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The Journal of Immunology, 1998, 160: 5154-5162.
Copyright © 1998 by The American Association of Immunologists

Syk Activation Is Required for Spreading and H2O2 Release in Adherent Human Neutrophils1

Rosemarie Fernandez*,{dagger} and Suzanne J. Suchard2,*,{dagger}

* Department of Pediatrics, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI 48109; and {dagger} Zeneca Pharmaceuticals, Wilmington, DE 19850


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cytoskeletally associated protein that is also tyrosine phosphorylated during PMN spreading and H2O2 release. Paxillin phosphorylation is kinetically slower than Syk phosphorylation and is inhibited with piceatannol, suggesting that paxillin is a substrate for Syk. An analysis of Syk immunoprecipitates indicated that Syk and paxillin associate during PMN spreading. This interaction is not mediated by the src kinases Lyn and Fgr, since neither kinase coprecipitated with Syk. Syk from FMLP-activated, adherent PMNs phosphorylated paxillin-glutathione S-transferase, suggesting that paxillin is a substrate for Syk in vivo. These results indicate that PMN spreading and H2O2 release require a Syk-dependent signaling pathway leading to paxillin phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The adhesion of polymorphonuclear leukocytes (PMNs)3 to extracellular matrix (ECM) proteins regulates a number of cell functions. The activation of adherent PMNs by inflammatory mediators such as FMLP and TNF-{alpha} results in cytoskeletal rearrangement, the formation of focal adhesion-like structures, cell spreading, and the release of specific granule constituents (1, 2, 3). With regard to adherent PMNs, both the release of lactoferrin, which is a component of specific granules, and the activation of the respiratory burst are kinetically slower but of greater magnitude when compared with PMNs in suspension, with a lag of ~30 to 45 min before the onset of these responses and a duration that is ~20 times longer than that seen with suspended PMNs (4, 5). While the signaling events leading either to adhesion or to the generation of oxygen radicals are currently under investigation, the mechanisms linking these two events in adherent PMNs have not been examined.

The most well-characterized cell-surface receptors for ECM proteins on PMNs are the ß2 integrins (6). In general, integrin-mediated cell adhesion to ECM proteins initiates a signaling cascade that involves receptor activation, increases in tyrosine kinase activity, protein phosphorylation, and reorganization of the actin-based cytoskeleton (7, 8, 9, 10). In PMNs that are adherent to serum proteins, activation by TNF-{alpha} leads to an increase in the phosphorylation of mitogen-activated protein kinase (11), paxillin (12), and the src-related kinases Lyn and Fgr (13, 14); this increase appears to coincide with H2O2 release. TNF-{alpha}-mediated PMN activation, cell spreading, and subsequent protein tyrosine phosphorylation are inhibited by treatment with the tyrosine kinase inhibitor genistein (9, 11). Furthermore, the inhibition of actin polymerization by treatment with either cytochalasin B or D blocks PMN spreading, oxidant generation, and associated protein phosphorylation in response to TNF-{alpha} or FMLP activation (12, 15).

The importance of tyrosine phosphorylation for cell spreading has been demonstrated in several different cell types. In some cells, integrin-dependent adhesion leads to the formation of specialized structures known as focal adhesions in regions in which the cell is in close contact with the ECM (16). Focal adhesions contain complexes of kinases, docking proteins, and cytoskeletal proteins that function both as structural links between the ECM and the cytoskeleton and as sites of intracellular signaling (17). Two proteins that are commonly found at focal adhesions are focal adhesion kinase (FAK) and paxillin (8, 18). FAK is a 125-kDa nonreceptor tyrosine kinase that associates with the cytoplasmic domain of integrins at focal adhesions (18) and is phosphorylated and activated in response to integrin ligation (8, 19, 20). FAK also contains binding sites for other signaling molecules, such as pp60src (21), paxillin (22), Grb2 (23), and pp77FAP (24), and may consequently act as a docking protein which facilitates the interactions of other proteins. Paxillin is a 68-kDa protein that binds to the COOH-terminus of FAK (22) and is required for FAK localization to focal adhesions (25). In addition, paxillin can be phosphorylated by FAK in vitro (26). Paxillin interacts with a number of other signaling molecules that localize to focal adhesions, including p210BCR/ABL (27) and pp60src (28), as well as the structural proteins vinculin and talin (29). It is currently thought that paxillin acts as a physical link between signaling molecules and the cytoskeleton.

Although PMNs do not form true focal adhesions (30), phosphorylated FAK and paxillin have been identified in adherent PMNs (12, 15). Recently, we have demonstrated that integrin-dependent adhesion results in a time-dependent phosphorylation of FAK and paxillin, and that this phosphorylation requires cell spreading (15). However, it is not clear whether integrin-mediated paxillin phosphorylation is FAK-dependent or -independent. FAK-independent paxillin phosphorylation occurs during the integrin-mediated adhesion of macrophages to vitronectin (31). Furthermore, p210BCR/ABL and v-src can phosphorylate paxillin in vitro (27). These findings suggest that there is an alternate, FAK-independent pathway that leads to paxillin phosphorylation.

