, 1 mM NaVO
,
1 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory
unit/ml aprotinin, and 20 µg/ml leupeptin) as previously
described(27) . Lysates from NIH 3T3 cells (500 µg) or Sf21
cells (100 µg) were immunoprecipitated by incubation with 3 µl
of various antisera for 1 h at 4 °C. Immune complexes were
collected on protein A-Sepharose and washed four times in lysis buffer.
They were then boiled for 3 min in SDS-sample buffer and resolved on
SDS-polyacrylamide gel electrophoresis. Western blotting was performed
with monoclonal anti-talin antibody (1:1000 dilution) or 12CA5 (1:1000
dilution) using the Amersham electrochemiluminescence system, as
previously described(27) .
Preparation of Recombinant FAK
Proteins
The various FAK mutants have been previously
described(28) . Two complementary synthetic oligonucleotides
encoding an epitope derived from the influenza virus hemagglutinin
sequence (YPYDVPDYA, which is recognized by the monoclonal antibody
12CA5) were ligated to the 5`-end of the FAK coding sequence, replacing
29 amino acid residues. Epitope-tagged FAK or its mutants were cloned
into expression vector pBlueBac2 (Invitrogen). These were then
transfected into Sf21 cells along with the linear genomic DNA of
baculoviruses, and the recombinant viruses were harvested and used to
infect Sf21 for recombinant protein production, as previously
described(27) .
Preparation of GST Fusion Proteins
The
cDNAs encoding chicken integrin 1 and the
4 mutant have been
previously described(29) . cDNA fragments containing the
cytoplasmic domains of the wild type or mutant chicken integrin
1
were excised by digestions with BclI and PvuII and
inserted into the cloning site of pGEX-3X (Pharmacia Biotech Inc.)
after digestions with BamHI and SmaI. The resulting
plasmids were used to generate in-frame fusion proteins
GST-cyto
1wt and GST-cyto
1
4, respectively, as previously
described(27) .
In Vitro Association Assays
For talin-FAK
binding assays, cellular talin from 500 µg of NIH 3T3 cell lysates
were immobilized on protein A beads by immunoprecipitation with
polyclonal anti-talin serum. Talin-bound beads were further incubated
with 100 µg of recombinant virus-infected Sf21 cell lysates in 1%
Nonidet P-40 lysis buffer for 2 h at 4 °C. The bound recombinant
FAK or mutants were eluted in SDS sample buffer, resolved on
SDS-polyacrylamide gel electrophoresis, and analyzed by Western
blotting with 12CA5. The membranes were then washed in stripping buffer
(62.5 mM Tris, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol) at 55 °C for 30 min and reblotted with
anti-talin antibody to show that similar amounts of talin were present
in the binding assays. Aliquots of lysates (15 µg) containing
recombinant proteins were also analyzed directly by Western blotting
with 12CA5 to show that similar amounts of recombinant FAK and mutants
were used in the binding assays.
Figure 1:
Coimmunoprecipitation of talin with FAK
in NIH 3T3 cells. Equal amounts of lysates from NIH 3T3 cells were
immunoprecipitated using preimmune serum (lane1) or
antisera against PDGF receptor (lane2), FAK (lane3), talin (lane4), or
vinculin (lane5). They were electrophoresed on an
SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and
probed with a monoclonal antibody against talin. The molecular mass
markers (kDa) are shown on the right. The arrow indicates the position of talin.
To determine the regions of FAK necessary
for association with talin, recombinant FAK and a series of its mutants
were produced in insect cells by baculovirus expression systems. These
mutants have been previously described (28) and are designated
here as kd (kinase-defective, Lys-454 mutated to Arg), N (deletion
of amino acids 50-376),
C1 (deletion of amino acids
721-857),
C2 (deletion of amino acids 853-963), and
C3 (deletion of amino acids 965-1012), respectively (Fig. 2A). To facilitate the detection and purification
of the recombinant proteins, an oligonucleotide encoding an epitope
derived from the influenza virus hemagglutinin sequence (YPYDVPDYA,
which is recognized by the monoclonal antibody 12CA5) was fused to the
amino terminus of FAK or its mutants, replacing 29 amino acid residues.
The epitope-tagged recombinant FAK and mutants were expressed in insect
Sf21 cells as described under ``Experimental Procedures.'' No
endogenous FAK was expressed in Sf21 cells (data not shown), and the
epitope-tagged recombinant proteins can be detected by
immunoprecipitation with anti-FAK serum followed by Western blotting
with 12CA5 (Fig. 2B). Immunoprecipitation of the
recombinant proteins followed by in vitro kinase assays
indicated that recombinant FAK and all deletion mutants exhibited
kinase activities (data not shown), indicating that gross alterations
in conformation were unlikely for these recombinant proteins. No kinase
activity was observed for kd, as expected (data not shown). However,
proper folding of this mutant is likely because it can bind to talin
(see Fig. 3and below).
