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Cell Growth & Differentiation Vol. 11, 41-48, January 2000
© 2000 American Association for Cancer Research


Articles

Phosphospecific Antibodies Reveal Focal Adhesion Kinase Activation Loop Phosphorylation in Nascent and Mature Focal Adhesions and Requirement for the Autophosphorylation Site1

Paul J. Ruest, Shyamali Roy, Ergang Shi, Raymond L. Mernaugh and Steven K. Hanks2

Departments of Cell Biology [P. J. R., S. R., S. K. H.] Biochemistry [R. L. M.], and The Vanderbilt-Ingram Cancer Center [E. S., R. L. M., S. K. H], Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Abstract

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.

Introduction

Cell adhesion to components of the ECM3 triggers signaling events affecting diverse cellular traits and activities including survival, proliferation, differentiation, and migration. FAK has emerged as a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin-ligand engagement promotes FAK tyrosine phosphorylation at multiple sites, which prompts FAK signaling activity (reviewed in Refs. 1 and 2 ). Phosphorylation of FAK Tyr-397, the only apparent autophosphorylation site, creates a high-affinity binding site for SH2 domains of Src-family kinases including c-Src and Fyn (3, 4, 5) . This interaction likely contributes both to the recruitment of Src-family kinases to sites of cell adhesion and to their catalytic activation through release of inhibitory intramolecular interactions (6) . Phosphorylated Tyr-397 also binds to SH2 domains from other effector molecules, including phosphatidylinositol-3-kinase (7) , phospholipase C-{gamma} (8) , and Shc (9) , indicating a multifunctional signaling role for this site. Tyr-397 is not strictly an autophosphorylation site but can also be phosphorylated by Src (10) . Other known FAK phosphoacceptor tyrosines are also phosphorylated by Src but are not sites of autophosphorylation (10) . Among these Src-specific sites are FAK tyrosines 576 and 577, which lie within the FAK kinase domain activation loop (10) , a region recognized as a general target for phosphorylation-mediated regulation of protein kinase activity. Consistent with Tyr-576/577 phosphorylation being required for maximal FAK kinase activity, phenylalanine mutations of these residues result in a 2–3-fold reduction of kinase activity (10, 11, 12) .

Evidence indicates that FAK signaling significantly influences cell behavior. FAK overexpression enhances migration of Chinese hamster ovary cells, requiring both FAK Tyr-397 (13) and a binding site for p130Cas (Crk-associated substrate; 14 ). A requirement for FAK Tyr-397 in a cell spreading response involving the substrate paxillin has been indicated (15) . The FAK activation loop tyrosines 576/577, in addition to Tyr-397, were found to be important for maximal cell spreading and migration responses in an inducible expression system (12) . FAK has been implicated in anchorage-dependent cell survival (16, 17, 18) , requiring both FAK kinase activity and Tyr-397 (16) in a signaling response that may suppress the p53-regulated cell death pathway (19) . Finally, FAK overexpression accelerates cell cycle progression through the G1 phase, and Tyr-397 phosphorylation has again been implicated in this response (20 , 21) .

In this report, we describe the production of phosphospecific polyclonal antibodies against the FAK activation loop and the use of these antibodies, together with another phosphospecific antibody against the FAK autophosphorylation site, to examine the mechanism of FAK activation and the localization of activated FAK within spreading and migrating fibroblasts. Our results support a model for signal amplification by reciprocal activation of FAK and Src-family kinases and indicate that FAK exists in its active signaling form at both nascent and mature focal adhesions.