Recent reports indicate that a unique family of nonreceptor tyrosine kinases, the Syk/ZAP-70 kinases, are crucial components of hemopoietic cell-signaling cascades. Syk has two tandem src homology (SH)2 domains that interact with the phosphorylated tyrosine residues on other proteins and stimulate kinase activity (32, 33), resulting in the phosphorylation of a number of different substrates, including {alpha}-tubulin (34), cortactin (35), and p50/HS1 (36). Unlike ZAP-70, which is restricted to T and B lymphocytes, Syk is found in B lymphocytes (34), mast cells (37), macrophages (38), platelets (39), and PMNs (40), and can be phosphorylated and activated in response to FcR cross-linking (41, 42), cytokines (43), and integrin receptor ligation (44, 45). In platelets, the ligation of ß1 integrins by collagen leads to Syk phosphorylation and to the subsequent activation of phospholipase C{gamma} (39). Monocyte adhesion to anti-ß1-coated surfaces also results in an increase in Syk activity, which is required for the nuclear translocation of NF-{kappa}B (46). Recently, Yan et al. (40) reported that TNF-{alpha}-stimulated PMN adhesion to fibrinogen (FG) resulted in an increase in Syk activity and in the formation of a complex containing Syk, Lyn or Fgr, and the ß2 integrins. In the present study, we demonstrate that paxillin is a substrate for Syk in vitro and provide evidence suggesting that Syk, not FAK, is responsible for the ß2 integrin-mediated phosphorylation of paxillin in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

FG was purchased from Chromogenix (Molindal, Sweden). Piceatannol was obtained from Biomol (Plymouth Meeting, PA). Anti-paxillin and anti-FAK mAbs were purchased from Transduction Labs (Lexington, KY). Anti-Syk polyclonal Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phosphotyrosine mAb (4G10) and the anti-FAK mAb used in the kinase assays were obtained from Upstate Biotechnology (Lake Placid, NY). The glutathione (GSH) S-transferase (GST) fusion proteins containing both the human paxillin sequence from 1 to 313 aa and the control GST sequence expressed in pGEX-2T (Pharmacia Biotech, Piscataway, NJ) were a generous gift of Christopher E. Turner (State University of New York Health Science Center, Syracuse, New York). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse and anti-rabbit Abs were obtained from Amersham (Arlington Heights, IL), and mouse and rabbit control IgG were purchased from Organon Teknika-Cappel (Malvern, PA). Reduced GSH was purchased from Sigma (St. Louis, MO). GSH-Sepharose and isopropyl thiogalactosidase were purchased from Pharmacia Biotech.

Cells

PMNs were isolated from human peripheral blood as previously described (47). Briefly, fresh whole blood was obtained by venipuncture from healthy volunteers or patients and immediately added to acid citrate dextrose. PMNs were purified by dextran sedimentation followed by hypotonic lysis to remove the majority of erythrocytes and then centrifuged through Ficoll-Hypaque (Pharmacia Biotech) to remove contaminating mononuclear cells. This purification process yielded >=98% PMNs and a viability of >95% as determined by trypan blue exclusion. PMNs were treated with diisopropyl fluorophosphate (5 mM) (Sigma) for 5 min on ice and washed three times with PBS.

Assays of H2O2 production

Assays were conducted in 24-well flat-bottom polystyrene Falcon Primaria tissue culture plates (Becton Dickinson, Lincoln Park, NJ) that had been coated with saturating concentrations of FG (50 µg/ml) (48). PMNs (105/well) that had been suspended in Krebs-Ringer phosphate buffer with glucose were added to FG-coated wells containing Krebs-Ringer phosphate buffer with glucose and 24 µM scopoletin, 5 µg HRP, 1 mM sodium azide (to prevent the destruction of H2O2 by myeloperoxidase and catalase), and the indicated agonists and/or inhibitors in a final volume of 1 ml. Before the addition of PMNs, plates were warmed to 37°C. PMN-containing plates were incubated under air at 37°C in a humidified incubator. Supernatants were removed from the wells at the indicated time points and centrifuged to remove nonadherent cells. A reduction in fluorescence as a result of H2O2 oxidation of scopoletin was determined by reading relative fluorescence in a fluorimeter at an excitation wavelength of 365 nm and an emission wavelength of 473 nm. A standard curve was generated using known amounts of H2O2 (49). To determine the degree of cell adhesion and spreading that coincided with H2O2 production, FG-coated wells were rinsed three times with PBS containing 1 mM Ca2+ and 1 mM Mg2+, fixed with 1% glutaraldehyde in PBS, and scored for adhesion and spreading. The number of attached and spread cells was quantitated as previously described (15).

Immunoblotting

After removing the supernatants for H2O2 determination, FG-coated wells were rinsed with PBS containing 1 mM Ca2+ and 1 mM Mg2+, and adherent cells were lysed by scraping in buffer containing 1% Triton X-100, 0.1% SDS, 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 0.1 µM PMSF, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin. We obtained 0-min time points by lysing PMNs just before they were added to coated plates. Lysates were clarified in a microfuge at 14,000 x g for 6 min, and protein concentrations were determined using a micro-Bradford assay (Pierce, Rockford, IL). PMN lysates were combined with 4x SDS-PAGE sample buffer containing Na3VO4, boiled for 5 min, and electrophoresed on 7.5% SDS-PAGE minigels. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Schleicher and Schuell, Keene, NH) and blocked with 2% BSA in PBS containing 1 mM EDTA, 0.05% Tween-20, and 1 mM Na3VO4 (blocking buffer). The membrane was probed with the anti-phosphotyrosine Ab 4G10 (1:1500) in blocking buffer, washed three times with 0.2% Tween-20 in 50 mM Tris (pH 8.0) and 100 mM NaCl, and then incubated with HRP-conjugated sheep anti-mouse Ab (1:10,000) in wash buffer containing 5% nonfat dry milk. Phosphorylated bands were visualized using the enhanced chemiluminescence system and Hyperfilm enhanced chemiluminescence (Amersham).

