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G_i-Mediated Activation of the Syk Kinase by the Receptor Mimetic Basic Secretagogues of Mast Cells: Role in Mediating Arachidonic Acid/Metabolites Release¹

Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

	Abstract

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Syk kinase is essential for FcRI-mediated signaling and release of inflammatory mediators from mast cells. We now show that activation of rat peritoneal mast cells by the nonimmunological, G_i-mediated pathway also results in the activation of Syk. We show that compound 48/80 (c48/80), a receptor analogue that activates directly G proteins, activates Syk in a pertussis toxin-sensitive fashion. We further show that Syk activation by c48/80 is blocked by the protein kinase C inhibitor GF109203X, by the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, by EGTA, and by the selective src-like kinase inhibitor PP1. These results suggest that in the nonimmunological, G_i-mediated pathway, Syk is located downstream from phospholipase C and phosphatidylinositol 3-kinase. However, in common with the FcRI-mediated pathway, activation of Syk by c48/80 is dependent on a src-like protein tyrosine kinase. Finally, we show that in the nonimmunological pathway, Syk plays a central role in the release of arachidonic acid/eicosanoid metabolites, but not in the release of prestored mediators such as histamine.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mast cells play a central role in inflammatory and immediate-type allergic reactions. These cells are specialized secretory cells, which release, in response to activation by external stimuli, a variety of inflammatory mediators. Mediators released upon mast cell activation fall into three classes. Class I mediators, such as histamine, are prestored in secretory granules and released from the cell within minutes of activation. Class II mediators include metabolites of arachidonic acid (AA),³ such as the prostaglandins and leukotrienes, and are generated de novo from their precursors as a result of cell activation. Finally, class III include cytokines and chemokines that are expressed and released within the first few hours after activation (1, 2).

The major pathway of activating mast cells is the immunological trigger brought about by the aggregation of high affinity receptors (FcRI) for IgE, by corresponding Ags (3). The earliest event in this pathway is the activation of src-like cytosolic protein tyrosine kinases (PTKs), p53/56lyn or p62c-yes, leading to the rapid tyrosine phosphorylation of the FcRI and subunits (4). These phosphorylations then enable the recruitment and activation of additional cytosolic PTKs, such as the Syk kinase (5, 6, 7) and the tyrosine phosphorylation of a number of protein substrates, including phospholipase C (PLC), Vav, Nck, and paxillin (reviewed in Ref. 8). Syk activation is essential for FcRI-induced activation of mitogen-activated protein kinase (MAPK) (9) probably through phosphorylation of shc and triggering of the Grb2/Sos/Ras cascade (10).

An alternative way of activating mast cells is by a large number of polycationic molecules collectively known as the basic secretagogues of mast cells (11). The latter include positively charged neuropeptides such as substance P and bradykinin, various amines such as the synthetic compound 48/80 (c48/80), and naturally occurring polyamines (11). Basic secretagogues activate connective tissue type, but not mucosal mast cells, in an IgE-independent manner. These agonists act as receptor mimetic agents, which trigger mast cell exocytosis by activating directly pertussis toxin (Ptx)-sensitive G_i proteins that control exocytosis (12, 13, 14, 15). The mechanism by which basic secretagogues activate exocytosis and release of the preformed mediators appears to be distinct from that of the immunological trigger. Unlike the immunological evoked response, release by basic secretagogues is inhibited by Ptx, does not require the presence of external Ca²⁺, and is completed within <1 min (12, 16). However, despite these marked differences, we have recently demonstrated similarities in the mechanisms by which the immunological or the G protein-mediated trigger stimulates the production and release of class II mediators. We have shown that c48/80 stimulates the activity of PTKs, leading to the enhanced tyrosine phosphorylation of a number of cellular proteins and to the activation of both the p42 and p44 MAPKs (17, 18). Activation of the PTK pathway occurred via a mechanism that involves protein kinase C (PKC), phosphatidylinositol 3-kinase (PI-3K), and Ca²⁺ as intermediates (17, 18). We have further demonstrated that this signaling pathway importantly contributes to the production and release of class II mediators, while it is dispensable for histamine release. In the present work, we have identified the Syk kinase as one of the PTKs, which are activated by c48/80. We also demonstrate that Syk activation occurs via a mechanism, which involves PKC, Ca²⁺, and PI-3K as well as a src-like PTK. Finally, we show that in the nonimmunological pathway, Syk mediates the release of AA/metabolites, but not degranulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