Results

FAK Phosphospecific Antibody Characterization and Analysis of FAK Phosphorylation in Cos-7 Cells Coexpressing Activated Src.
Polyclonal antibodies were prepared from rabbits immunized with peptides, derived from the FAK activation loop, in which phosphotyrosines were incorporated in place of either Tyr-576 alone or both Tyr-576 and Tyr-577 (see "Materials and Methods"). To determine the specificity of these pFAK576 and pFAK576/577 antibodies, as well as a commercial polyclonal antibody generated against the FAK phosphoTyr-397 site (pFAK397), immunoblot analyses were performed on whole-cell lysates of Cos-7 cells expressing WT FAK with a COOH-terminal HA epitope tag (WT-FAKHA) versus various FAKHA mutants (F397-FAKHA, F576-FAKHA, F577-FAKHA, and FF576/F577-FAKHA) in which phosphoacceptor tyrosines were substituted with phenylalanine (Fig. 1)Citation . The FAKHA variants were coexpressed with a "kinase active" form of c-Src in which the COOH-terminal regulatory tyrosine was changed to phenylalanine (KA Src; Fig. 1ECitation ) to achieve high levels of total FAK phosphotyrosine, as assessed using the 4G10 anti-phosphotyrosine antibody (Fig. 1Citation D, compare Lanes 2 with 3 where a "kinase dead" (KD) Src, lacking the conserved lysine important for ATP binding, was coexpressed). Equal loading of the lysates was shown by immunoblotting with the FAK C-20 antibody (Fig. 1G)Citation , which recognizes endogenous Cos-7 FAK but does not recognize the FAKHA variants because of disruption of the C-20 epitope by the HA tag. The near-equal expression of the FAKHA variants was shown by immunoblotting with the 12CA5 antibody against the HA epitope (Fig. 1F)Citation .



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Fig. 1. Immunoblot characterization of FAK phosphospecific antibodies. HA epitope-tagged WT mouse FAK (WT-FAKHA) was transiently coexpressed in Cos-7 cells together with either "kinase active" (KA) or "kinase dead" (KD) mouse c-Src mutants (Src), and whole-cell lysates were subjected to immunoblot (IB) analysis using antibodies raised against phosphotyrosine-containing peptides representing either the FAK activation loop or autophosphorylation sites. Also analyzed were mock-transfected cells and cells expressing KA-Src together with an HA-tagged FAK mutant (either F397-FAKHA, F576-FAKHA, F577-FAKHA, or F576F577-FAKHA) in which phosphoacceptor tyrosine(s) were changed to phenylalanine(s). Seven separate blots containing near-equal amounts of each of the lysates were prepared for detection with either pFAK576/577 polyclonal antiserum (A), pFAK 576 polyclonal antiserum (B), pFAK397 polyclonal antibody (C), anti-phosphotyrosine monoclonal antibody 4G10 (D), Src monoclonal antibody 327 (E), anti-HA monoclonal antibody 12CA5 (F), or FAK polyclonal antibody C-20, which only recognizes the endogenous FAK (G). Bound antibodies were visualized using enhanced chemiluminescence with exposure times ranging from 5–10 s (D and E) to 1–5 min (B, F, and G) to 10 min (A and C). The panels show only regions of the developed film corresponding to the position of either FAK (A–D, F, and G) or Src (E).

 
Each of the three pFAK antibodies detected WT-FAKHA in a manner that was enhanced by coexpression of KA Src (Fig. 1, A–CCitation , compare Lanes 2 and 3), indicating that the antibodies recognize FAK tyrosine sites phosphorylated by Src. Although total cell lysates were used in this analysis, at the exposures shown in Fig. 1Citation the immunoreactivity of each of the three pFAK antibodies was limited to the region of the blots corresponding to the position of FAKHA, and no signal was detected in the mock-transfected sample (Lane 1), although the endogenous Cos-7 FAK could be weakly detected upon prolonged exposures. The specificities of the pFAK antibodies for the individual phosphotyrosine sites were demonstrated by lack of recognition of the relevant FAKHA mutants. Thus, the pFAK397 antibody failed to detect F397-FAKHA, although coexpression of KA Src resulted in some tyrosine phosphorylation of this variant, as detected by 4G10 (Fig. 1, CCitation and D, Lane 5). Similarly, the pFAK576 antibody failed to recognize either F576-FAKHA or F576/F577-FAKHA but still recognized F577-FAKHA (Fig. 1Citation B, Lanes 4, 6, and 7). The pFAK576/577 antibody provided no detectable signal when either WT-FAKHA was coexpressed with KD Src or when any of the phosphorylation site mutants was expressed with KA Src (Fig. 1A)Citation . The signal generated by the pFAK576/577 antibody was weak, relative to that of pFAK576. This may be attributable in part to the lower stoichiometry of dually phosphorylated Tyr-576 and Tyr-577 residues compared with singly phosphorylated Tyr-576 (10) .