Immunoprecipitation

PMNs were lysed in lysis buffer containing 1% Triton X-100 (paxillin and Syk) or 1% SDS (FAK) along with 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 0.1 µM PMSF, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin. Lysates were clarified, and protein concentrations were determined as outlined above. Lysates (100–200 µg protein) were precleared with protein Sepharose A or G for 30 min and incubated overnight at 4°C with 3 µg of anti-paxillin or anti-FAK mAbs, 1 µg of anti-Syk Ab, or 3 µg of mouse IgG. Protein A-Sepharose (Syk) or protein G-Sepharose (FAK and paxillin) was added to each sample and incubated for 2 h with rotation at 4°C. The beads were washed thoroughly with lysis buffer, and adsorbed proteins were solubilized in sample buffer and separated on 7.5% SDS-PAGE minigels. Transfer to PVDF and subsequent immunoblotting with 4G10 anti-phosphotyrosine Ab was conducted as outlined above. For coprecipitation assays, anti-Syk immunoprecipitates were processed as described above, except the transferred proteins were probed with anti-paxillin mAb. PVDF membranes were stripped with 100 mM ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris (pH 6.7) at 50°C and reprobed with anti-Syk Ab to demonstrate equivalent amounts of immunoprecipitated protein. Alternatively, samples were immunoprecipitated and Western blotted with anti-paxillin or anti-FAK mAbs to demonstrate equal loading.

Isolation of paxillin-GST fusion proteins

Both pGEX-2T containing the amino acids (aa) 1 to 313 from chicken paxillin and pGEX-2T without an inserted sequence were expressed and purified as previously described (50). Fusion proteins were incubated with GSH-Sepharose beads and eluted with 5 mM reduced GSH in 50 mM Tris-HCl (final pH 7.5). The eluate was diluted in 50 mM HEPES (pH 7.6) and concentrated in either a Centricon-10 (GST only) or a Centricon-30 (paxillin-GST 1–313) microconcentrator (Millipore, Bedford, MA). Proteins were stored at 4°C in the presence of 0.02% NaN3.

Kinase assays

PMNs were lysed in lysis buffer containing 1% Triton X-100, 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 0.1 µM PMSF, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin. Lysates were clarified, and protein concentrations were determined as outlined above. Cleared lysates were incubated with 1 µg of anti-Syk Ab or anti-FAK mAb (Upstate Biotechnology) overnight, with rotation at 4°C. Protein A/G-Sepharose was added to each sample and incubated for 2 h with rotation at 4°C. For autophosphorylation assays, beads were washed three times with cold lysis buffer and three times with 50 mM HEPES (pH 7.6), 10 mM MnCl2, 2 mM MgCl2, 10 µM Na3VO4, and 1 mM 4-nitrophenyl phosphate. Beads were resuspended in kinase buffer containing 50 mM HEPES (pH 7.6), 10 mM MnCl2, 2 mM MgCl2, 10 µM Na3VO4, 1 mM 4-nitrophenyl phosphate, 2 µM ATP, and 10 µCi/sample [{gamma}-32P]ATP. The samples were incubated for 10 min at 30°C, and the reaction was terminated by the addition of sample buffer. Proteins were separated on 10% SDS-PAGE minigels and transferred to PVDF membranes. Phosphorylated proteins were visualized by autoradiography. In kinase assays in which GST proteins were used as substrates, 5 µM paxillin-GST or control GST protein was added to the kinase assay mixture and incubated as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein tyrosine phosphorylation increases in parallel with H2O2 release in adherent PMNs

As we have previously shown (3), PMNs plated onto FG in the presence of FMLP showed a lag of ~30 to 45 min before the onset of H2O2 release (Fig. 1GoB). A maximal response was achieved by 90 to 120 min. In unactivated cells, H2O2 release was barely detectable by 120 min. The kinetics of H2O2 release are closely correlated with the kinetics of adhesion and spreading (Fig. 1GoA). PMNs plated onto FG under conditions identical with those used for H2O2 measurements began adhering by 30 min, with maximal adhesion occurring between 60 and 90 min. Cell spreading lagged slightly behind adhesion, with spreading occurring by 45 and maximal spreading by 90 min. In unactivated cells, spreading was observed by 120 min, which correlated with the initiation of H2O2 release.



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  		FIGURE 1. Kinetics of PMN adhesion, spreading, and H2O2 generation on FG-coated plastic (A and B). A, PMNs (1 x 105) were added to FG-coated 24-well plates containing either buffer (control) or FMLP (10-7 M) and then incubated at 37°C. Nonadherent cells were subsequently removed at the indicated times, the wells were washed, and adherent cells were fixed with 1% glutaraldehyde. The number of PMNs that attached and spread was quantitated as previously described (15). B, The kinetics of H2O2 release were determined by analyzing the levels of H2O2 in the supernatants from each well. Values represent the means ± SEM of four separate experiments performed in duplicate. C and D indicate changes in protein tyrosine phosphorylation during the H2O2 release of FMLP-activated PMNs that had been plated onto FG. 6-well tissue culture dishes were coated with 50 µg/ml FG. PMNs (5 x 105) were added to each well in the presence of buffer alone (C) or 10-7 M FMLP (D) and incubated at 37°C. At the indicated times, the supernatants were removed from the wells and analyzed for H2O2 production as described in Materials and Methods (see B). Attached cells were lysed in lysis buffer containing 1% Triton X-100 and 0.1% SDS. An aliquot of PMNs in suspension was removed and lysed just before the addition of PMNs to plates; this aliquot was designated as time 0. Cell lysates were analyzed by 7.5% SDS-PAGE and transferred to PVDF membranes, and blots were probed with 4G10 anti-phosphotyrosine mAb.