Figure 2:
Expression of recombinant FAK and mutants. A, schematic representation of the recombinant FAK proteins. wt, wild type; kd, kinase-defective mutant with
Lys-454 changed to Arg; N, deletion at amino acid
residues 50-376;
C1, deletion at amino acid
residues 721-857;
C2, deletion at amino acid
residues 853-963;
C3, deletion at amino acid
residues. 965-1012. Opencircles at the amino termini
represent the epitope tag. B, lysates from Sf21 insect cells
infected with recombinant viruses encoding FAK and its mutants (lanes2-7) or uninfected cells (lane1) were immunoprecipitated using antiserum against FAK.
They were electrophoresed on an SDS-polyacrylamide gel, transferred to
nitrocellulose membrane, and probed with 12CA5. The molecular mass
markers (kDa) are shown on the right.
Figure 3:
In vitro association of talin and
recombinant FAK. A, aliquots of lysates from infected insect
cells were analyzed by Western blotting using 12CA5 to verify that
similar amounts of input were used in binding assays for all samples. B, cellular talin was immobilized on protein A-Sepharose beads
by anti-talin immunoprecipitation and then incubated with various
recombinant FAK proteins. The bound proteins were eluted in SDS sample
buffer and analyzed by Western blot with 12CA5. C, the same
membrane was stripped and reprobed using anti-talin monoclonal antibody
to verify that similar amounts of talin were present in all samples in
the binding experiments. Abbreviations used are as described in the
Fig. 2 legend.
In vitro binding assays were
performed using cell lysates prepared from Sf21 cells infected with
various recombinant viruses encoding FAK or its mutants. An aliquot of
each sample was analyzed by Western blotting with 12CA5 to show that a
similar amount of recombinant proteins was expressed in insect cells (Fig. 3A). The remaining samples were incubated with
cellular talin immobilized on protein A-Sepharose beads by
immunoprecipitation with polyclonal anti-talin serum. After washing,
the bound proteins were resolved on SDS-polyacrylamide gel
electrophoresis and detected by Western blotting with 12CA5. As shown
in Fig. 3B, all recombinant proteins except C3
bound to talin. Western blotting of the same membranes showed that
similar amounts of talin were present in all samples (Fig. 3C). None of the recombinant proteins bound to
control beads where normal rabbit serum was used for talin
immunoprecipitation (data not shown). These data indicated that amino
acids 965-1012 of FAK (deleted in
C3) were necessary for binding
talin immobilized on beads.
-actinin and paxillin could not be detected in anti-talin
immunoprecipitates even before the treatment under our experimental
conditions (data not shown). It is also unlikely that any other
proteins were still associated with talin after boiling in SDS of the
cell lysates. Nevertheless, recombinant FAK bound to talin-coated beads
prepared from boiled lysates as efficiently as that from untreated
lysates (compare lanes4 and 6). The
recombinant FAK did not bind to control beads where normal rabbit serum
was used for talin immunoprecipitation (lanes3 and 5). Taken together, these results suggest that FAK likely
binds to talin directly in vitro and possibly to a contiguous
stretch of amino acids on talin.
Figure 4:
Direct association of talin with FAK in vitro. Talin from SDS-denatured (+, lanes1, 3, and 4) or untreated (-, lanes2, 5, and 6) cell lysates
were immobilized on protein A-Sepharose beads by anti-talin
immunoprecipitation (lanes1, 2, 4,
and 6). Control beads were generated by using normal rabbit
serum in immunoprecipitations (lanes3 and 5). They were then incubated with recombinant FAK, and the
bound proteins were eluted in SDS sample buffer and analyzed by Western
blot with 12CA5 (lanes3-6). The beads with
immobilized talin were also analyzed by Western blotting with
anti-vinculin to detect any associated vinculin (lanes1 and 2).
