©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Tyrosine Phosphorylation of Paxillin in Vitro by Focal Adhesion Kinase (*)

(Received for publication, February 28, 1995; and in revised form, May 3, 1995)

Susan L. Bellis (§) , John T. Miller , Christopher E. Turner (¶)

From the Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse, New York 13210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.


INTRODUCTION

Cell adhesion to an extracellular matrix plays a fundamental role in regulating cellular behaviors such as migration, proliferation, and differentiation(1, 2) . The organization of cell adhesion sites is directed by a family of transmembrane receptors known as integrins(3) . The binding of integrins to extracellular matrix ligands catalyzes the recruitment of multiple cytoskeletal associated proteins to the cytoplasmic face of cell attachment sites (focal adhesions) and thereby organizes the actin cytoskeleton(4, 5) . The molecular mechanisms by which cell adhesion triggers various cellular events have not yet been defined. However, evidence is emerging that implicates the tyrosine phosphorylation of certain focal adhesion proteins in at least one aspect of integrin-meditated signaling, that of cytoskeleton reorganization. In particular, the binding of integrins to extracellular matrix ligands results in an increase in tyrosine-phosphorylated forms of several focal adhesion proteins(6, 7) . Two focal adhesion proteins that demonstrate a high stoichiometry of tyrosine phosphorylation upon integrin activation are the nonreceptor tyrosine kinase, focal adhesion kinase (FAK)()(8, 9, 10, 11, 12) , and the 68-kDa protein, paxillin(8, 13) . The tyrosine phosphorylation of these two proteins has been suggested to be involved in both the formation of focal adhesions and the assembly of actin stress fibers(8) .

The coordinate phosphorylation of paxillin and FAK on tyrosine residues has also been observed in other systems. Cells transformed by Rous sarcoma virus (14) and tissues undergoing embryogenesis (15, 16) have been shown to contain enriched populations of tyrosine-phosphorylated forms of paxillin and FAK. Additionally, tyrosine-phosphorylated paxillin and FAK have been detected in cultured cells that have been treated with a variety of stimuli, including lysophosphatidic acid (17) , angiotensin II(18) , sphingosine(19) , low doses of platelet-derived growth factor(20) , and the neuropeptides bombesin, endothelin, and vasopressin(21, 22) . The multiple observations of concomitant phosphorylation of FAK and paxillin suggest that tyrosine phosphorylation of paxillin may be a direct consequence of FAK activity. Additional support for this hypothesis is provided by the finding that purified paxillin can be phosphorylated in vitro by a FAK immunoprecipitate(16) .

The paxillin cDNA has been recently cloned(23, 24) , and a region of the paxillin molecule has been identified that supports the binding of both vinculin and FAK(24) . An analysis of the paxillin amino acid sequence reveals the presence of additional domains, which are thought to function in protein-protein interactions. Among these domains are a proline-rich, putative SH3 binding motif(25, 26, 27) , four LIM domains (28) , and multiple tyrosine residues, which are likely to represent sites of phosphorylation, given that these residues lie within good consensus sequences for binding to proteins that contain SH2 domains (29) . In the present study, we have determined that one of the tyrosine residues that potentially comprises part of an SH2 binding domain for the adapter protein, Crk, is a primary site of paxillin phosphorylation by FAK in vitro.


MATERIALS AND METHODS

Synthesis of GST-Paxillin Fusion Proteins

Selected regions of paxillin cDNA were amplified from paxillin clone 10 (24) by polymerase chain reaction (PCR) using oligonucleotides containing 5`-BamHI or 3`-EcoRI restriction sites. The PCR products were subcloned into BamHI/EcoRI-digested pGEX-2T (Pharmacia Biotech Inc.). Verification that paxillin was subcloned into pGEX-2T in the correct reading frame was provided by dideoxy chain termination sequencing (Sequenase 2, U. S. Biochemical Corp.). The GST-paxillin fusion proteins were expressed and purified using glutathione (GSH)-Sepharose as described elsewhere(30) . For use in kinase assays, fusion proteins were eluted from GSH-Sepharose beads with 20 mM GSH in 50 mM Tris-HCl buffer (pH 8.0), then dialyzed overnight into kinase buffer (10 mM HEPES, 3 mM MnCl, pH 7.3).