The Cos-7 immunoblot analyses also provided evidence for FAK activation loop and autophosphorylation sites being regulated in a manner partially dependent upon one another. Thus, pFAK576 and pFAK576/577 antibody recognition of the F397-FAKHA variant was much reduced, relative to WT-FAKHA (Fig. 1, ACitation and B, compare Lanes 2 and 5). Similarly, pFAK397 antibody recognition of FAKHA was reduced by the F576 and/or F577 mutations (Fig. 1Citation C, compare Lane 2 with Lanes 4, 6, and 7).

FAK Activation Loop Phosphorylation Is Elevated in Src-transformed Cells.
Our pFAK576 and pFAK576/577 antibodies were next used to determine whether the FAK activation loop sites were phosphorylated to higher levels in Src-transformed cells. Immunoprecipitates were prepared from lysates of normal NIH 3T3 mouse fibroblasts versus their counterparts transformed by F527-Src using antibody C-20 against the FAK COOH terminus, and immunoblot analyses were performed using either of the phosphospecific antibodies. Control immunoblots revealed the equal recovery of FAK from the lysates and the expected elevation of total FAK phosphotyrosine and retarded electrophoretic mobility in the transformed cells (Fig. 2, C and D)Citation . Both the pFAK576 and the pFAK576/577 antibodies gave significantly stronger signals in the Src-transformed cells relative to nontransformed cells (Fig. 2, A and B)Citation , indicating that FAK activation loop phosphorylation is elevated by expression of the constitutively active Src mutant. The pFAK576/577 antibody was unable to detect FAK from the nontransformed cells (Fig. 2A)Citation , suggesting that very little dual phosphorylation of the activation loop occurs in these cells under normal growth conditions.



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Fig. 2. Elevated FAK activation loop phosphotyrosine content in Src-transformed NIH 3T3 fibroblasts. FAK was immunoprecipitated (IP) from RIPA buffer lysates (260 µg of total protein) obtained from either normal mouse NIH 3T3 fibroblasts or NIH 3T3 fibroblasts transformed with F527 chicken c-Src. Four separate blots containing equal amounts of the two immunoprecipitates were prepared for immunoblot (IB) detection with either pFAK576/577 polyclonal antiserum (A), pFAK 576 polyclonal antiserum (B), anti-phosphotyrosine monoclonal antibody 4G10 (C), or FAK polyclonal antibody C-20 (D). Bound antibodies were visualized using enhanced chemiluminescence with exposure times ranging from 15 s (C and D) to 2 min (A and B).

 
FAK Activation Loop Phosphorylation Is an Early Response to Integrin-mediated Cell Adhesion.
To examine the effects of integrin stimulation on FAK activation loop phosphorylation, a fibronectin replating experiment with NIH 3T3 fibroblasts was performed. FAK immunoprecipitates were prepared with C-20 antibody from RIPA buffer lysates of cells either attached under normal growth conditions, trypsinized and held in suspension for 30 min, or replated onto fibronectin-coated dishes and allowed to respread for various amounts of time (Fig. 3)Citation . Near-equal FAK recovery from the samples was shown by immunoblot analysis of the immunoprecipitates using the C-20 antibody (Fig. 3D)Citation . Immunoblot detection with the pFAK576 antibody indicated that FAK Tyr-576 is phosphorylated under attached conditions, becomes dephosphorylated upon cell suspension, and then becomes rapidly rephosphorylated, recovering to the levels of the attached growing cells within 30 min of fibronectin replating (Fig. 3B)Citation . The dynamics of Tyr-576 phosphorylation during this experiment was similar to that of total FAK tyrosine phosphorylation, as assessed by analysis of the same samples with 4G10 antibody (Fig. 3C)Citation . The pFAK576/577 antibody detected FAK poorly, but weak reactivity was observed in the adherent samples (Fig. 3A)Citation . These results indicate that FAK activation loop phosphorylation, primarily of the Tyr-576 site, is part of the initial rapid FAK tyrosine phosphorylation in response to integrin-mediated cell adhesion.



