Activation of Protein-tyrosine Kinase Pyk2 Is Downstream of Syk in FcεRI Signaling*

Aggregation of the FcεRI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosine-phosphorylated and activated after FcεRI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by FcεRI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the FcεRI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.

Aggregation of the Fc⑀RI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosinephosphorylated and activated after Fc⑀RI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by Fc⑀RI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the Fc⑀RI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.
The aggregation of the Fc⑀RI 1 on basophils or mast cells initiates a cascade of biochemical events that results in degranulation and the release of inflammatory mediators (1,2). Tyrosine phosphorylation of proteins plays a critical role in this signal transduction pathway (3)(4)(5)(6)(7)(8). Among the signal transduction molecules that are tyrosine-phosphorylated are the ␤ and ␥ subunits of the receptor, Lyn, Syk, phospholipase C-␥1 and -␥2, Vav, Btk, and the focal adhesion kinase FAK (9 -15). One of the earliest events after aggregation of Fc⑀RI is the activation of protein-tyrosine kinases, probably Lyn or another Src family kinase, that results in the tyrosine phosphorylation of the ␤ and ␥ subunits of the receptor (9,16). The proteintyrosine kinase Syk is then recruited by the tyrosine-phosphorylated receptor and is critical for the downstream activation signals (11,(17)(18)(19). For example, Fc⑀RI aggregation in a Sykdeficient mast cell line does not mobilize Ca 2ϩ from intracellular and extracellular sources and fails to propagate downstream signaling events (20).
Previously we observed that Fc⑀RI aggregation results in the tyrosine phosphorylation of ϳ115-kDa proteins in the rat ba-sophilic leukemia RBL-2H3 mast cell line (21,22). Two of these proteins were identified as FAK and the cell surface adhesion molecule CD31 (15,23). Recently Pyk2 was identified as another member of the FAK family of protein-tyrosine kinases (24 -26). Pyk2 (also called RAFTK for related adhesion focal tyrosine kinase, CAK␤ for cell adhesion kinase ␤, CADTK and FAK2) is a cytoplasmic protein-tyrosine kinase that, like FAK, lacks a transmembrane region, myristoylation sites, and Src homology 2 and 3 domains. Both FAK and Pyk2 have a central kinase region flanked by large N-terminal and C-terminal domains. Pyk2 is expressed in neuronal cells, CD34 ϩ bone marrow cells, primary bone marrow megakaryocytes, platelets, and T and B cells (24,26,27).
The stimulation of many different cell surface receptors results in the tyrosine phosphorylation and activation of Pyk2 (24, 26, 28 -31). These stimuli include carbachol acting through nicotinic acetycholine receptors, stress signals, membrane depolarization, cytokines, and molecules that activate G-proteincoupled receptors. Recently, Pyk2 was found to be tyrosinephosphorylated after integrin or immune receptor activation (27,(32)(33)(34)(35). Pyk2 is also activated by addition of the calcium ionophore and PMA, suggesting that the activation of Pyk2 is downstream of the increase in intracellular calcium and the activation of protein kinase C (24,36).
Here we report that Pyk2 was present in RBL-2H3 cells. Stimulation of the cells with different stimuli induced the tyrosine phosphorylation of Pyk2. In a Syk-deficient cell line, G-protein-coupled receptors, but not Fc⑀RI aggregation, still induced tyrosine phosphorylation of Pyk2. Therefore, there are Syk-dependent and -independent pathways for Pyk2 activation.

EXPERIMENTAL PROCEDURES
Materials and Antibodies--Mouse monoclonal anti-Pyk2 was from Transduction Laboratories (Lexington, KY). The horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody, 4G10, was from Upstate Biotechnology Inc. (Lake Placid, NY). Affinity-purified rabbit anti-mouse Igs were obtained from Jackson ImmunoResearch (West Grove, PA). All other antibodies have been described previously (11). The materials for electrophoresis were purchased from Novex (San Diego, CA), and the source of other materials was as described previously (11). N 6 -2-(4-Aminophenyl)ethyladenosine (APNEA) was from Research Biochemicals International (Natick, MA), and fibronectin was from Calbiochem (La Jolla, CA).