 
The initiation of cell spreading and the generation of H2O2 in PMNs appears to require the activation of several different tyrosine kinases (11, 51, 52). This is supported by the finding that pretreating PMNs with the tyrosine kinase inhibitors genistein or erbstatin blocks cell spreading and H2O2 production (9). Overall tyrosine phosphorylation has been shown to increase in FMLP-activated PMNs, but the kinetics of phosphorylation in FMLP-activated, adherent PMNs has not been evaluated. Therefore, we examined the changes in the pattern of protein tyrosine phosphorylation in PMNs plated onto FG in the absence (Fig. 1GoC) and presence (Fig. 1GoD) of FMLP. There was a low level of tyrosine phosphorylation at time 0. In the absence of FMLP, PMNs showed relatively low levels of tyrosine phosphorylation until 90 to 120 min, which coincided with the slight increases seen in PMN spreading and H2O2 release (see Fig. 1GoA). In FMLP-stimulated cells, the tyrosine phosphorylation of several proteins increased over time, with maximal phosphorylation occurring between 45 and 60 min, coinciding with the onset of H2O2 production and cell spreading. Pretreating cells with the tyrosine kinase inhibitor genistein blocked H2O2 production, cell spreading, and associated changes in tyrosine phosphorylation (data not shown). These data suggested a relationship between the tyrosine phosphorylation of specific proteins, cell spreading, and H2O2 release.

Tyrosine phosphorylation of FAK, Syk, and paxillin correlates with H2O2 release and cell spreading

Several signaling proteins are phosphorylated during integrin-mediated adhesion in PMNs (14, 53, 54). FAK and Syk are nonreceptor tyrosine kinases that are phosphorylated and activated during integrin-mediated signaling (15, 39, 40, 55). Furthermore, paxillin, a potential substrate for FAK and/or Syk (38, 50), appears to be critical for the cytoskeletal rearrangement that occurs during PMN spreading on ECM proteins (15). To determine whether these proteins were also important for H2O2 release from adherent PMNs, PMNs were plated onto FG-coated plates in the presence of FMLP and examined for the tyrosine phosphorylation of these proteins. At the indicated times, cells were lysed and immunoprecipitated with anti-FAK mAb (Fig. 2GoA), anti-Syk Ab (Fig. 2GoB), or anti-paxillin mAb (Fig. 2GoC). The immunoprecipitates were electrophoresed on a 7.5% SDS-PAGE minigel, transferred to PVDF membranes, and probed with an anti-phosphotyrosine mAb. Neither FAK, Syk, nor paxillin were phosphorylated in PMNs before their addition to FG-coated plates (Fig. 2Go, A–C, lane 0). PMNs plated onto FG showed a time-dependent phosphorylation of FAK, Syk, and paxillin, with maximal phosphorylation seen by 45 min. These immunoprecipitations were specific, since nonspecific IgG did not bring down protein bands of similar m.w. (Fig. 2Go, A–C, left panel, IgG). The phosphorylation of FAK and Syk appeared to precede the phosphorylation of paxillin, since there was detectable FAK and Syk tyrosine phosphorylation by 15 min in the absence of detectable paxillin phosphorylation (data not shown). This observation suggested that either FAK and/or Syk may lie upstream of paxillin in the signaling cascade. The tyrosine phosphorylation of all three proteins was an adhesion-dependent event, since FMLP-activated PMNs that were incubated with FG in suspension for 45 min did not show a similar increase in the tyrosine phosphorylation of these proteins (Fig. 2Go, A–C, S45').



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  		FIGURE 2. Time course of tyrosine phosphorylation of FAK (A), Syk (B), and paxillin (C) during PMN release of H2O2. We coated 6-well tissue culture plates with 50 µg/ml FG, added 5 x 105 PMNs to each well, and incubated the plates at 37°C. At the indicated times, attached PMNs were lysed in lysis buffer containing either 1% Triton X-100 and 0.1% SDS (paxillin and Syk) or 1% SDS (FAK). Just before their addition to plates, aliquots of cells in suspension were removed, lysed, and designated as time 0. For receptor ligation in the absence of adhesion, PMNs in suspension were also incubated with 50 µg/ml FG for 45 min at 37°C and then lysed (S45'). Cell lysates were incubated with anti-FAK mAb (A), polyclonal anti-Syk Ab (B), or anti-paxillin mAb (C) and subsequently incubated with either protein A- or G-Sepharose. Identical samples were also incubated with nonspecific rabbit (Syk) or mouse (paxillin, FAK) IgG to demonstrate the specificity of the precipitating Abs. Immunoprecipitates were analyzed by 7.5% SDS-PAGE and transferred to PVDF membranes, and blots were probed with 4G10 anti-phosphotyrosine mAb.