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Fig. 3. Integrin stimulation enhances FAK activation loop phosphorylation in NIH 3T3 fibroblasts. FAK was immunoprecipitated (IP) from RIPA buffer lysates (340 µg of total protein) obtained from nontransformed NIH 3T3 cells either adherent on tissue culture dishes (Attached), trypsinized and held in suspension for 30 min (Suspended), or replated onto fibronectin (FN) for 0.5, 1, 2, or 4 h as indicated. Four separate blots containing equal amounts of the immunoprecipitates were prepared for immunoblot (IB) detection with either pFAK576/577 polyclonal antiserum (A), pFAK 576 polyclonal antiserum (B), anti-phosphotyrosine monoclonal antibody 4G10 (C), or FAK polyclonal antibody C-20 (D). Bound antibodies were visualized using enhanced chemiluminescence with exposure times ranging from 1 min (C and D) to 10 min (A and B).

 
Adhesion-dependent FAK Activation Loop Tyrosine Phosphorylation Is Largely Dependent on Tyr-397.
The pFAK antibodies were next used in conjunction with Tet-FAK(WT) and Tet-FAK(F397) mouse fibroblasts, which inducibly express either WT-FAK or F397-FAK, respectively, in a FAK-null background (12) .

These cells allow a direct test of the hypothesis that FAK Tyr-397 phosphorylation and subsequent Src recruitment to this site are required to achieve phosphorylation of the FAK activation loop during an integrin-stimulated signaling response. The Tet-FAK cells were induced to express WT-FAK or F397-FAK to near-equal levels; then the FAK variants were immunoprecipitated from lysates of attached, suspended, and fibronectin-replated cells, and immunoblot analyses were performed (Fig. 4)Citation . Similar to results obtained from NIH 3T3 fibroblasts, WT-FAK expressed in the Tet-FAK cells exhibited adhesion-dependent elevation of phosphotyrosine content including the Tyr-576 site in the activation loop (Fig. 4Citation , right panel). In striking contrast, F397-FAK accumulated little or no phosphotyrosine, including at the activation loop site, under adherent conditions (Fig. 4Citation , left panel). These results provide convincing evidence that FAK Tyr-397 phosphorylation and Src-family kinase recruitment to this site are necessary steps leading to phosphorylation of other FAK tyrosine sites, including those in the activation loop.



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Fig. 4. FAK Tyr-397 is required for adhesion-regulated phosphorylation of activation loop tyrosines. FAK was immunoprecipitated (IP) from RIPA buffer lysates (260 µg of total protein) obtained from induced Tet-FAK(F397) or Tet-FAK(WT) cells under attached, suspended, or fibronectin-replated conditions and characterized by immunoblot (IB) analysis as described in the legend to Fig. 3Citation . Bound antibodies were visualized using enhanced chemiluminescence with exposure times ranging from 10 s (D) to 3 min (C) to 10 min (A and B).

 
Maximal Adhesion-dependent FAK Tyr-397 Phosphorylation Requires Tyr-576/577.
Tet-FAK(F576/F577) cells, which inducibly express F576/F577-FAK (12) , were similarly used together with the pFAK397 antibody to assess the role of FAK activation loop phosphorylation in regulating adhesion-dependent FAK Tyr-397 phosphorylation. In parallel experiments, WT-FAK and F576/F577-FAK were induced to comparable levels, and FAK was immunoprecipitated from lysates of attached, suspended, and fibronectin-replated cells (Fig. 5)Citation . Consistent with our previous report (12) , F576/F577-FAK shows reduced phosphotyrosine content in adherent cells, relative to WT-FAK (Fig. 5B)Citation . This is most evident for the early time points after fibronectin replating. Immunoblot analysis of the immunoprecipitates with the pFAK397 antibody indicated that Tyr-397 phosphorylation is also reduced in the F576/F577-FAK mutant, relative to WT-FAK. These results further support the hypothesis that FAK activation loop phosphorylation elevates autophosphorylation activity as part of an integrin signaling response.



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Fig. 5. FAK activation loop tyrosines are required for maximal adhesion-regulated phosphorylation of Tyr-397. FAK was immunoprecipitated (IP) from RIPA buffer lysates (200 µg of total protein) obtained from induced Tet-FAK(WT) or Tet-FAK(F576F577) cells under attached, suspended, or fibronectin-replated conditions as described in the legend to Fig. 3Citation and then characterized by immunoblot (IB) analysis using either pFAK397 polyclonal antibody (A), anti-phosphotyrosine monoclonal antibody 4G10 (B), or FAK polyclonal antibody C-20 (C). Bound antibodies were visualized using enhanced chemiluminescence with exposure times ranging from 10 s (C) to 2 min (A) to 10 min (B).