Cell Activation and Preparation of Cell Lysates-RBL-2H3, the Syknegative variant TB1A2, and the Syk-transfected cells were maintained as monolayer cultures (20,37). Cells were stimulated either with antigen after overnight culture in the presence of antigen-specific IgE, or with different stimuli essentially as described previously (3). The concentration of the stimuli were: 30 ng/ml dinitrophenyl coupled to human serum albumin, 0.5 M calcium ionophore A23187, 40 nM PMA, 1 unit/ml human plasma thrombin, or 10 M APNEA. After stimulation for the indicated times, the medium was removed for histamine analysis. The monolayers were then rinsed with ice-cold phosphate-buffered saline containing protease inhibitors and sodium orthovanadate with the same concentrations as in the lysis buffer described below and * 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.
Immunoprecipitation-Lysates were precleared by mixing for 1 h at 4°C with protein A-agarose beads. The lysates were then incubated with 3 g of mouse IgG or anti-Pyk2 antibody that had been preincubated with 10 g of rabbit anti-mouse Ig and 25 l of protein A-agarose beads. After gentle rotation at 4°C for 1 h, the beads were washed three times with 1 ml of wash buffer (cell lysis buffer with detergent concentration decreased by 50%), once with 150 mM NaCl, 50 mM Tris, pH 7.4, and the proteins eluted by boiling for 5 min with Laemmli's sample buffer as described previously (17).
Immunoblotting-Samples from the immunoprecipitations were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, electrotransferred to polyvinylidene difluoride membranes (Millipore), and tyrosine-phosphorylated proteins were detected with monoclonal antibody 4G10 conjugated to horseradish peroxidase as described previously (4). Proteins were visualized using the enhanced chemiluminescence kit from DuPont and Kodak X-Omat radiographic film (Eastman Kodak Co.). Antibodies were stripped from the membranes, and then membranes were reprobed with anti-Pyk2 antibodies.
In Vitro Kinase Reaction-Pyk2 immunoprecipitated as described above was further washed with kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl 2 , and 2 mM MnCl 2 ), and resuspended in 30 l of kinase buffer. The reactions were started by the addition of 5 Ci of [␥-32 P]ATP and 5 M ATP. After 30 min of incubation at room temperature, the reactions were stopped by the addition of 50 l of 2 ϫ Laemmli's sample buffer and boiling for 5 min. Following centrifugation, the eluted proteins were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis (4 -20% gels), electrotransferred to membranes, and visualized by autoradiography. In some experiments, the kinase reaction buffer contained as substrate 20 g of poly(Glu-Tyr) (4:1) (20 -50 kDa). Scanning densitometry was with a Pharmacia LKB Imagemaster.

Pyk2 Is Tyrosine-phosphorylated and Activated after Fc⑀RI
Aggregation-We and others have reported that several ϳ115-kDa proteins including FAK are tyrosine-phosphorylated after stimulation of RBL-2H3 cells (6,15). Since Pyk2 is another ϳ115-kDa molecule with homology to FAK, we examined whether Pyk2 was tyrosine-phosphorylated. By immunoblotting, the Pyk2 protein-tyrosine kinase was detectable in RBL-2H3 cells (see below). There was constitutive low level tyrosine phosphorylation of Pyk2 (Fig. 1A). Fc⑀RI aggregation induced a dramatic increase in the tyrosine phosphorylation of Pyk2 that was dependent on the extent of the stimulation with different concentrations of antigen. In time-course experiments, the tyrosine phosphorylation of Pyk2 was apparent at 1 min after stimulation and peaked by 10 min (Fig. 1B). This tyrosine phosphorylation paralleled the release of histamine from the cells. These results demonstrate that there is rapid tyrosine phosphorylation of Pyk2 after Fc⑀RI aggregation.