 
Piceatannol, an inhibitor of Syk activation, inhibits H2O2 release and the tyrosine phosphorylation of FAK and paxillin

The kinetics of protein tyrosine phosphorylation and H2O2 release suggested that the activation of Syk and/or FAK may be required for cell spreading and H2O2 release. To determine whether Syk activity was required for H2O2 production, H2O2 assays were conducted in the presence of various concentrations of piceatannol, a Syk-selective kinase inhibitor (56). At the 120-min time point, samples were taken for H2O2 determination, and PMNs were evaluated for cell spreading. Piceatannol inhibited cell spreading and H2O2 release in a dose-dependent manner (Fig. 3GoA). Both spreading and H2O2 release were inhibited by >90% following treatment with 10 µM piceatannol. In contrast, 10 µM piceatannol did not affect adhesion. To verify that piceatannol was inhibiting Syk phosphorylation, PMNs were incubated in the presence or absence of 10 µM piceatannol, and H2O2 assays were set up as outlined above. After 45 min, cells were lysed and analyzed for Syk phosphorylation. Treatment with piceatannol completely inhibited Syk phosphorylation as compared with control cells incubated with DMSO alone (Fig. 3GoB). PVDF membranes were stripped and reprobed with anti-Syk Ab to demonstrate equal loading in treated vs untreated lanes (Fig. 3GoB, right panel).



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  		FIGURE 3. Piceatannol inhibition of cell spreading and H2O2 release by PMNs adherent to FG (A). PMNs were incubated with various concentrations of piceatannol or control vehicle (DMSO) for 10 min at room temperature before addition to the FG-coated 24-well plates. Plates were incubated at 37°C for 2 h, at which time H2O2 production and cell spreading were determined as outlined in Materials and Methods. Piceatannol inhibition of tyrosine phosphorylation of FAK (B), Syk (C), and paxillin (D). PMNs were incubated with piceatannol (10 µM) and added to 6-well FG-coated plates as described above. Plates were incubated at 37°C for 45 min, at which time attached cells were lysed for immunoprecipitation as described in Materials and Methods. Lysates were immunoprecipitated with anti-Syk (B), anti-FAK (C), or anti-paxillin mAb (D), separated by SDS-PAGE, transferred to PVDF membranes, and probed with 4G10 anti-phosphotyrosine mAb ({alpha}PY). Immunoprecipitates from treated and untreated cells were also probed with anti-FAK (C, right panel) or anti-paxillin mAb (D, right panel) to demonstrate equivalent protein load. Alternately, anti-Syk membranes were stripped and reprobed to demonstrate equal loading (B, right panel). Aliquots of the supernatants were analyzed for H2O2 production to verify that 10 µM piceatannol was effective (>95% inhibition) in these experiments (data not shown).

 
As stated earlier, the time courses of Syk, FAK, and paxillin phosphorylation suggested that paxillin may lie downstream of Syk and/or FAK signaling. We have recently demonstrated that paxillin can be phosphorylated in the absence of FAK activation in adherent PMNs (15). To determine whether Syk is in the same pathway and is upstream of paxillin and/or FAK, we evaluated the tyrosine phosphorylation of these proteins in PMNs treated with piceatannol (10 µM). Neither FAK nor paxillin were tyrosine phosphorylated in PMNs incubated with piceatannol (Fig. 3Go, C and D). Normal levels of tyrosine phosphorylation were detected in cells treated with DMSO alone. To demonstrate equivalent protein loading, parallel samples were run and immunoblotted with the immunoprecipitating Ab (Fig. 3Go, C and D, right panel). These data suggest that Syk lies upstream of both FAK and paxillin. However, realizing the limitations of inhibitors, we cannot rule out the possibility that FAK is also inhibited by piceatannol. Our interpretation of these data are further complicated by the fact that integrin-mediated FAK and paxillin phosphorylation appear to require cell spreading (8, 15). Thus, it is unclear whether the inhibition of FAK and paxillin phosphorylation in piceatannol-treated cells results from the disruption of specific signaling events or from a general absence of cell spreading.

Syk forms a complex with paxillin in vivo

In PMNs, Syk forms protein complexes with a number of different signaling molecules, including Lyn, Fgr, ß2 integrins, and the G-CSFR (40, 43). Syk has two tandem SH2 domains that interact with the phosphorylated tyrosine residues on other proteins and positively regulate kinase activity (32, 33) Paxillin has binding sites for SH2 and SH3 domain-containing proteins and consequently may act as a docking site for multiple signaling molecules (57). To determine whether Syk formed complexes with paxillin during PMN spreading and H2O2 release, we examined Syk immunoprecipitates for the presence of paxillin. Paxillin coprecipitated with Syk in PMNs that were plated onto FG in the presence of FMLP (Fig. 4GoA, upper right panel). This interaction was specific, since paxillin was not detected in the nonspecific rabbit IgG lanes (Fig. 4GoA, upper left panel). These results suggested a direct interaction between Syk and paxillin. However, the possibility that both proteins are linked by an "adaptor" molecule cannot be ruled out. Yan et al. (40) reported that Syk forms immunocomplexes with the src family kinases Lyn and Fgr during integrin-mediated adhesion in PMNs (40). These complexes, however, dissociate under conditions similar to those used in our assays; i.e., lysis buffer containing 1% Triton X-100. To verify that Lyn and Fgr were not present in the complexes with Syk, we probed Syk immunoprecipitates with Abs specific for Lyn and Fgr. Neither Lyn nor Fgr were present in Syk immunoprecipitates (Fig. 4Go, B and C, upper panels). All membranes were stripped and reprobed with anti-Syk Ab to demonstrate equal amounts of Syk on the blots (Fig. 4Go, lower panels).