GF109203X, Go 6976, wortmannin, and PD98059 were purchased from Calbiochem (La Jolla, CA). Piceatannol was obtained from Sigma (St. Louis, MO). The protease inhibitor cocktail complete was obtained from Boehringer Mannheim (Indianapolis, IN), and the src inhibitor PP1 was obtained from Alexis (San Diego, CA). [-³²P]ATP (3000 Ci/mmol) and [³H]AA (60–100 Ci/mmol) were obtained from DuPont-NEN (Boston, MA).

The plasmid encoding the hemopoietic lineage cell-specific protein (HS1) fused with GST (GST-HS1) was kindly provided by U. Blank (Immuno-Allergie, Institute Pasteur, Paris, France), and the protein was expressed in Escherichia coli and was affinity purified on GST beads.

Antibodies

mAbs against phosphotyrosine (P-Tyr; PY20) were obtained from Transduction Laboratories (Lexington, KY); anti-active MAPK Abs were purchased from Promega (Madison, WI); Syk Ab directed against a peptide comprising the 11 C-terminal aa of Syk was kindly provided by U. Blank. Peroxidase-conjugated Affinipure goat anti-mouse or anti-rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Isolation and purification of mast cells

Rat peritoneal mast cells were obtained from Wistar rats by a peritoneal lavage and purified as previously described (12). Briefly, a suspension of washed peritoneal cells was layered over a cushion of 30% Ficoll 400 (Pharmacia Biotech, Uppsala, Sweden) in buffered saline supplemented with 0.1% BSA, and centrifuged at 150 x g for 15 min. The purity of mast cells recovered from the bottom of the tube was 90% as assessed by toluidine blue staining.

Triggering of intact cells

Purified mast cells (5 x 10⁵ cells/ml in a final volume of 0.5 ml) were incubated in Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 20 mM HEPES, pH 7.4, 1 mM CaCl₂, 5.6 mM glucose, 1 mg/ml BSA) with buffer or with the indicated stimuli for 20 min. Reactions were terminated by placing the tubes in ice, followed by a brief spin (12,000 x g, 20 s) at 4°C.

In vitro kinase assay

Cells were washed three times with cold PBS and subsequently lysed in lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaF, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM -glycerophosphate, and 40 µg/ml protease inhibitor mixture) for 20 min on ice. Cell lysates were cleared by centrifugation at 12,000 x g for 15 min at 4°C. For immunoprecipitation, cell lysates were incubated for 2 h at 4°C with the primary anti-Syk Ab, which was preconjugated to protein A-Sepharose beads. The immune complexes were washed once with 0.5 M LiCl/0.1 M Tris-HCl, at pH 8, three times with lysis buffer, once with lysis buffer without Triton X-100, and once with buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM sodium orthovanadate, and 1 mM PMSF. The immune complexes were subsequently incubated for 10 min at 30°C with 2 µg GST-HS1 in 40 µl kinase buffer containing 10 mM MgCl₂, 2 mM MnCl₂, 30 mM HEPES, pH 7.4, 1 µM ATP, 1 mM sodium orthovanadate, and 10 µCi [-³²P]ATP. Reactions were terminated by the addition of 5-fold concentrated Laemmli sample buffer (19). Samples were boiled for 5 min, centrifuged for 1 min at 14,000 x g, and resolved by SDS/12% PAGE under reducing conditions.

Determination of protein tyrosine phosphorylation

Cell lysates prepared in lysis buffer comprising 150 mM sucrose, 80 mM -glycerophosphate, 2 mM EDTA, 2 mM EGTA, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1% Triton X-100, 1 mM PMSF, and 40 µg/ml protease inhibitor cocktail were centrifuged for 15 min at 12,000 x g, and the supernatants were mixed with 5x concentrated Laemmli sample buffer. Samples were boiled, resolved by SDS/10% PAGE under reducing conditions, and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with mAbs directed against P-Tyr (PY20, 1 µg/ml). Bound Abs were visualized by ECL detection with the use of goat antiserum to mouse coupled to HRP (Jackson ImmunoResearch Laboratories).