In Vitro Kinase Assays

14-16-day-old embryonic chicken gizzards (a source of smooth muscle focal adhesion proteins) were homogenized in 10 volumes of buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Triton X-100, 0.1% deoxycholate, 10 µg/ml leupeptin, and 50 µM sodium orthovanadate (lysis buffer). Lysates were centrifuged at 100,000 g to remove insoluble material. FAK was immunoprecipitated by incubating the lysates for 90 min at 4 °C with anti-FAK monoclonal antibody 2A7 (gift from Dr. J. T. Parsons, University of Virginia), followed by the addition of rabbit, anti-mouse IgG complexed to protein A-Sepharose. The immune complexes were pelleted, washed extensively with lysis buffer, washed once with 10 mM HEPES, 3 mM MnCl, pH 7.3 (kinase buffer), then resuspended into 20 µl of kinase buffer. Approximately 5-20 µg of GST-paxillin fusion protein (previously dialyzed into kinase buffer) were added to the FAK immunoprecipitate with 10 µCi of [-P]ATP. In some kinase assays, 100 µM tyrphostin (Life Technologies, Inc.) was also added. Following a 20-min incubation at room temperature, kinase reactions were terminated by boiling the samples in SDS-PAGE sample buffer. Samples were resolved on 10% acrylamide gels by SDS-PAGE (31) and then visualized by autoradiography. Protein amounts were determined by using a Bio-Rad protein assay kit, as well as by Coomassie staining of SDS-PAGE gels.

To perform kinase assays with native paxillin, anti-paxillin monoclonal antibody 165 (32) and anti-FAK monoclonal antibody 2A7 were added simultaneously to gizzard lysates. Following a 90-min incubation at 4 °C, paxillin and FAK were coprecipitated by the addition of rabbit anti-mouse saturated protein A-Sepharose. The precipitates were washed and then resuspended in kinase buffer containing 10 µCi of [-P]ATP. As described above, kinase reactions proceeded for 20 min at room temperature, and the phosphorylated samples were resolved by electrophoresis.

Site-directed Mutagenesis

Phenylalanine substitutions for tyrosine residues were introduced into the paxillin sequence using a PCR method described by Landt et al.(33) . Mutated paxillin cDNA was subcloned into pGEX-2T as described above. The correct sequence of the mutated GST-paxillin fusion protein was verified by sequencing.

Western Blotting

GST, GST-paxillin 54-313, and GST-paxillin 54-313/Y118F, each coupled to GSH-Sepharose beads(30) , were incubated with embryonic chicken gizzard lysates for 90 min at 4 °C. Samples were pelleted and washed extensively with lysis buffer. Samples were resolved by electrophoresis on 10% acrylamide gels and transferred to nitrocellulose using standard procedures(34) . FAK and FRNK, an alternately spliced FAK isoform(6) , were detected by incubating nitrocellulose blots with anti-FAK polyclonal antibody, BC3 (gift from Dr. Tom Parsons, University of Virginia), followed by incubation with I-labeled goat anti-rabbit IgG.

Phosphopeptide Mapping

P-Labeled fusion proteins were eluted from dried SDS-PAGE gels, trypsinized, and subjected to two-dimensional phosphopeptide mapping according to standard methods(35) . Phosphopeptides were separated in the first dimension by thin layer electrophoresis in pH 1.9 buffer (35) and then resolved by thin layer chromatography in isobutyric acid buffer(35) . Performic acid oxidation of phosphopeptides was not performed prior to the two-dimensional analysis.

Transfection and Immunofluorescence

Chicken paxillin cDNA encoding amino acid residues 54-559 was amplified by PCR using paxillin clone 10 (24) as a template. PCR primers contained 5`-BamHI and 3`-EcoRI restriction sites to permit force cloning into the BamHI/EcoRI restriction sites of the eukaryotic expression vector, pcDNA3 (Invitrogen), a plasmid which contains a cytomegalovirus promoter. In addition, residue 58 of the paxillin sequence was mutated to methionine since transcription from the pcDNA3 vector is initiated at the first methionine residue within the insert. Mutation of tyrosine 118 to phenylalanine was as described above.

Transient transfections of NIH 3T3 cells, plated on coverslips, were performed using the CaPO precipitation method of Solowska et al.(36) . 24 h post-transfection, the cells were fixed and processed for immunofluorescence microscopy as previously described(32) . Expression and localization of transfected paxillin were detected using a chicken-specific polyclonal antisera, Pax 2(24) . Cells were double-labeled with rhodamine-phalloidin to visualize actin-containing stress fibers.

Nomenclature

Following our initial report of the cloning of paxillin and the identification of FAK binding to a partial paxillin GST-fusion protein(24) , we have identified, using 5`-rapid amplification of cDNA ends, an additional chicken cDNA sequence encoding 5 amino acids (MDDLD) at the amino terminus. Thus, all nomenclature used in this report is based on a full-length protein of 559 amino acids. The additional cDNA sequence (including 5`-untranslated sequence) has been deposited in GenBank. An identical 5-amino acid sequence was reported for human paxillin (23) following submission of this manuscript.