 
Phosphospecific FAK Antibodies Prominently Stain Both Newly Forming and Mature Focal Adhesions.
To further characterize the phosphospecific FAK antibodies and to examine possible differences in subcellular localization of tyrosine-phosphorylated FAK relative to total FAK, we carried out indirect immunofluorescence microscopic analysis of induced Tet-FAK(WT) fibroblasts. The analyses were carried out on either early adherent cells, allowed to attach and spread for 30 min in serum-free medium after plating onto fibronectin-coated coverslips, or on growing cells incubated on coverslips overnight in serum-containing medium. The cells were fixed and costained with a phosphospecific FAK antibody (pFAK576 or pFAK397) together with a monoclonal FAK antibody (FAK-TL), which does not show a preferential recognition of tyrosine-phosphorylated FAK (22 , 23) . In cells actively spreading on fibronectin, the pFAK576 antibody (Fig. 6A)Citation or pFAK397 antibody (not shown) readily detected the nascent punctate adhesive contacts enriched at the cell periphery. In overnight cultures, fully spread cells commonly exhibited the characteristic polarized migrating morphology, and both the pFAK576 (Fig. 6C)Citation and pFAK397 (Fig. 6E)Citation antibodies stained the prominent peripheral focal adhesions of these cells. Notably, the pFAK antibodies did not distinguish between focal adhesions in the broad leading edge lamellipodia and those in the narrower trailing tails. In all cells examined, tyrosine-phosphorylated FAK recognized by the pFAK antibodies had a cellular localization closely resembling that of total FAK detected by the FAK-TL antibody (Fig. 6Citation , compare panels A, C, and E with B, D, and F). Similar data were obtained when the pFAK antibodies were used to stain NIH-3T3 cells (data not shown). In uninduced Tet-FAK(WT) cells not expressing FAK, neither the pFAK576 nor pFAK397 antibodies detected nascent or mature focal adhesions, although these structures were readily recognized by an antibody against vinculin (data not shown). Taken together, these results provide further evidence for the specificity of the pFAK antibodies and indicate that tyrosine-phosphorylated forms of FAK (those likely to be actively engaged in signaling functions) are present in both newly forming and mature focal adhesions.



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Fig. 6. Immunolocalization of tyrosine-phosphorylated FAK. Induced Tet-FAK(WT) cells were either plated on fibronectin-coated coverslips and allowed to spread for 30 min in the absence of serum (A and B) or cultured overnight on glass coverslips in the presence of serum (C–F). Double-label indirect immunofluorescence was carried out by using a monoclonal antibody against FAK (B, D, and F) and either polyclonal antibody pFAK576 (A and C) or polyclonal antibody pFAK397 (E).

 
Discussion

In this report, we describe phosphospecific polyclonal antibodies generated against the FAK kinase domain activation loop and their use in experimental procedures involving immunoblot analysis and cell immunostaining. The antibodies, designated pFAK576 and pFAK576/577, were generated by immunizing rabbits with conjugated peptides corresponding to mouse FAK residues 571–582 (MEDSTYYKASKG), in which phosphotyrosines were incorporated in place of either Tyr-576 or both Tyr-576 and Tyr-577 residues. This sequence is absolutely conserved in all known FAK orthologues described in other vertebrate species including Xenopus, chicken, rat, and human. Thus, antibodies recognizing this region should detect phosphorylated FAK from a variety of vertebrate species. Moreover, the antibodies may not cross-react with the FAK-related kinase Pyk2/Cakß/RAFTK/CADTK, which is significantly divergent in this region, with the corresponding sequence being IEDEDYYKASVT. Because FAK Tyr-576 and Tyr-577 have been recognized as sites of phosphorylation by Src (10) , rather than autophosphorylation sites, phosphospecific antibodies targeting these residues will detect FAK molecules that have been activated in their signaling capacity through their association with and phosphorylation by Src-family kinases.

