©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Requirement for Syk in the Activation of the Microtubule-associated Protein Kinase/Phospholipase A Pathway by FcR1 Is Not Shared by a G Protein-coupled Receptor (*)

Noriyasu Hirasawa (1), Andrew Scharenberg (2), Hirohei Yamamura (3), Michael A. Beaven (1)(§), Jean-Pierre Kinet (2)

From the (1) Laboratory of Molecular Immunology, NHLBI, and the (2) Molecular Allergy and Immunology Section, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and the (3) Department of Biochemistry, Fukui Medical School, Matsuoka, Fukui 910-11, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric () immunoglobulin E receptor (FcR1) leads to the activation of cytosolic phospholipase A and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A pathway is linked to FcR1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the FcR1 motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the FcR1 motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A pathway and demonstrate the existence of the FcR1-Syk-MAP kinase pathway.


INTRODUCTION

Antigen-stimulated mast cells release a variety of inflammatory mediators through the release of secretory granules and the generation of arachidonic acid and cytokines (1) . The multivalent binding of antigen to receptor-bound IgE and the ensuing aggregation of receptors for IgE (FcR1) provides the trigger for this release. FcR1 is a multimeric receptor with , , and a homo-dimeric subunits (2) . Both the and chains contain the motif, ITAM()(3, 4, 5) , which allows recruitment of tyrosine kinases and the tyrosine phosphorylation of various proteins (6, 7, 8) , including phospholipase C1 (9) . The sustained activation of phospholipase C (10, 11, 12) and D (13, 14) , along with sustained elevation of diglycerides (14) and mobilization of Ca from intracellular and extracellular sources (15, 16, 17, 18) , results in the activation of protein kinase C. Activation of MAP kinases is also apparent from an increase in MAP kinase activity (19, 20) and the tyrosine phosphorylation and shift in electrophoretic migration of p42 and, much less so, of p44(21) .

Studies in a cultured mast cell (RBL-2H3) cell line indicate that the phospholipase C and the MAP kinase pathways subserve different functions. For example, elevation of [Ca]and the activation of certain isozymes of protein kinase C provide sufficient signals for maximal secretory reponses (22) , whereas release of arachidonic acid does not require protein kinase C. This release is dependent instead on MAP kinase for the phosphorylation and activation of a cytosolic phospholipase A and an increase in [Ca]for the binding of the phospholipase A with the membrane fraction (20) .

The tyrosine kinases, Lyn (p56) and Syk (p72), probably link aggregated FcR1 to phospholipase C, although this has not been unequivocally established. In unstimulated RBL-2H3 cells, Lyn is normally associated with the chain of FcR1 (23, 24, 25, 26) . Studies with chimeras of the extracellular and transmembrane domains of the subunit of the IL-2 receptor and the cytosolic, carboxyl-terminal portion of the (TT) or the cytosolic domain of the (TT) chains of FcR1 (25) , as well as other studies (26, 27) , indicate that aggregation of FcR1 causes tyrosine phosphorylation of the and chains of FcR1, and the recruitment of Syk and additional Lyn. Cross-linking of the TT chimeras with biotinylated anti-Tac antibody and avidin suffice for full expression of responses in RBL-2H3 cells including the activation of Syk (25) , mobilization of Ca, and release of secretory granules (25) .

Here we examine whether or not the Lyn/Syk kinases have an essential role in the activation of the MAP kinase/phospholipase A pathway in RBL-2H3 cells. An early step in this pathway in many types of cells is the association of Raf-1 with Ras at the plasma membrane (28, 29, 30) . The serine phosphorylation of MEK-1 by Raf-1 and, finally, the tyrosine/threonine phosphorylation of MAP kinases by MEK-1 results in activation of MAP kinase activity and, in turn, phospholipase A (reviewed in Ref. 31). This same pathway is stimulated by a variety of stimulants in RBL-2H3 cells through different mechanisms (20) . We have determined that subunits of FcR1 and Syk transduce signals for activation of this pathway by expression of the aforementioned Tac chimeras, a porcine Syk, and a truncated Syk, Syk(T), which lacks the kinase domain, in RBL-2H3 cells. As a control, we have also examined the activation of the MAP kinase/phospholipase A2 pathway via a G protein-coupled receptor, the muscarinic m1 receptor, which has been stably transfected in RBL-2H3 cells (16) .


MATERIALS AND METHODS

Reagents

These were obtained from the following sources: avidin from Sigma, carbachol from Aldrich, thapsigargin from LC Services Corp., Woburn, MA; [1-C]arachidonic acid, from DuPont NEN, reduced streptolysin O from Burroughs Wellcome Co., phenyl-Sepharose from Pharmacia Biotech, Inc., MAP kinase substrate (residues 94-102 of myelin basic protein) from Upstate Biotechnology Inc., Lake Placid, NY, and ECL detection kit from Amersham Corp. The antigen, DNP-BSA, and DNP-specific monoclonal IgE were kindly supplied by Dr. Henry Metzger (NIAMS, National Institutes of Health). Other reagents and materials were from the sources listed previously (22, 32, 33, 34) .

Antibodies were from the following sources; antibody against phosphotyrosine (PY-20 HRP) from ICN, biotinylated anti-Tac B1.49.9 from Amac, Westbrook, ME; polyclonal antibody against the carboxyl-terminal peptide of rat MAP kinase R2 (Erk1-CT) from Upstate Biotechnology Inc., polyclonal antibody against a synthetic peptide of Vav from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and the polyclonal antibody to synthetic peptide derived from Syk was prepared as described previously (25) .