RESULTS AND DISCUSSION

Previously, a GST-paxillin fusion protein containing paxillin sequence from amino acids 54 through 313 was shown to support the binding of FAK (24) (see ``Materials and Methods'' regarding a change in nomenclature). This same fusion protein, GST-paxillin 54-313 (Fig. 1), was used in in vitro kinase assays with FAK immunoprecipitates. Phosphorylation of the GST-paxillin fusion protein (Fig. 2A, lane3), but not GST alone (Fig. 2A, lane2), was observed, confirming that the phosphorylation of the fusion protein was specific to the paxillin domain. Phosphorylation of immunoprecipitated FAK (i.e. no fusion protein added) was also observed (Fig. 2A, lanes4 and 5). The phosphorylation of FAK most likely represents an autophosphorylation event(37) . Excision of phosphorylated GST-paxillin 54-313 from SDS-PAGE gels and subsequent phosphoamino acid analyses revealed that the phosphorylation of the fusion protein was restricted to tyrosine residues (data not shown). Additionally, the phosphorylation of both paxillin and FAK was eliminated by the inclusion of a tyrosine kinase inhibitor, tyrphostin(38) , in the assay buffer (data not shown). Tyrphostin has been previously shown to inhibit FAK activity in vitro and in vivo(39) .


Figure 1: Synthesis of GST-paxillin 54-313. Schematic representation of the paxillin molecule and the region of the protein incorporated into a GST-paxillin fusion protein(24) . Numbers correspond to amino acid position. All of the tyrosine residues included within this fusion protein are indicated.




Figure 2: A, phosphorylation of GST-paxillin 54-313 and truncated variants of GST-paxillin 54-313. FAK was immunoprecipitated from embryonic chicken gizzard lysates with monoclonal antibody 2A7. After washing the immunoprecipitates, fusion proteins (previously dialyzed into kinase buffer) were added along with 10 µCi of [-P]ATP. Kinase reactions proceeded for 20 min at room temperature and were terminated by boiling in SDS-PAGE sample buffer. Proteins were resolved by electrophoresis on 10% acrylamide gels. The following protein samples were added to the FAK immunoprecipitates: GST (lane2), GST-paxillin 54-313 (lane3), no fusion protein added (lanes4 and 5), and truncated variants of GST-paxillin 54-313 (lane1 and lanes6-12). B, summary of results from kinase assays with truncated GST-paxillin. Kinase assays performed with truncated variants of GST-paxillin 54-313 indicated that the primary site of phosphorylation lies within amino acids 114-133.



To map the site(s) of paxillin phosphorylation by FAK, a series of truncated GST-paxillin fusion proteins was synthesized and used in in vitro kinase assays with FAK immunoprecipitates (Fig. 2, A and B). The original fusion protein, GST-paxillin 54-313, contained four tyrosine residues, Tyr-76, Tyr-88, Tyr-118, and Tyr-182. In vitro kinase assays using truncated variants of this original fusion protein indicated that phosphorylation of the fusion protein by the FAK immunoprecipitate occurred within a region of the paxillin molecule spanning amino acids 114-133. This finding implicated Tyr-118 as the most likely site of phosphorylation by the FAK immunoprecipitate.

To confirm that tyrosine 118 was the primary site of phosphorylation by the FAK immunoprecipitate, site-directed mutagenesis was used to alter individual tyrosine residues within the fusion protein to phenylalanine. Mutated fusion proteins were then used in in vitro kinase assays with one additional modification. All of the mutated fusion proteins contained paxillin sequence from amino acids 54-191 instead of the 54-313 fusion protein used in Fig. 1. The truncation of paxillin sequence from amino acid 313 to 191 was instituted because fusion protein 54-313 migrated to the same position as IgG heavy chain on SDS-PAGE gels. This factor made it difficult to quantify the fusion protein by Coomassie staining and thereby ensure that equivalent amounts of mutated fusion proteins were added to each kinase assay. GST-paxillin 54-191 contained all of the tyrosine residues that were present in 54-313 and, furthermore, no diminution of phosphorylation of 54-191 was observed with respect to 54-313 (Fig. 2A, lanes6 and 3, respectively).