The specificity of the pFAK576 and pFAK576/577 polyclonal antibodies was demonstrated in Cos-7 cell expression experiments, where enhanced immunoblot recognition of HA-tagged FAK was observed upon coexpression of a catalytically active Src kinase and by the complete loss of this immunoreactivity when the relevant FAK activation loop tyrosines were mutated to phenylalanine. Data from Src-transformed NIH 3T3 cells further demonstrated a relationship between Src activity and pFAK576 and pFAK576/577 antibody recognition. In Cos-7 cells, the pFAK576 and pFAK576/577 antibodies failed to recognize the FAK activation loop mutants, although these mutants were significantly phosphorylated on other tyrosine sites, as shown by their efficient detection with the 4G10 anti-phosphotyrosine antibody. Thus, the FAK phosphospecific antibodies do not recognize phosphotyrosine-containing motifs in a nonspecific manner. Further indicating specificity of the pFAK576 antibody was the observation that no significant immunoreactivity was observed when the antibody was used in immunostaining experiments of uninduced Tet-FAK cells in which FAK was not expressed. This is also evidence for a lack of cross-reactivity with Pyk2/Cakß/RAFTK/CADTK, which is highly expressed and tyrosine phosphorylated in these cells (12) . In immunoblot detection experiments, the pFAK576 antibody consistently demonstrated a much greater recognition of FAK as compared with the pFAK576/577 antibody. This is likely a reflection of higher in vivo stoichiometric levels of singly phosphorylated Tyr-576 relative to doubly phosphorylated Tyr-576/Tyr-577. This conclusion is consistent with FAK phosphopeptide mapping studies, which indicated that Tyr-576 is a major site of FAK phosphorylation in vivo and a preferred in vitro Src site, relative to Tyr-577 (10) . The pFAK576/577 antibody may also have a relatively low affinity for the doubly phosphorylated activation loop site, which could contribute to the weak signal observed.

Our data showing a striking reduction of pFAK576 and pFAK576/577 antibody immunoreactivity with FAK from both Cos-7 and Tet-FAK cells expressing the F397-FAK mutant indicate that FAK activation loop phosphorylation is largely dependent on the autophosphorylation site. This observation, considered together with our other findings that immunoblot detection with the pFAK576 and pFAK576/577 antibodies is enhanced under conditions of high Src kinase activity, strongly indicate that in vivo FAK activation loop phosphorylation is primarily achieved through the direct SH2-mediated association of Src-family kinases with the Tyr-397 site, as speculated previously (10) . Our data from Cos-7 and Tet-FAK cells expressing the F576/F577-FAK mutant further indicate that the FAK activation loop tyrosines are required to achieve maximal Tyr-397 phosphorylation. The dependency of Tyr-397 phosphorylation upon the activation loop tyrosines was not as dramatic as that of activation loop phosphorylation dependency on Tyr-397. This is consistent with past observations that FAK autophosphorylation activity is only partially lost in F576/F577-FAK mutants (12) . FAK detection with the pFAK397 antibody could also reflect the phosphorylation of this site by Src-family kinases bound elsewhere within the complex.

In fibronectin-replating experiments, both pFAK576 and pFAK397 antibodies detected FAK from spreading cells with kinetics indistinguishable from total FAK tyrosine phosphorylation, as detected by the 4G10 antibody. Moreover, both pFAK576 and pFAK397 antibodies recognized the early focal contacts apparent during initial cell spreading on fibronectin. Thus, phosphorylation of both the FAK activation loop and autophosphorylation sites appear to be part of the initial integrin-stimulated FAK signaling response that occurs at new sites of cellular contact with the ECM. Our results also indicate that FAK phosphorylated at both the activation loop and autophosphorylation sites is present in mature focal adhesions, as evidenced by immunostaining analyses of growing polarized cells, where both pFAK576 and pFAK397 antibodies detected focal adhesions at both the leading and trailing edges of apparently motile cells. These findings indicate that FAK signaling complexes are not uniquely distributed among the contacts of polarized migrating fibroblasts. Rather, they raise the possibility that FAK signaling occurs at many areas of cell-ECM contact, including new contacts made as lamellipodia extend from the leading edge and at fully developed contacts at both the leading edges and trailing tails. This is consistent with previous studies suggesting a role for FAK signaling in regulating both focal adhesion formation associated with cell spreading and focal adhesion turnover associated with cell motility (12 , 24 , 25) . Further studies will be needed to determine whether distinct FAK signaling functions occur during focal adhesion assembly and maturation as a result of differences in stoichiometry of phosphorylation at the activation loop, autophosphorylation, and other FAK tyrosine phosphoacceptor sites and their association with downstream effector proteins.