Determination of MAPK activation

Cell lysates, prepared as described above for the determination of protein tyrosine phosphorylation, were resolved by SDS/10% PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with polyclonal Abs directed against the active phosphorylated form of p42/p44 MAPKs (1/20,000 dilution). Bound Abs were visualized by ECL detection with the use of goat antiserum to rabbit coupled to HRP (Jackson ImmunoResearch Laboratories).

Determination of AA release

Purified mast cells (1 x 10⁶ cells/ml) were incubated with 2–5 µCi/ml [³H]AA for 2 h at 37°C. The cells were subsequently washed three times in Tyrode’s buffer, resuspended in Tyrode’s buffer at 5 x 10⁵ cells/ml, and triggered for 20 min. Reactions were terminated by placing the tubes in ice, followed by a brief centrifugation (12,000 x g, 20 s at 4°C). Supernatants were collected and used to determine the amount of radiolabeled AA released by liquid scintillation.

Determination of histamine release

The amount of histamine released was determined, as previously described (12), using the O-phthaldialdehyde fluorometric method (20).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
c48/80 induces activation of Syk in rat peritoneal mast cells

To investigate whether c48/80 can activate the Syk kinase, rat peritoneal mast cells were incubated for 20 min in the absence or presence of c48/80 (5 µg/ml), and Syk was subsequently immunoprecipitated. Initially, we attempted to determine whether c48/80 stimulated tyrosine phosphorylation of Syk. However, the low cell number did not allow detection of P-Tyr or Syk protein by Western blot analyses. Therefore, we used instead an in vitro kinase assay, using the Syk substrate GST-HS1 (21). As shown in Fig. 1, the activity of Syk derived from c48/80-treated cells was markedly higher, as evidenced by its increased potency to phosphorylate the exogenous substrate (lane 2 vs lane 1). This ability to phosphorylate GST-HS1 was completely abolished when piceatannol (50 µg/ml), a specific inhibitor of Syk (22), was included in the kinase assay (lane 3 vs lane 2). Therefore, these results have indicated that c48/80 was able to activate Syk in intact rat peritoneal mast cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Activation of Syk by c48/80. Purified mast cells (5 x 105 cells/ml) were incubated in the presence of vanadate (0.1 mM) with buffer (lane 1) or c48/80 (5 µg/ml, lanes 2 and 3) for 20 min at 37°C. At the end of incubation, cells were lysed and Syk was immunoprecipitated. The immune complexes were preincubated with buffer (lanes 1 and 2) or piceatannol (50 µg/ml, lane 3) for 10 min at 30°C and incubated in the presence of GST-HS1 (2 µg) in an in vitro kinase assay, as described in Materials and Methods. Proteins were analyzed by SDS-PAGE, followed by autoradiography.

To determine whether the activation of Syk by c48/80 was dependent on G_i proteins, we examined the effect of Ptx on the state of Syk activation. As shown in Fig. 2, pretreatment of the cells with Ptx (300 ng/ml for 2 h) has completely eliminated the ability of c48/80 to activate Syk. Therefore, c48/80-induced activation of Syk is indeed mediated by Ptx-sensitive G_i protein(s).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Effect of Ptx on Syk activation induced by c48/80. Purified mast cells (5 x 105 cells/ml) were incubated for 2 h at 37°C with (lanes 3 and 4) or without (lanes 1 and 2) Ptx (300 ng/ml). Cells were subsequently incubated in the presence of vanadate (0.1 mM) with buffer (lanes 1 and 3) or c48/80 (5 µg/ml, lanes 2 and 4) for an additional 20 min. At the end of incubation, cells were lysed and Syk was immunoprecipitated. Syk activation was assayed as described under Fig. 1.