Since tyrosine kinase activity is important for signal transduction, we next determined whether Pyk2 was activated by stimulation of Fc⑀RI. By in vitro kinase reaction there was increased autophosphorylation of Pyk2 within 2 min after Fc⑀RI aggregation (Fig. 2). There was also increased kinase activity as determined by phosphorylation of the poly(Glu-Tyr) substrate. Therefore, aggregation of Fc⑀RI induced increased tyrosine phosphorylation and kinase activity of Pyk2.
Characteristics of the Tyrosine Phosphorylation of Pyk2-Following Fc⑀RI aggregation, some proteins are tyrosine-phosphorylated early, whereas others are phosphorylated at later stages after a rise in intracellular calcium or the activation of protein kinase C (3,4,8,21). Furthermore, activation and tyrosine phosphorylation of Pyk2 by stimulation of several cell-surface receptors may be due to the increase in intracellular calcium (24). In the RBL-2H3 cells, addition of calcium ionophore A23187 to directly increase intracellular calcium induced an increase in the tyrosine phosphorylation of Pyk2, which was similar to that by Fc⑀RI activation (Fig. 3). There was also strong tyrosine phosphorylation of Pyk2 when cells were stimulated with PMA, an activator of protein kinase C. Under these conditions, there was histamine release with the calcium ionophore A23187 but no degranulation with PMA. These experiments suggested that the increased tyrosine phosphorylation of Pyk2 could be due to the rise in intracellular calcium and/or the activation of protein kinase C.
Fc⑀RI aggregation results in the release of Ca 2ϩ from intracellular stores followed by the influx of Ca 2ϩ from the medium. Since calcium ionophore induced the tyrosine phosphorylation of Pyk2, we examined the role of extracellular Ca 2ϩ in the Fc⑀RI-mediated tyrosine phosphorylation of Pyk2. RBL-2H3 cells were washed and then stimulated in a Ca 2ϩ -free medium (Fig. 4). The tyrosine phosphorylation of Pyk2 was decreased when the cells were stimulated in the absence of Ca 2ϩ in the medium. Therefore, at least part of the tyrosine phosphorylation of Pyk2 was independent of the large increase in intracellular Ca 2ϩ that occurs by influx of Ca 2ϩ from the medium.
Cell Adhesion Regulated the Tyrosine Phosphorylation of Pyk2 after Cell Stimulation-Previously we observed that in- tegrin-mediated adherence of RBL-2H3 cells to fibronectin resulted in the tyrosine phosphorylation of FAK (15). Cell adhesion also enhanced the Fc⑀RI-induced secretion and proteintyrosine phosphorylation of FAK (15,38). For the experiments described so far, the cells were adherent as monolayers and there was always some constitutive tyrosine phosphorylation of Pyk2. We therefore investigated whether adherence by integrins regulated the tyrosine phosphorylation of Pyk2 (Fig. 5). When RBL-2H3 cells were added to either BSA-or fibronectincoated surfaces, more than 90% of the cells adhered to fibronectin-coated surfaces, but none attached to BSA-coated surfaces. After plating the cells for 20 min at 37°C, there was a slight increase in the tyrosine phosphorylation of Pyk2 in adherent compared with nonadherent cells (data not shown). Cell stimulation had dramatically different effects in nonadherent as compared with adherent cells. In the nonadherent cells, there was a very slight increase in the tyrosine phosphorylation of Pyk2 after Fc⑀RI aggregation but no detectable change with the calcium ionophore or with PMA. In contrast, adherence dramatically enhanced the tyrosine phosphorylation of Pyk2 by stimulation with antigen, calcium ionophore, and PMA. Therefore, cell activation by adherence and other receptors synergistically regulate the tyrosine phosphorylation of Pyk2.
Syk Dependence of the Fc⑀RI-induced Tyrosine Phosphorylation of Pyk2-In a Syk-deficient variant of the RBL-2H3 cell line, some proteins including the ␤ and ␥ subunits of Fc⑀RI are still tyrosine-phosphorylated, but there is no release of Ca 2ϩ from intracellular sources and no influx of Ca 2ϩ (20). We therefore used these Syk-deficient cells to evaluate the role of Syk in tyrosine phosphorylation of Pyk2 (Fig. 6). Although there was less Pyk2 in the Syk-negative cells, its constitutive tyrosine phosphorylation was similar to that in the wild type RBL-2H3 cells. Fc⑀RI aggregation did not induce an increase in the tyrosine phosphorylation of Pyk2 in the Syk-negative cells. However, in the cells that had been stably transfected with Syk, there was reconstitution of the Fc⑀RI-induced tyrosine phosphorylation of Pyk2. Therefore, the Fc⑀RI-induced tyrosine phosphorylation of Pyk2 requires Syk.