Previous data from our laboratory
and others have implicated that the carboxyl-terminal amino acids in
the cytoplasmic domain of integrin 1 is responsible for
intracellular signaling, resulting in tyrosine phosphorylation of
FAK(4, 33) . To test if the ability of integrin to
activate FAK is correlated with its binding to talin, two integrin
1 cytoplasmic domain-derived GST fusion proteins were produced in
bacteria, including an intact cytoplasmic domain (GST-cyto
1wt) and
a truncated mutant (GST-cyto
1
4). The mutant lacked 13 amino
acids from the carboxyl terminus of integrin
1 cytoplasmic domain
and was unable to induce FAK tyrosine phosphorylation(4) . The
GST fusion proteins were immobilized on glutathione-agarose beads and
further incubated with NIH 3T3 cell lysates. After washing, the bound
protein complexes were eluted in SDS sample buffer and analyzed by
Western blot with anti-talin. As shown in Fig. 5A, talin
was precipitated by GST-cyto
1wt but not GST alone or
GST-cyto
1
4. Ponceau S staining of the same membranes showed
that similar amounts of the fusion proteins were present in all samples (Fig. 5B). These results correlate integrin binding to
talin and its ability to induce FAK phosphorylation, which is
consistent with the notion that talin-FAK interaction plays a role in
integrin-mediated signaling.
Figure 5:
In vitro association of talin and
GST-integrin 1 cytoplasmic domain fusion proteins. A,
bacterially expressed GST fusion proteins were immobolized on
glutathione beads and then incubated with lysates from NIH 3T3 cells in
1% Nonidet P-40 lysis buffer for overnight at 4 °C. The bound
complexes were eluted in SDS sample buffer and analyzed by Western blot
with anti-talin monoclonal antibody (lanes2-4). An aliquot of the input lysate was analyzed
directly (lane1). B, the membranes were
then stained with Ponceau S to verify that similar amounts of fusion
proteins were present in all samples in the binding experiments (lanes2-4). The molecular mass markers (kDa)
are shown on the right. The arrow indicates the
position of talin.
1 interacts with talin (Ref. 25 and Fig. 5). The binding of talin to the kinase-deficient FAK is as
effective as that to the wild type FAK (Fig. 3), indicating that
the possible association of talin with FAK is independent of FAK
activation and phosphorylation. This is consistent with the proposal
that talin is an upstream mediator of FAK activation by integrins but
not a downstream effector of FAK activation.
1
mutants expressed in NIH 3T3 cells, Lewis and Schwartz (34) have
recently shown in vivo the correlated localization of talin,
FAK, and actin, as induced by microbeads coated with anti-integrin
antibodies. The colocalization of these three proteins with integrin
all requires the presence of amino acids 791-799 in the
cytoplasmic domain of
1 integrin. These observations are
consistent with our present results, suggesting a role for talin in FAK
activation by integrins. Indeed, talin did not bind the GST fusion
protein containing the cytoplasmic domain of integrin mutant
4
(the same mutant as
790 used by Lewis and Schwartz; see Ref. 29),
which lacked amino acids 791-803, under our experimental
conditions that allowed its efficient binding to the GST fusion protein
containing the entire cytoplasmic domain of
1 integrin (Fig. 5). Previous studies also showed that the same mutant was
neither localized to focal contacts (29) nor induced FAK
activation(4) .
1 integrin could bind to talin in vitro. This sequence
lies adjacent to amino acids 791-799 discussed above, and as
Lewis and Schwartz suggested(34) , amino acids from both
segments could form one talin binding site in vivo. Removal of
amino acids 790-799 may weaken the affinity sufficiently to
prevent association in vivo. This may account for the
inability of the
4 mutant to activate FAK in
vivo(4) .
C3) necessary for
talin binding. These amino acids have also been found previously to be
necessary for FAK localization to focal contacts(28) .
Therefore, talin may be one of the FAK binding proteins responsible for
its targeting to the focal contacts. However, interaction with talin
alone may not be sufficient for FAK localization to focal contacts
because FAK
C2 was not localized to focal contacts(28) ,
although it could bind talin in vitro (Fig. 3). It is
possible that interaction of FAK with more than one protein in
multimolecular complexes in vivo is necessary for its proper
localization in the focal contacts and/or for its activation and
signaling. Interestingly, Miyamoto et al. (36) have recently
shown that integrin clustering by a non-inhibitory antibody coated on
the beads induced colocalization of tensin and FAK but not other
cytoskeletal proteins. It is unclear, however, which region of FAK is
responsible for its association with tensin. Recently, FAK has also
been shown to bind to another cytoskeletal protein,
paxillin(37) . Although paxillin can interact with talin via
vinculin (32), it is unlikely that FAK associates with talin via its
binding to paxillin because FAK
C2, which does not bind
paxillin,
(
)could associate with talin
efficiently (Fig. 3). Experiments are in progress to determine
more precisely the binding sites on FAK for these various cytoskeletal
proteins and to determine relative contributions of these interactions
to the activation and tyrosine phosphorylations of FAK.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.