We have shown previously that the enhancement in protein tyrosine phosphorylation and MAPK activation, effected by c48/80, is dependent on the presence of external Ca²⁺ and on the activation of PI-3K and PKC (17, 18). Therefore, we investigated whether Syk activation also requires these intermediates. As shown in Fig. 3A, in cells treated with c48/80 in the presence of EGTA, activation of Syk was abolished. Similarly, exposing the cells to GF109203X (100 nM), which is considered a specific inhibitor for the PKC isozymes , , , , and , has completely abolished Syk activation (Fig. 3B). In contrast, Go 6976, which blocks specifically the activity of the PKC and isoforms, failed to affect Syk (Fig. 3B). Hence, although not proven, these findings strongly implicate the Ca²⁺-independent PKC isozymes PKC and in mediating Syk activation by c48/80.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. The role of Ca2+ PKC and PI-3K in the activation of Syk. A, Purified mast cells (5 x 105 cells/ml) incubated in the presence of vanadate (0.1 mM) and Ca2+ (1 mM, lanes 1 and 2) or without Ca2+ and with EGTA (0.1 mM, lanes 3 and 4) with buffer (lanes 1 and 2) or c48/80 (5 µg/ml, lanes 2 and 4) for 20 min. At the end of the incubation, cells were lysed and Syk was immunoprecipitated. Syk activation was assayed as described in Fig. 1. B, The cells were incubated in the presence of vanadate (0.1 mM) for 15 min at 37°C with buffer (lanes 1 and 2), wortmannin (100 nM, lane 3), GF109203X (100 nM, lane 4), or Go 6976 (20 nM, lane 5). At the end of incubation, buffer (lane 1) or c48/80 (5 µ g/ml, lanes 2–5) was added for an additional 20 min. At the end of incubation, cells were lysed and Syk activation was assayed, as described in A.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. TPA-induced activation of Syk kinase. Purified mast cells (5 x 105 cells/ml) were incubated in the presence of vanadate (0.1 mM) for 15 min at 37°C with buffer (lanes 1–5) or wortmannin (100 nM, lane 6). At the end of incubation, buffer (lane 1), Ca2+ ionophore (A23187, 1 µM, lane 2), TPA (5 nM, lanes 3), TPA (100 nM, lane 4), or the combination of TPA (5 nM) and Ca2+ ionophore (A23187, 1 µM, lanes 5 and 6) was added and incubation continued for an additional 20 min. Cells were subsequently sedimented, the supernatants were collected, and the cell pellets were lysed. Syk activation (A) was assayed as described in Fig. 1. The intensities of the bands corresponding to the phosphorylated GST-HS1 were quantified by densitometry and are presented in B. The amount of histamine present in the cell supernatants was determined as described in Materials and Methods and is presented in arbitrary units (C).

Activation of Syk is dependent on a src-like PTK, but not on MAPK activation

Recently, we have shown that c48/80 activates the MAPKs by a mechanism, which involves PTKs, Ca²⁺, PKC, and PI-3K as intermediates (18). Because activation of Syk included similar intermediates, we investigated whether Syk was localized downstream from MAPKs. For this purpose, the effect of PD98059, a specific inhibitor of mitogen-activated protein/extracellular signal-related kinase kinase, was evaluated. As shown in Fig. 5, PD98059 had no effect on Syk activation at concentrations that we have previously shown to block MAPK activation (18). These results have indicated that MAPKs were not involved in the pathway leading to Syk activation.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Effect of PP1 and PD98059 on c48/80-induced Syk activation. Purified mast cells (5 x 105 cells/ml) were incubated in the presence of vanadate (0.1 mM) for 15 min at 37°C with buffer (lanes 1 and 2), PP1 (5 µM, lane 3), or PD98059 (50 µM, lane 4). Buffer (lane 1) or c48/80 (5 µg/ml, lanes 2–4) was added for another 20-min incubation. At the end of the incubation, cells were lysed and Syk was immunoprecipitated. Syk activation was assayed as described in Fig. 1.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Dose-response relationships of PP1. Purified mast cells (5 x 105 cells/ml) were incubated for 15 min at 37°C in the presence of vanadate (0.1 mM) with the indicated concentrations of PP1. Buffer, c48/80 (5 µg/ml), or TPA (100 nM) was subsequently added, and incubation continued for an additional 20-min incubation. At the end of incubation, cells were sedimented and lysed. Syk activation (A and D) was assayed as described in Fig. 4. The level of protein tyrosine phosphorylation (B) was determined by subjecting the cell lysates to SDS-PAGE, followed by Western blot analysis with anti-P-Tyr Abs. MAPK activation (C) was determined by subjecting the cell lysates to SDS-PAGE, followed by Western blot analysis with anti-active MAPK Abs.