In summary, our findings provide new experimental support for a model of reciprocal catalytic activation of FAK and Src-family kinases (12) . According to this model, association with the FAK autophosphorylation site promotes catalytic activation of Src-family kinases, leading to phosphorylation of the FAK activation loop. This in turn enhances FAK kinase activity and intermolecular autophosphorylation, resulting in signal amplification at sites of integrin-mediated cell adhesion. FAK activation loop phosphospecific antibodies and antibodies directed against other sites of FAK phosphorylation represent a new tool for study of FAK signaling. The antibodies should be particularly useful in revealing areas of active FAK signaling in intact tissues including developing embryos, tumors, and other normal or diseased tissues undergoing dynamic changes resulting from cell adhesion and other extracellular stimuli.

Materials and Methods

Antibodies.
Anti-FAK rabbit polyclonal antibody C-20 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FAK monoclonal antibody, designated FAK-TL, was obtained from Transduction Laboratories (Lexington, KY). Anti-Src monoclonal antibody 327 was kindly provided by A. Reynolds, Vanderbilt University. Anti-phosphotyrosine monoclonal antibody 4G10 was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-FAK pTyr-397 (pFAK397) was from Biosource International, Inc. (Hopkinton, MA). Polyclonal antibodies, recognizing phosphorylated FAK activation loop tyrosines, were raised in rabbits immunized with peptides conjugated to diphtheria toxoid. The peptide immunogens were either MEDSTpYYKASKGC or MEDSTpYpYKASKGC (single letter code, pY standing for phosphoTyr) for production of pFAK576 or pFAK576/577 antisera, respectively. Rabbits were initially injected s.c. with 100 µg of peptide conjugate in RIBI (ImmunoChem Research, Inc., Hamilton, MT) adjuvant and subsequently boosted (biweekly or monthly) with 100 µg of peptide conjugate in PBS.

Cells and Cell Culture.
Cos-7 cells were obtained from Steve Hann, Vanderbilt University. NIH 3T3 and F527 Src-transformed NIH 3T3 fibroblasts were obtained from A. Reynolds, Vanderbilt University. These cell lines were cultured in DMEM supplemented with 10% calf serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin and were maintained at 37°C in a humidified 5% CO2 incubator.

Tet-FAK(WT), Tet-FAK(F397), and Tet-FAK(F576/577) are mouse embryo fibroblasts derived from FAK-null embryos and engineered to inducibly express either WT-FAK, F397-FAK, or F576/577-FAK, respectively, under the control of the tetracycline repression system (12) . All Tet-FAK cells were maintained at 37°C in a humidified 5% CO2 incubator in DMEM containing 4500 mg/l D-glucose and 584 mg/l L-glutamine and was further supplemented with 1 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Amphotericin B, 1 mM nonessential amino acids (all from Life Technologies, Inc., Grand Island, NY), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA). Tetracycline (Calbiochem, La Jolla, CA) was included in the culture media to maintain Tet-FAK cells in the "uninduced" state. FAK expression was induced by removal of tetracycline from the culture medium. FAK expression levels were controlled by varying the duration cells were cultured in the absence of tetracycline. Tet-FAK(WT) and Tet-FAK(F576/577) cells were induced for ~2 days to achieve maximal expression levels of the FAK variants, which are 2–3-fold higher than FAK levels observed in normal mouse fibroblasts (12) . Tet-FAK(F397) cells were induced for only 12–14 h to achieve F397-FAK expression levels comparable with WT-FAK.