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  		FIGURE 4. Syk forms protein complexes with paxillin (A), but not with Lyn (B) and Fgr (C) in FMLP-treated PMNs plated onto FG. We coated 6-well tissue culture plates with 50 µg/ml FG, added 5 x 105 PMNs to each well, and incubated the plates for 45 min at 37°C. Following incubation, attached PMNs were lysed in lysis buffer containing 1% Triton X-100 and 0.1% SDS. Just before addition to plates, aliquots of cells in suspension were removed, lysed, and designated as time 0. Cell lysates were incubated with polyclonal anti-Syk Ab followed by incubation with A-Sepharose. Identical samples were also incubated with nonspecific rabbit IgG to demonstrate the specificity of the precipitating Abs (A–C, upper left panels). Immunoprecipitates were separated by 7.5% SDS-PAGE and transferred to PVDF membranes, and blots were probed with anti-paxillin mAb (A, upper right panel), polyclonal anti-Lyn Ab (B, upper right panel), or polyclonal anti-Fgr Ab (C, upper right panel). Membranes were stripped and reprobed with anti-Syk Ab to demonstrate equal loading (A–C, lower panels).

 
Syk, but not FAK, is activated in PMNs during cell spreading and H2O2 production

In platelets, increased tyrosine phosphorylation correlates with increased Syk and FAK activity (39, 55). However, there is evidence indicating that phosphorylating FAK may positively or negatively regulate kinase activity depending upon the specific tyrosine residue involved. To verify that increased tyrosine phosphorylation correlated with increased activity, we performed in vitro kinase assays. For these experiments, PMNs were plated onto FG in the presence of FMLP and incubated at 37°C for 45 min. Cells were then lysed, immunoprecipitated with anti-Syk or anti-FAK Abs, and incubated with kinase buffer. Autophosphorylation was used as an indication of kinase activity. As shown in Figure 5GoA, increased Syk activity was detected in lysates from PMNs that were plated onto FG (Fig. 5GoA, lane 45') as compared with control cells lysed before their addition to coated plates (Fig. 5GoA, lane 0). As expected, preincubating PMNs with piceatannol before plating completely inhibited Syk activity (Fig. 5GoA, P45'). The absence of a band in the nonspecific IgG lane indicated that the immunoprecipitations were specific for Syk. PMNs incubated with FMLP and FG for 45 min in suspension (Fig. 5GoA, S45') showed no increase in kinase activity, supporting our earlier finding that cell spreading is required for Syk activity. These results are similar to what has been reported for TNF-{alpha}-stimulated, adherent PMNs (40). No kinase activity was detected in FAK immunoprecipitates from adherent PMNs (Fig. 4GoB). To demonstrate that this absence of FAK activity was not related to tyrosine phosphorylation, a parallel sample was electrophoresed and immunoblotted with an anti-phosphotyrosine mAb (Fig. 4GoB, right panel). The level of FAK phosphorylation was comparable with that seen in previous experiments. This demonstrated that FAK activity is not increased during PMN adhesion even though there is an increase in FAK tyrosine phosphorylation.



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  		FIGURE 5. Changes in Syk (A) and FAK (B) kinase activity during spreading and H2O2 release from PMNs. We coated 6-well tissue culture plates with 50 µg/ml FG and pretreated 5 x 105 PMNs for 10 min with piceatannol (P45') or buffer alone and added them to each well. Following a 45-min incubation, attached PMNs were lysed in lysis buffer containing 1% Triton X-100. For receptor ligation in the absence of adhesion, PMNs were incubated with 50 µg/ml FG in suspension for 45 min at 37°C and then lysed (S45'). Just before addition to plates aliquots of cells in suspension were removed, lysed, and designated as time 0. Cell lysates were incubated with either polyclonal anti-Syk Ab (A) or anti-FAK mAb (B) and subsequently incubated with protein A-Sepharose (Syk) or protein G-Sepharose (FAK). Identical samples were incubated with nonspecific rabbit (Syk) or mouse (FAK) IgG to demonstrate the specificity of the precipitating Abs. Sepharose beads were washed, resuspended in kinase buffer, incubated for 10 min at 30°C, and solubilized in sample buffer. Proteins were separated on 10% (Syk) or 7.5% (FAK) gels and transferred to PVDF membranes. Phosphorylated proteins were detected by autoradiography. To demonstrate the presence of FAK phosphorylation in the absence of FAK activity, a parallel sample was run on a 7.5% gel and Western blotted with 4G10 anti-phosphotyrosine mAb (B, right panel). Membranes were stripped and reprobed with anti-Syk Ab to demonstrate equivalent protein load (A, lower panel). C and D indicate the phosphorylation of paxillin-GST fusion protein by Syk and FAK in vitro, respectively. Syk (C) and FAK (D) were immunoprecipitated as described above and incubated in kinase buffer containing 5 µM paxillin-GST (Pax-GST) or GST only. Proteins were separated on 10% (Syk) or 7.5% (FAK) gels and transferred to PVDF membranes. Phosphorylated proteins were visualized by autoradiography. FAK phosphorylation was verified by Western blotting with 4G10 anti-phosphotyrosine mAb (B, right panel). Membranes were stripped and reprobed with anti-Syk Ab to demonstrate equivalent protein loading (C, lower panel).