Next we examined whether activation of Syk by TPA was also dependent on a src-like PTK. Indeed, as shown in Fig. 6D, PP1 also inhibited TPA-induced activation of Syk. Taken together, our results indicate that both src-like and Syk kinases are located downstream from PKC in the G_i-mediated signaling pathway.

Syk activation is not dependent on trans activation of the receptor for epidermal growth factor (EGFR)

Trans activation of the EGFR is one pathway by which G protein-coupled receptors activate protein tyrosine and MAP kinases (25, 26). Although the mechanism of trans activation remains obscure, it involves activation of the src kinase (27). Therefore, we explored the possibility that activation of Syk by c48/80 was mediated by the EGFR. For this purpose, we used AG1478, a specific inhibitor of the EGFR tyrosine kinase. As shown in Fig. 7, incubation with AG1478 had no effect on c48/80-induced activation of Syk, therefore, ruling out the possibility of trans activation of the EGFR. As a matter of fact, incubation of mast cells with EGF (10 ng/ml) failed to evoke Syk activation, excluding further involvement of the EGFR (Fig. 7).

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Effect of AG1478 on c48/80-induced Syk activation. Purified mast cells (5 x 105 cells/ml) were incubated in the presence of vanadate (0.1 mM) for 15 min at 37°C with the indicated concentrations of AG1478. Buffer (lane 1), c48/80 (5 µ g/ml, lanes 2–4), or EGF (10 ng/ml, lane 5) was added for an additional 20-min incubation. At the end of incubation, cells were lysed and Syk activation was assayed, as described in Fig. 1.

We have previously shown that c48/80-induced release of AA/metabolites is dependent on PTKs, but not on the activation of the MAPKs (17, 18). Because activation of Syk was also dependent on PTKs, but not on MAPKs, we investigated whether Syk played a role in the stimulation of AA/metabolites release. For this purpose, we made use of piceatannol, an inhibitor of the Syk kinase. As shown in Fig. 8, incubation of the cells with piceatannol inhibited the release of AA/metabolites in a dose-dependent manner. Half-maximal inhibition was achieved at 20 µg/ml and maximal at 50 µg/ml. In contrast, histamine release induced by c48/80 under the identical experimental conditions was unaffected by piceatannol (Fig. 8). Therefore, these results have implicated Syk in mediating c48/80-induced release of class II, but not class I, mediators.

View larger version (19K): [in this window] [in a new window]   FIGURE 8. Effect of piceatannol on release of histamine and AA/metabolites induced by c48/80. Purified mast cells (5 x 105 cells/ml) were incubated with 3H-labeled AA for 2 h at 37°C. At the end of incubation, the cells were washed three times with Tyrode’s buffer and incubated for an additional 15 min with buffer or with the indicated concentrations of piceatannol. Buffer or c48/80 (5 µg/ml) was subsequently added, and the cells were incubated for an additional 20 min. At the end of incubation, cells were sedimented, and the amount of [3H]AA/metabolites and histamine released was determined as described in Materials and Methods. The results are presented as the percentage of the maximal response obtained in the absence of inhibitor, for [3H]AA/metabolites released, and the percentage of total for histamine.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Syk kinases play a critical role in the signal transduction pathways elicited by immune receptors. In mast cells, expression and activation of Syk are essential for FcRI-mediated signaling. Indeed, in Syk-deficient mast cells, aggregation of the FcRI fails to induce mast cell activation (9, 28). In a similar fashion, inhibitors, such as piceatannol (22) or ER-27319 (29), which inhibit the Syk kinase, inhibit Ag-induced release of allergic mediators. In this study, we show that activation of mast cells by the nonimmunological, G_i-dependent pathway also results in the activation of Syk (Fig. 1). As expected, activation of Syk by c48/80, a receptor analogue that activates directly G_i proteins (12, 13, 14, 15), is Ptx sensitive (Fig. 2). It is presently unknown which G protein, or which subunits, or , mediate the activation of the Syk kinase by c48/80. However, based upon our previous results (30), rat peritoneal mast cells express only two Ptx-sensitive G proteins. These include G_i2 and G_i3, whereby G_i3 serves as the principal mediator of exocytosis (30). Therefore, the finding that Ptx treatment prevents Syk activation limits the G proteins, which are possibly involved, to G_i2 or G_i3.