In vitro kinase assays performed with mutated fusion proteins revealed that the phosphorylation of paxillin by the FAK immunoprecipitate was eliminated by introducing a phenylalanine substitution at tyrosine 118 (Fig. 3). In contrast, no diminution in the level of phosphorylation was observed in any of the other mutated proteins, indicating that the phosphorylation of GST-paxillin by FAK occurred on a single tyrosine residue, Tyr-118. It is unlikely that the inhibition in phosphorylation of the Y118F mutant was due to a mutation-induced perturbation in the folding of the paxillin molecule because the Y118F mutant was shown by Western blot analysis to bind both FAK and FRNK, a truncated variant of FAK(6) , equally as well as the wild type fusion protein (Fig. 4).


Figure 3: In vitro kinase assays with mutated GST-paxillin. Site-directed mutagenesis was used to alter individual tyrosine residues within GST-paxillin 54-191 to phenylalanine. Four mutated fusion proteins were synthesized, corresponding to the four tyrosine residues within GST-paxillin 54-191, Y76F, Y88F, Y118F, and Y182F. Mutated fusion proteins were used in in vitro kinase assays with FAK immunoprecipitates as described under ``Materials and Methods.''




Figure 4: Western blot of FAK precipitated by GST-paxillin fusion proteins. GST (lane2), GST-paxillin 54-313 (lane3), and GST-paxillin 54-313 containing a phenylalanine substitution at tyrosine 118 (lane4) were incubated with embryonic chicken gizzard lysates for 90 min at 4 °C. GST and GST-fusion proteins were precipitated with GSH-Sepharose, washed, and resolved by SDS-PAGE. A sample of the embryonic chicken gizzard lysate was also electrophoresed (lane1). Following electrophoresis, proteins were transferred to nitrocellulose, and both FAK and FRNK, a truncated variant of FAK(6) , were detected by Western blotting using anti-FAK polyclonal antisera (BC3). The position of molecular weight standards are indicated on the left.



The segment of paxillin used in the previous experiments represents approximately 50% of the paxillin sequence (the expression of full-length paxillin as a GST-fusion protein resulted in an insoluble product). Because a significant portion of the paxillin sequence was excluded from the fusion proteins used in our assays, it was important to determine if the phosphorylation of the fusion protein by the FAK immunoprecipitate reflected the in vitro phosphorylation of native paxillin by FAK. To address this question, both GST-paxillin 54-313 and native paxillin were phosphorylated in vitro by the FAK immunoprecipitate. The phosphorylated proteins were excised from SDS-PAGE gels, trypsinized, and subjected to two-dimensional phosphopeptide analysis. As shown in Fig. 5, multiple radiolabeled spots were observed in the maps of both native paxillin and GST-paxillin fusion protein. Given that previous data ( Fig. 2and 3) suggested that phosphorylation occurred at a single site (Tyr-118), it is likely that multiple spots, at least in the case of the fusion protein, resulted from incomplete tryptic digests and/or differences in the oxidation state of a single phosphopeptide. A comparison of the two phosphopeptide maps revealed that the patterns of spots were superimposable, confirming that the phosphorylation of native paxillin occurred at essentially the same site(s) as the fusion protein. Variation in the relative intensities of individual phosphopeptides are likely due to the differential accessibility of trypsin sites in the native paxillin versus the GST-paxillin fusion protein. Alternatively, these differences may reflect the phosphorylation of paxillin at a second site, which was labeled more efficiently in the native molecule.


Figure 5: Phosphopeptide analysis of native paxillin and GST-paxillin fusion protein. Both native paxillin and GST-paxillin 54-313 were phosphorylated in vitro by FAK immunoprecipitates. The phosphorylated proteins were excised from SDS-PAGE gels and subjected to thin layer electrophoresis (E) in pH 1.9 buffer(35) , followed by thin layer chromatography (C) in isobutyric acid buffer(35) .



The phosphopeptide analysis yielded two important findings. First, phosphopeptide 2 represented one of the most intensely labeled phosphopeptides in both maps, implicating the same tyrosine residue, Tyr-118, as a principle site of phosphorylation in both native paxillin and the GST-paxillin fusion protein. Second, no novel phosphopeptides were noted in the map of native paxillin as compared with the fusion protein, suggesting that all of the primary sites of paxillin phosphorylation by the FAK immunoprecipitate were present within fusion protein 54-313. To further consider the possibility that tyrosine residues not included in GST-paxillin 54-313 might be substrates for phosphorylation by FAK, we performed in vitro kinase assays with several additional fusion proteins: GST-paxillin 11-79 (proline-rich region), GST-paxillin 323-383 (LIM 1), GST-paxillin 382-445 (LIM 2), GST-paxillin 439-496 (LIM 3), and GST-paxillin 501-559 (LIM 4). No significant phosphorylation of any of these fusion proteins by FAK immunoprecipitates was observed (data not shown), supporting our hypothesis that Tyr-118 is the primary site of phosphorylation by FAK invitro. While this manuscript was under review, the identification of Tyr-118 as a substrate for phosphorylation by FAK was corroborated by a report indicating that Tyr-118 of paxillin is phosphorylated in cultured cells that overexpress FAK(40) . In this same report, however, Tyr-31 was also implicated as a potential phosphorylation site. In contrast with this finding, we have not detected any phosphorylation of paxillin on Tyr-31 under the conditions used in our in vitro kinase assays.