 
Paxillin is a substrate for Syk in vitro

In smooth muscle cells and fibroblasts, paxillin is thought to be a substrate for FAK during cell adhesion (50). However, our results indicated that an alternate kinase is responsible for paxillin phosphorylation during integrin-mediated PMN activation. Because paxillin was found in protein complexes with Syk, we evaluated whether paxillin could serve as a substrate for Syk in in vitro kinase assays. PMNs were plated onto FG-coated plates under the same conditions used for H2O2 assays. After 45 min, PMNs were lysed, and immunoprecipitations were performed as outlined in Materials and Methods. A GST fusion protein containing the sequence for paxillin (1–313 aa) (paxillin-GST) (50) or GST alone was used as a substrate in immunocomplex kinase assays. Syk that was immunoprecipitated from adherent cells phosphorylated paxillin-GST but not GST alone (Fig. 5GoC). This kinase activity was absent in cells lysed before their addition to the plates (Fig. 5GoC, lane 0). The immunoprecipitations were specific for Syk, since no activity was detected in lysates treated with IgG (Fig. 5GoC, IgG lane). Similar experiments were conducted using FAK immunoprecipitates to verify that FAK activity was not present in the lysates. As in the autophosphorylation assays, no kinase activity, as indicated by the absence of paxillin-GST phosphorylation, was detected with FAK immunoprecipitates (Fig. 5GoD, right panel), even though normal levels of FAK phosphorylation were observed (Fig. 5GoB, right panel).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study makes several novel observations. First, we demonstrated that in PMNs that are adherent to FG, the tyrosine phosphorylation of Syk, paxillin, and FAK correlated with the initiation of cell spreading and H2O2 release, with Syk and FAK phosphorylation slightly preceding that of paxillin. Second, Syk, but not FAK activity was required for cell activation and corresponding protein tyrosine phosphorylation. Third, Syk forms a complex with paxillin and is capable of phosphorylating a paxillin-GST substrate in in vitro kinase assays. These results suggested that PMN spreading on FG requires Syk activation and initiates Syk-dependent, FAK-independent, paxillin phosphorylation.

PMNs that are adherent to ECM proteins undergo a respiratory burst of increased magnitude and duration in comparison with cells in suspension (4, 5). This response is dependent upon the activation of protein tyrosine kinases (9, 58) and cell spreading (3). The importance of tyrosine kinases in PMN spreading and degranulation has been demonstrated in studies using PMNs that were isolated from mice lacking the Hck and Fgr src kinases (13). These PMNs fail to spread on FG-coated surfaces and do not release H2O2 in response to FMLP or TNF-{alpha}. Consistent with this finding, we saw an increase in protein tyrosine phosphorylation in FMLP-activated adherent PMNs that correlated with the initiation of cell spreading and H2O2 generation. In cells that were not treated with FMLP, the onset of tyrosine phosphorylation was much slower but still correlated with the start of cell spreading and H2O2 release.

Much of the initial work evaluating signal transduction in adherent cells has focused on the role of integrin receptors. In fibroblasts, platelets, and tumor cells, FAK and paxillin are tyrosine phosphorylated during integrin-mediated adhesion (8, 18, 55). Similarly, we demonstrated that FAK and paxillin were phosphorylated during FMLP-stimulated PMN adhesion to FG. The kinetics of phosphorylation closely paralleled cell spreading and H2O2 release, with the phosphorylation of FAK slightly preceding that of paxillin. In contrast, FAK and paxillin phosphorylation did not occur in PMNs that were incubated with FG in suspension, indicating that receptor ligation alone was not sufficient for integrin signaling. In PMNs adherent to laminin, the kinetics of FAK phosphorylation also precede the kinetics of paxillin phosphorylation (15). In those studies, both spreading and FAK/paxillin phosphorylation were reduced in the presence of the cytoskeletal-disrupting agent cytochalasin D, suggesting that these events are strongly coupled.

A recent study by Yan et al. (40) indicates that Syk is phosphorylated in TNF-{alpha}-activated adherent PMNs, and that this phosphorylation is dependent upon cell spreading. We saw similar phosphorylation of Syk in FMLP-stimulated PMNs that were plated onto FG. The kinetics for Syk phosphorylation were similar to those for FAK, with Syk phosphorylation paralleling FAK phosphorylation and slightly preceding paxillin phosphorylation. Syk phosphorylation required PMN adhesion, since incubating PMNs with FG in suspension did not activate Syk phosphorylation. This differs somewhat from monocytes, in which ß1 integrin-mediated adhesion is required for FAK and paxillin phosphorylation but only ß1 integrin ligation is necessary for Syk phosphorylation (46).

Our kinetic analyses of Syk, FAK, and paxillin phosphorylation suggested a link between the activation of these signaling molecules and the initiation of cell spreading and H2O2 generation. Piceatannol is a Syk-selective inhibitor that has been used to verify the role of Syk in collagen-mediated platelet aggregation and to sort out the sequence of kinases involved (39). Preincubating PMNs with piceatannol resulted in a dose-dependent inhibition of cell spreading and H2O2 generation. A concentration of piceatannol (10 µM) that completely blocked PMN spreading and H2O2 release also inhibited the phosphorylation of Syk, FAK, and paxillin. This concentration of inhibitor is similar to that used in other studies and was shown to be specific for Syk activity (39, 56). These results suggested that Syk activity is required for adhesion-dependent PMN activation and is involved in signaling events upstream from FAK and paxillin. However, FAK and paxillin phosphorylation are thought to require cell spreading (12, 15). Therefore, it is difficult to determine from these studies whether 1) Syk lies upstream from FAK and paxillin or whether 2) Syk is in a different pathway, and its inhibition by piceatannol indirectly affects FAK and paxillin phosphorylation by blocking cell spreading. We also cannot rule out the possibility that piceatannol is exerting a nonspecific effect on cell spreading, although past studies indicate that this is unlikely (39, 56).