Activation of the Syk kinase by c48/80 implicates Syk as one of the signaling molecules that are activated by either the immune receptor FcRI or upon activation of Ptx-sensitive G_i proteins by receptor analogues. However, the mechanism by which c48/80 activates Syk is different from its mode of activation by the FcRI. In the latter case, tyrosine phosphorylation of the activated FcRI by the lyn kinase creates the protein modules, which allow the activated receptor to bind and activate Syk. This interaction between the tyrosine-phosphorylated receptor and Syk is mediated by tandem SH2 groups located in the N-terminal region of Syk (31). Therefore, activation of Syk by FcRI reflects one of the early events following receptor aggregation. As such, activation of Syk precedes the phosphorylation of PLC and the subsequent mobilization of Ca²⁺ and activation of PKC. In contrast, activation of Syk by G_i occurs at a later step. We show in this study that in this signaling pathway, Syk is localized downstream from PKC. Two observations support this conclusion. First, GF109203X, an inhibitor that blocks specifically PKC, 1, 2, , , and , prevents activation of Syk by c48/80 (Fig. 3B). Second, the phorbol ester TPA, which activates directly PKC, also promotes Syk activation (Fig. 4). Interestingly, Go 6976, an inhibitor that selectively blocks the and , Ca²⁺-dependent PKC isozymes (reviewed in Ref. 32), has no effect (Fig. 3B), therefore implicating the Ca²⁺-independent PKC or PKC isozymes in mediating this response. Indeed, we have previously shown that stimulation of protein tyrosine phosphorylation and activation of the MAPKs by c48/80 are mediated by PKC isozymes sensitive to GF109203X, but resistant to Go 6976 (17, 18). Nevertheless, Ca²⁺ is required for activation of either the MAPKs (18) or Syk, as EGTA abolishes c48/80-induced activation of both (Fig. 3A). It is presently unknown at which step Ca²⁺ is required. Ca²⁺ does not interfere with the ability of c48/80 to activate G_i, as indicated by the fact that external Ca²⁺ is not required for the trigger of histamine release by c48/80 (33). The finding that Ca²⁺ ionophore neither activates Syk, nor does it synergize with a suboptimal dose of TPA may suggest that Ca²⁺ is involved at a step downstream to G_i activation by c48/80, but upstream to PKC activation. For example, Ca²⁺ may be required to activate phospholipase D (PLD) and produce diacylglycerol, which in turn activates PKC (34). Nevertheless, taken together our results clearly demonstrate that whereas in the immunological pathway Syk is located upstream to PLC and therefore also upstream to Ca²⁺ mobilization and PKC activation, in the G_i-mediated pathway, Syk is located downstream from PKC. These results are consistent with the finding that in a Syk-deficient mast cell line, G protein-coupled receptors (adenosine and thrombin), but not FcRI aggregation, could still induce tyrosine phosphorylation of Pyk2, a member of the focal adhesion kinase family, whose phosphorylation requires Ca²⁺ and PKC (35).

We have previously shown that two mechanistically different inhibitors of PI-3K, wortmannin and LY294002, prevent the stimulation of protein tyrosine phosphorylation and activation of the MAPKs by c48/80 (18). These findings indicated the involvement of PI-3K in mediating this signaling pathway. We now show that wortmannin (Fig. 3B) and LY294002 (data not shown) also prevent Syk activation, indicating that Syk also resides downstream from PI-3K. Moreover, the fact that wortmannin also prevents TPA-induced activation of Syk (Fig. 4) suggests that PI-3K is located downstream from PKC in the cascade leading to Syk activation (see model, Fig. 9). However, it should be noted that heterotrimeric G proteins, belonging to the G_i family, can activate PI-3K via their subunits (36). Therefore, we cannot exclude the possibility that activation of Syk by c48/80 may be mediated by PI-3K(s) activated simultaneously by subunits and by PKC (see model, Fig. 9).