In view of the previous observation that focal adhesion formation is correlated with the tyrosine phosphorylation of paxillin and FAK(8) , we were interested in determining if the phosphorylation of paxillin on tyrosine 118 was necessary for the localization of paxillin to focal adhesions. Consequently, paxillin cDNA and paxillin cDNA containing a phenylalanine substitution at tyrosine 118 were transfected into NIH 3T3 cells (Fig. 6). These cDNA constructs contained chicken paxillin sequence coding for amino acids 54-559. Similar to nonmutated paxillin (panelA), the Y118F mutant paxillin was detected in focal adhesions by the chicken-specific antibody (panelC), indicating that phosphorylation at tyrosine 118 was not required for the recruitment of paxillin to adhesion sites. It is also noteworthy that the proline-rich, putative SH3 binding domain of paxillin (amino acids 46-55) does not appear to be necessary for localization, since the amino acids coding for this domain were not contained within the transfected cDNA constructs. This is of interest because previous studies have shown an interaction between paxillin and the SH3 domain of Src(41) , a tyrosine kinase that has been identified in focal adhesions(42) . Paxillin is a multi-domain protein containing binding sites for several focal adhesion proteins. It is therefore likely that the transfected paxillin constructs are recruited to focal adhesions through associations with vinculin and/or FAK or via interactions involving the paxillin LIM domains. Clearly, the phosphorylation of paxillin on Tyr-118 is not essential for the localization of paxillin to pre-existing focal adhesion sites; however, the possible requirement for phosphorylation of Tyr-118 in the initial assembly of focal adhesions remains to be determined, given that endogenous paxillin is present in NIH 3T3 cells.


Figure 6: Transfection of paxillin containing a phenylalanine substitution at Tyr-118. NIH 3T3 cells were transiently transfected with either chicken paxillin cDNA encoding amino acids 54-559 (panelsA and B) or with the same construct containing a phenylalanine substitution at tyrosine 118 (Y118F) (panelsC and D). PanelsE and F represent nontransfected NIH 3T3 cells. Cells in panelsA, C, and E were labeled with chicken-specific anti-paxillin antisera (see ``Materials and Methods''), followed by incubation with a fluorescein-conjugated secondary antibody. Cells in panelsB, D, and F were double labeled with rhodamine-conjugated phalloidin to visualize actin-containing stress fibers. Bar, 5 µm.



Another potential function of paxillin phosphorylation on Tyr-118 may involve the recruitment of other signaling molecules to focal adhesion sites. Phosphotyrosine residues typically serve to provide docking sites for proteins containing SH2 domains and thereby facilitate the formation of signaling complexes. It has been established that the three amino acids immediately following a phosphotyrosine residue confer a specificity for binding to particular SH2 domains(29) . An examination of the amino acid sequence following tyrosine 118 of paxillin (Y118SFP) suggests that phosphorylation at this site is likely to be responsible for mediating the known association between paxillin and the SH2 domain of the oncogenic protein, v-Crk(43) . This hypothesis is supported by a recent report demonstrating an interaction between paxillin phosphorylated on Tyr-118 and the SH2 domain of Crk (40) .

In summary, we have identified a specific tyrosine residue within paxillin that is a primary target for phosphorylation by FAK and have further demonstrated that the phosphorylation of this residue is not essential for the localization of paxillin to focal adhesion sites. Additional experiments will be required to elucidate signaling events that may lie downstream of paxillin phosphorylation by FAK.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM47607 and a grant from the Muscular Dystrophy Association. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L30099[GenBank® Link].

§
An American Heart Association Postdoctoral Fellow.

An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, State University of New York Health Science Center, 750 E. Adams St., Syracuse, NY 13210. Tel.: 315-464-8598; Fax: 315-464-8535.

The abbreviations used are: FAK, focal adhesion kinase; GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are indebted to Dr. J. Tom Parsons (University of Virginia) for the generous gift of antibodies against FAK. We also thank Dr. Michael Brown for critical reading of this manuscript.


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