G-protein-coupled Receptor-induced Tyrosine Phosphorylation of Pyk2 Does Not Require Syk-In different cell types, stimulation of G-protein-coupled receptors such as those for bradykinin, thrombin, or lysophospatidic acid results in the tyrosine phosphorylation of Pyk2 (24,26,31). Stimulation of thrombin and adenosine G-protein-coupled receptors in RBL-2H3 cells results in transient mobilization of intracellular calcium (39,40). Thrombin stimulation is mediated by a pertussis toxin-insensitive G-protein, whereas adenosine is inhibited by this toxin, suggesting that it is probably mediated by G i (39,41). Stimulation with thrombin of both the RBL-2H3 and the Syk-negative TB1A2 cells induced the rapid tyrosine phosphorylation of Pyk2 (Fig. 7A). This tyrosine phosphorylation in both the wild type and in the Syk-negative cells was detectable within 30 s of stimulation. Similarly the stimulation of adenosine receptors induced Pyk2 tyrosine phosphorylation in both the RBL-2H3 and the Syk-negative cells (Fig. 7B). Therefore, Syk is not required for tyrosine phosphorylation of Pyk2 induced by G-protein-coupled receptors. DISCUSSION These studies indicate that Pyk2 was present in mast cells and was tyrosine-phosphorylated and activated after cell stimulation. There were at least two different pathways that led to Pyk2 activation. The pathway from Fc⑀RI aggregation required Syk, whereas that from G-protein-coupled receptors was Sykindependent. Pyk2 was also tyrosine-phosphorylated either by the addition of calcium ionophore A23187 to raise intracellular calcium or when protein kinase C was activated with PMA. These results strongly suggest that, similar to results in other cells, the tyrosine phosphorylation and activation of Pyk2 in mast cells was due to the rise in intracellular calcium (24,28,31).
Syk plays a major role in Fc⑀RI-mediated activation of mast cells (20). In this pathway, receptor aggregation activates a protein-tyrosine kinase, probably Lyn, which results in tyrosine phosphorylation of the receptor subunits. Syk then binds to the tyrosine-phosphorylated receptor subunits and is activated to propagate downstream signals including the tyrosine phosphorylation of phospholipase C-␥, the release of calcium from intracellular sources, and the influx of calcium from the extracellular medium. Although Fc⑀RI aggregation in the Sykdeficient cells results in the tyrosine phosphorylation of the ␤ and ␥ subunits of the receptor and of several proteins (20,42), it did not induce the activation or tyrosine phosphorylation of Pyk2. In contrast, activation of the T cell receptor, another member of the immune receptor family, results in tyrosine phosphorylation of Pyk2 that is selectively mediated by the Src family kinase Fyn (32,33). These seemingly contradictory observations can be explained if, in mast cells, Fc⑀RI aggregation by a Syk-dependent pathway results in an increase in intracellular calcium, which then utilizes a Src family kinase to tyrosine-phosphorylate Pyk2.