Syk activation is thought to promote the association of Syk with other signaling molecules (34, 35, 36, 40, 43, 59). This may facilitate their phosphorylation by Syk or bring them in close proximity to other cellular components, such as the plasma membrane or cytoskeleton. Paxillin contains several domains that are capable of interacting with other signaling proteins, including three SH2 binding sites, an SH3 binding site, and four tandem LIM domains (57). We identified paxillin in Syk immunoprecipitates isolated from FMLP-stimulated PMNs that were adherent to FG. This coprecipitation was not seen in PMNs before activation, suggesting that ß2 integrin ligation and/or FMLP activation were required. In similar adhesion assays, Syk has been shown to form a complex with the src family kinases Lyn and Fgr (40). Lyn has been shown to link Syk and the G-CSFR in a three-component signaling complex that is formed upon receptor ligation (43). In addition, paxillin contains a src-binding site and is capable of interacting with v-crk in vitro (27, 60). When we evaluated Syk immunoprecipitates for the presence of Lyn or Fgr, neither protein was detected, indicating that these proteins probably do not mediate the interaction between Syk and paxillin. This observation is in agreement with the report by Yan et al. (40) that states that Syk/src kinase complexes are dissociated in lysis conditions similar to those used in our assays and supports the possibility that paxillin and Syk interact through direct binding; however, it does not preclude the involvement of an as yet unidentified "linker" protein.

In platelets, the increased tyrosine phosphorylation of Syk and FAK has been shown to correlate with increased kinase activity in vitro (55, 61). However, there is evidence indicating that FAK phosphorylation may either positively or negatively regulate its kinase activity depending upon the specific tyrosine residue involved (30). We performed autophosphorylation assays on Syk and FAK immunoprecipitates to determine whether increased tyrosine phosphorylation correlated with an increase in kinase activity. Syk activity was markedly increased in lysates from adherent PMNs, and this activity was inhibited in the presence of piceatannol. As expected from our phosphotyrosine analyses, neither untreated PMNs nor PMNs incubated with FMLP and FG in suspension demonstrated increases in Syk kinase activity. Interestingly, identical assays performed with FAK immunoprecipitates demonstrated an absence of kinase activity in lysates from adherent PMNs. Levels of phosphotyrosine, however, were increased in a manner similar to those seen with Syk. FAK also failed to phosphorylate a GST fusion protein containing 1 to 313 aa of paxillin. This fusion protein has been used successfully as a substrate in similar kinase assays involving smooth muscle FAK (50). Our results indicate a dissociation between FAK tyrosine phosphorylation and kinase activity in vitro, and suggest that FAK kinase activity is not required for PMN adhesion and H2O2 release.

Initially, tyrosine phosphorylation of FAK was thought to directly activate FAK activity (55, 62). However, studies by Eide et al. (63) demonstrate that substituting the primary FAK tyrosine phosphorylation site with Phe does not alter tyrosine kinase activity but does affect FAK binding to src kinase. In addition, the dephosphorylation of FAK by alkaline phosphatase treatment had no affect on in vitro kinase activity (30). This finding is consistent with a report demonstrating that Fc{epsilon}RI aggregation in mast cells induced FAK phosphorylation but not FAK activity (24). It may be that there are multiple phosphorylation sites on FAK that regulate protein binding and/or kinase activity during cell activation. A similar model is suggested by Guan and Shalloway (18) in a report demonstrating that src-induced phosphorylation of FAK adds to that induced by ß1 integrin ligation in adherent cells. Clearly, additional study is required to determine the role of FAK tyrosine phosphorylation during FG-mediated PMN adhesion.

The substrate for Syk in adherent PMNs has not previously been identified. Several different Syk substrates that are activated through immunoreceptor ligation have been identified in lymphocytes (34, 36, 64). In our assay system, the kinetics of Syk and paxillin phosphorylation, coupled with the ability of Syk and paxillin to form complexes during PMN adhesion, suggested that paxillin may be a substrate for Syk. To determine whether this was the case, we used a GST fusion protein containing the amino terminal 313 aa of paxillin in Syk immunocomplex kinase assays. This portion of paxillin is considered important for interaction with other signaling molecules. Syk that was isolated from FG-adherent PMNs phosphorylated paxillin-GST in vitro. This phosphorylation was specific to one of the six tyrosines located in the 1 to 313 aa region of paxillin, since GST protein alone was not phosphorylated in these assays. However, we do not know which tyrosine residue(s) is/are phosphorylated by PMN Syk. Our current data demonstrate that paxillin is a substrate for Syk and is likely to be an intermediary of Syk signaling in PMNs.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant HL53074 (to S.J.S.). R.F. was a Howard Hughes Medical Institute medical student research training fellow and the recipient of a Minority Graduate Research Supplement for National Institutes of Health Grant HL53074. Back

2 Address correspondence and reprint requests to Dr. Suzanne J. Suchard, Zeneca Pharmaceuticals, 1800 Concord Pike, P.O. Box 15437, Wilmington, DE 19850-5437. E-mail address: Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; FG, fibrinogen; ECM, extracellular matrix; FAK, focal adhesion kinase; PVDF, polyvinylidene fluoride; aa, amino acid; SH, src homology; GSH, glutathione; GST, glutathione S-transferase; HRP, horseradish peroxidase. Back

Received for publication October 23, 1997. Accepted for publication January 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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