©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Focal Adhesion Kinase with Cytoskeletal Protein Talin (*)

Hong-Chen Chen (1), Paul A. Appeddu (1), J. Thomas Parsons (2), Jeffrey D. Hildebrand (2), Michael D. Schaller (2)(§), Jun-Lin Guan (1)(¶)

From the (1)Cancer Biology Laboratories, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 and the (2)Department of Microbiology and Cancer Center, School of Medicine, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Focal adhesion kinase (FAK)()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) .

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 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.

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 -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.


EXPERIMENTAL PROCEDURES

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-cyto1wt and GST-cyto14, 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.

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.


RESULTS

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.


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.

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 -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-cyto1wt) and a truncated mutant (GST-cyto14). 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-cyto1wt but not GST alone or GST-cyto14. 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.




DISCUSSION

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 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.

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 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) .

Tapley et al.(35) have identified that amino acids 780-789 of the cytoplasmic domain of 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) .

We have identified amino acids 965-1012 in the carboxyl-terminal domain of FAK (deleted in 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 FAKC2 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 FAKC2, 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.


FOOTNOTES

*
This research was supported by National Institutes of Health grants (to J.-L. G., and J. T. P). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cell Biology and Anatomy, School of Medicine, University of North Carolina, Chapel Hill, NC 27599.

To whom correspondence should be addressed. Tel.: 607-253-3586; Fax: 607-253-3317.

The abbreviations used are: FAK, focal adhesion kinase; PDGF, platelet-derived growth factor; GST, glutathione S-transferase.

Hildebrand, J. D., Schaller, M. D., and Parsons, J. T. (1995) Mol. Biol. Cell.6, in press


ACKNOWLEDGEMENTS

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.


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