Cos-7 Cell Expression and Immunoblot Analysis of HA-tagged FAK.
Plasmids for expression of mouse FAK variants tagged at their COOH termini with the HA epitope and mouse c-Src variants have been described previously (12 , 26) . Plasmid DNA (2 µg total) was transfected with Lipofectamine (Life Technologies) into subconfluent Cos-7 cells, and cells were maintained in culture 2 days prior to lysis in 2x SDS-PAGE sample buffer. Proteins in the whole-cell lysates were resolved on 7% polyacrylamide SDS-PAGE, transferred to Immobilon-P (Millipore Corp., Bedford, MA), and subjected to immunoblot analysis with C-20, 4G10, 12CA5, Src-327, pFAK397, pFAK576, or pFAK576/577 antibodies. Blots were blocked 2 h in Tris-buffered saline containing 0.2% Tween 20 (TBST) and either 3% BSA/1% ovalbumin (for 4G10 antibody) or 5% nonfat dry milk (for all others). After blocking, the blots were incubated 2 h in primary antibody solutions prepared in blocking buffer and then washed extensively in TBST prior to detection by enhanced chemiluminescence using horseradish peroxidase-conjugated secondary antibodies and detection reagents from Amersham Pharmacia Biotech (Piscataway, NJ). Concentrations of primary antibodies were as follows: C-20, 0.4 µg/ml; 4G10, 1.0 µg/ml; 12CA5, 1.0 µg/ml; Src-327, 1.0 µg/ml; pFAK397, 0.5 µg/ml; pFAK576 and pFAK576/577, 1:200 dilution of whole antiserum.

Analysis of FAK Phosphorylation in Src-transformed Cells and in Cells Plated onto Fibronectin.
Subconfluent cultures of NIH 3T3 and F527 Src-transformed NIH 3T3 fibroblasts were maintained in serum-containing medium prior to lysis. Fibronectin-replating experiments were performed essentially as described (12) , except that cells (NIH 3T3 or Tet-FAK) were maintained in the absence of serum for 2 h prior to suspension and were replated onto fibronectin for times ranging from 0.5 to 4 h. Cells were lysed in RIPA buffer [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1% aprotinin, and 0.1 mM Na3VO4], and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL) and adjusted to equal amounts of total protein for all samples within a given experiment in 1 ml of final volume. FAK was immunoprecipitated from the lysates using 1 µg of C-20 antibody, resolved by 7% polyacrylamide SDS/PAGE, transferred to Immobilon-P transfer membrane (Millipore Corp., Bedford, MA), and subjected to immunoblot analysis with C-20, 4G10, pFAK397, pFAK576, or pFAK576/577 antibodies, as indicated above.

Immunofluorescence Microscopy.
Tet-FAK(WT) fibroblasts were induced for 2 days and then either plated onto glass coverslips and allowed to adhere and grow overnight in serum-containing medium or plated onto coverslips coated with 5 µg/ml fibronectin and allowed to adhere and spread for 30 min. The cells were fixed 10 min in 3.7% formaldehyde in PBS, permeabilized 10 min in 0.5% Triton X-100 in PBS, and then blocked 1–2 h in 5% milk in PBS. After blocking, the coverslips were incubated 1 h at 37°C in primary antibody mixtures prepared in 5% milk/PBS. The primary antibody mixtures consisted of FAK-TL (25 µg/ml) and either pFAK576 (1:20 dilution of whole antiserum) or pFAK397 (5 µg/ml). After repeated washes with PBS, secondary antibodies of rhodamine-conjugated goat antimouse IgG and FITC-conjugated goat antirabbit IgG (Molecular Probes, Eugene, OR) were applied at 7–10 µg/ml each in 5% milk/PBS and incubated for 1 h at 37°C. After repeated washes with PBS, coverslips were mounted onto glass microscope slides and viewed with a Zeiss Axioplan photomicroscope.

Acknowledgments

We thank Eric Schaeffer for providing the pFAK397 antibody, Tara Gower and Barney Graham for assistance with immunofluorescence microscopy, and Samyuka Reddy for general technical assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants GM49882 (to S. K. H.) and P03-CA68485 (funding the Molecular Recognition Shared Resource of the Vanderbilt-Ingram Cancer Center). Back

2 To whom requests for reprints should be addressed, at Department of Cell Biology, Vanderbilt University School of Medicine, Medical Center North, 1161 21st Avenue South, Nashville, TN 37232. Phone: (615) 343-8502; Fax: (615) 343-4539; E-mail; hankss@ctrvax.vanderbilt.edu. Back

3 The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; SH2, Src homology 2; WT, wild type. Back

Received for publication 9/13/99. Revision received 12/ 1/99. Accepted for publication 12/ 2/99.

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