The integrin-mediated adherence of RBL-2H3 cells to fibronectin results in cell spreading, reorganization of the cytoskeleton, and a redistribution of the granules to the periphery of the cells (38,43). Adhesion also results in tyrosine phosphorylation of proteins such as FAK and the cytoskeletal protein paxillin (15,44,45). Adherence of platelets, megakaryocytes, and T and B cells, but not fibroblasts, results in the tyrosine phosphorylation of Pyk2 (25,27,35,46). Similarly, in the present experiments, there was a slight increase in tyrosine phosphorylation of Pyk2 after adherence to fibronectin, a reaction that is probably mediated by ␤ 1 integrins. The aggregation of ␤ 1 integrins in B cells and ␤ 3 integrins in T cells results in tyrosine phosphorylation of Pyk2 (34,35). Pyk2 localizes to sites of cell-to-cell contact and to focal adhesion like structures (25,27). At these sites, the cytoplasmic domains of the integrins form focal adhesion complexes that contain cytoskeletal proteins such as talin, vinculin, ␣-actinin, filamin, FAK, and other phosphoproteins (27,44,47). Although RBL-2H3 cells do not form classical focal adhesion complexes, stimulation of the cells results in actin plaques and tyrosine phosphorylation of FAK (15,48). Here we observed that the stimulated tyrosine phosphorylation of Pyk2, as was previously reported for FAK, was dramatically enhanced by the adhesion of the cells to fibronectin (15). Therefore, adhesion to fibronectin modulates the activation of both Pyk2 and FAK. There is also enhanced secretion from adherent cells. Therefore, adherence by regulating the level of the tyrosine phosphorylation of Pyk2, FAK, and other proteins could control the extent of degranulation in these cells.
Pyk2 interacts with signaling molecules and may therefore play a role in signal transduction in mast cells. Pyk2 associates through its C-terminal region with paxillin, a 68-kDa cytoskeletal protein that is tyrosine-phosphorylated after Fc⑀RI aggregation (36,49). Paxillin also accumulates at focal adhesion sites and binds to pp60 src , Lyn, Crk, vinculin, and talin. Pyk2 also associates with Grb2, which by binding to Shc and Sos may activate the Ras pathway (24,32). Although Src family kinases such as Fyn, Lck, and c-Src associate by their SH2 domains with Pyk2 (31, 32), we could not detect association of Pyk2 with the Src family kinase Lyn (data not shown). There is also binding of p130 cas to Pyk2, probably by the SH3 domain of p130 cas binding to the C-terminal proline-rich region of Pyk2 (35). Pyk2 tyrosine-phosphorylates the potassium channel and suppresses channel currents (24) and also acts as an upstream regulator for stress signal activation of c-Jun N-terminal kinase (28). As many of these pathways are activated in stimulated mast cells, tyrosine phosphorylation of Pyk2 may play a role in the signals that lead to generation of mediators. Stimulation of cells by many G-protein-coupled receptors results in the tyrosine phosphorylation of Pyk2 (24,26,30,31). Mast cells have thrombin receptors that couple to G q , a pertussis toxin-insensitive G-protein ␣-subunit and A 3 adenosine receptors that couple to a pertussis toxin-sensitive ␣-subunit G i . The stimulation of these receptors in RBL-2H3 cells results in a transient increase in intracellular Ca 2ϩ (39,40). Here we observed that stimulation of the cells with either thrombin or an adenosine analog induced the rapid tyrosine phosphorylation of Pyk2. Interestingly the extent of this phosphorylation was not dependent on the presence of Syk in the cells. These results demonstrate that there are Syk-independent pathways that link G-protein-coupled receptors to Pyk2 activation.
In summary, these experiments indicate that stimulation of mast cells with different stimuli induces the tyrosine phosphorylation of Pyk2. The Fc⑀RI-mediated phosphorylation was downstream of Syk and probably secondary to the mobilization of intracellular Ca 2ϩ . In contrast, G-protein-coupled receptors induced tyrosine phosphorylation of Pyk2, which was independent of Syk. Adherence of cells to fibronectin regulated the tyrosine phosphorylation of Pyk2, similar to that which we had observed previously for FAK (15). Since there are two different focal adhesion kinases in RBL-2H3 cells, it will be important to clarify the function of these two molecules in the signaling cascade.
FIG. 7. G-protein-coupled receptorinduced tyrosine phosphorylation of Pyk2 by a pathway independent of Syk. RBL-2H3 and TB1A2 (SykϪ) cells were stimulated for the indicated times with either thrombin (A) or the adenosine analog APNEA (B). Cell lysates were then immunoprecipitated with anti-Pyk2 antibody and analyzed by immunoblotting with anti-phosphotyrosine and anti-Pyk2 antibodies.