View larger version (13K): [in this window] [in a new window]   FIGURE 9. According to this model, a src-like PTK is one of the PTKs that are activated by c48/80-activated Gi, by a mechanism that involves Ca2+, PKC, and PI-3K. A src-like PTK then activates Syk, which mediates release of AA/metabolites. The src-like PTK also negatively regulates an alternative, Syk-independent pathway, of an as yet unknown nature, which also leads to AA release.

In contrast to its marked effect on Syk, PP1 exerted only a moderate inhibitory effect on the total cell protein tyrosine phosphorylation (Fig. 6B) and had no effect on MAPK activation (Fig. 6C). Therefore, these results indicate that the signaling pathway initiated by c48/80-activated G_i diverges downstream from PKC, Ca²⁺, and PI-3K to a MAPK cascade and to a src-like PTK/Syk pathway (see model, Fig. 9). Activation of neither of these pathways is mediated by trans activation of the EGFR (Fig. 7).

A clue to what might possibly be the function of Syk was obtained from the observation that like Syk also, release of AA/eicosanoid metabolites from c48/80-activated cells is dependent on PTKs, but not on MAPKs (18). Therefore, in the present study, we explored the possibility that Syk may mediate release of class II mediators. Indeed, the Syk inhibitor piceatannol inhibits AA/eicosanoid metabolites release in a dose-dependent manner (Fig. 8). Surprisingly, however, PP1 was ineffective and could not reproduce the inhibitory action of piceatannol (data not shown). Whereas the reason for this discrepancy is presently unknown, we would like to propose a model (Fig. 9), according to which two distinct pathways can stimulate AA/eicosanoid metabolite release. One pathway is mediated by Syk, whereas the other is Syk independent, but negatively regulated by a src-like PTK. Therefore, under conditions in which the src-like PTK is blocked (e.g., by PP1), the alternative, Syk-independent pathway dominates. Both phospholipase A₂ and PLD contribute to AA release in mast cells (37) and, therefore, may represent the two distinct pathways. Consistent with this model are the findings that in the mucosal mast cell line (rat basophilic leukemia, RBL-2H3), activation of phospholipase A₂ by transfected, G protein-coupled muscarinic m1 receptor is not influenced by overexpression of a dominant-negative form of Syk (38), whereas activation of PLD was recently shown to depend on Syk (39).

When triggered via the FcRI, Syk mediates release of both AA/eicosanoid metabolites and class I mediators stored in the secretory granules (9). However, when triggered by c48/80, Syk is dispensable to histamine release (Fig. 8). Therefore, these results indicate that the immune and the G_i protein-mediated signaling pathways converge to stimulate production and release of class II and possibly class III mediators. In contrast, the signaling pathways evoking degranulation are essentially different.

In summary, in the present study we have identified src-like and Syk kinases as two of the PTKs, which are activated by the nonimmunological, G_i-mediated pathway. Although the mechanism underlying this activation remains obscure, we have shown that it includes PKC, Ca²⁺, and PI-3K as intermediates, and that it does not involve trans activation of the receptor for EGF. Finally, we show that although the mechanisms by which the immunological and nonimmunological triggers elicit degranulation and release of the prestored class I mediators are tremendously different, the signaling pathways responsible for release of class II mediators are similar or at least share common signaling molecules.

	Acknowledgments

 
We thank Dr. Ulrich Blank for his generous gifts of Abs and cDNA.

	Footnotes

 
¹ Supported by grants from the Israel Science Foundation, the Israel Ministry of Health, and the Rekanati Foundation (to R.S.-E.).

² Address correspondence and reprint requests to Dr. Ronit Sagi-Eisenberg, Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel. E-mail address: histol3{at}post.tau.ac.il

³ Abbreviations used in this paper: AA, arachidonic acid; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PI-3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PTK, protein tyrosine kinase; Ptx, pertussis toxin; P-Tyr, phosphotyrosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received for publication November 6, 2000. Accepted for publication April 25, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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