Preparation of Vaccinia Virus/SC-65 Constructs of Syk and Truncated Syk: Cell Infection

Constructs for expression of porcine Syk tyrosine kinase and the truncated Syk kinase (Syk(T)) were prepared by subcloning the respective cDNAs into the pSC-65 vaccinia recombination plasmid. This plasmid places expression under the control of a strong synthetic early/late promoter.() Established techniques and the WR wild type vaccinia strain (American Type Culture Collection) were used to select for recombinant TK(-)/-galactosidase-expressing plaques, to amplify the recombinant plaques into crude stocks, and for establishing titers of the crude stocks (35) . The porcine Syk cDNA was prepared as described previously (36) . The Syk(T) was constructed from the porcine Syk cDNA by using polymerase chain reaction to delete an adenosine at base number 1481, resulting in a frameshift mutation causing isoleucine 396 to change to serine and leucine 397 to a stop codon. The sequence of the Syk(T) cDNA construct was confirmed by dideoxy chain termination sequencing.

Cell Culture and Permeabilization: Measurement of Release of Arachidonic Acid and Secretion of Hexosaminidase

Experiments were performed with a subline of RBL-2H3 cells that had been transfected with the gene for muscarinic m1 receptors (RBL-2H3(m1), Ref. 16) or the Tac chimeras (25) , TT (RBL-TT) and TT (RBL-TT). Cultures were incubated overnight with DNP-specific IgE (0.5 µg/ml), [C]arachidonic acid (0.1 µCi/ml) in complete growth medium in 24-well plates (2 10 cells/400 µl of medium/well) (22) . For the experiments with RBL-TT and RBL-TT, the IgE or biotinylated anti-Tac antibody (2.5 µg/ml) was added to the medium as required 2 h before the experiment (25) . Cultures were washed, and experiments were performed with the following buffers: a glucose/saline/PIPES-buffered medium (pH 7.2) that contained 1 mM Ca for experiments with intact cells (37) ; a potassium glutamate/PIPES-buffered medium (pH 7.2) that contained 5 mM glucose, 7 mM magnesium acetate, 5 mM ATP, 1 mM EGTA, and sufficient Ca to give the indicated [Ca](33) for studies with permeabilized cells. Cells were permeabilized with streptolysin O (33) in the presence of stimulant. Stimulants included DNP-BSA, carbachol, and thapsigargin, at the indicated concentrations, as well as avidin (15 µg/ml). For measurement of release of hexosaminidase (22) and [C]arachidonic acid (34) , reactions were terminated (on ice) 10 min after the addition of stimulant except where indicated. For measurement of MAP kinase activity and detection of proteins by immunoblotting techniques, the reactions were terminated at 2.5 or 5 min as indicated.

Partial Purification and Assay of MAP Kinase Activity

Procedures were based on those described by Offermanns et al.(38) . After each experiment, medium was removed from cultures (in 6-well cluster plates). The cells were harvested in 0.5 ml of a Tris buffer as follows: 25 mM Tris, pH 7.5, 25 mM NaCl, 0.1 mM NaVO, 2 mM EGTA, 1 mM dithiothreitol, 1 mM p-nitrophenyl phosphate, and 20 µg/ml leupeptin. Cells were disrupted by freezing and thawing three times before centrifugation. To the supernatant fraction was added ethylene glycol (10% final concentration) and then phenyl-Sepharose. The mixture was centrifuged, and the sedimented phenyl-Sepharose was washed with Tris buffer that contained 10% (v/v) ethylene glycol. The process was repeated, but with Tris buffer that contained 30% (v/v) ethylene glycol. MAP kinase was eluted from the phenyl-Sepharose with 75 µl of Tris buffer that contained 60% (v/v) ethylene glycol. After centrifugation, 15 µl of the supernatant fraction was incubated (15 min, 37 °C) in a solution that contained 50 mM Tris, pH 7.5, 10 mM MgCl, [-P]ATP (10 Ci/mmol, 37 kBq/tube), 25 µg of a MAP kinase substrate (peptide 94-102 of bovine myelin basic protein). The phosphorylated peptide was isolated by centrifugation of the incubation mixture through phosphocellulose membrane (SpinZyme, Pierce), which was then washed twice with 500 µl of 75 mM HPO for assay of radioactivity.

Electrophoretic Separation and Immunoblotting of MAP Kinase and Other Proteins

After the experiment, cells were lysed in ice-cold lysis buffer (20 mM HEPES, pH 7.3, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 2.5 mM p-nitrophenyl phosphate, 1 mM NaVO, 10 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10% glycerol). Immunoprecipitation of Vav was performed with the antibodies listed under Reagents as recommended by the manufacturer. Proteins in the cell lysate or immunoprecipitated proteins were separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Proteins were detected by the immunoblotting technique with the antibodies listed above. The antibodies were used as recommended by the manufacturer along with alkaline phosphatase-conjugated antibody to rabbit IgG for the second antibody. Tyrosine-phosphorylated proteins were detected by the ECL technique as described previously (21) .

Expression of Data

Mean values (±S.E. for three cultures) from a single experiment are presented where these are representative of at least two experiments. Unless stated otherwise, values were corrected for spontaneous release of either hexosaminidase (<1.0 for intact cells and <7% for permeabilized cells) or arachidonic acid (<1.0% for intact cells and <5.0% for permeabilized cells) in unstimulated cells. MAP kinase activity was expressed as a percent of the activity in unstimulated cells (typically 4500-6500 dpm of P incorporated/µg of isolated protein).


RESULTS

Aggregation of TT but Not of TT Chains in RBL-2H3 Cells Results in Activation of the MAP Kinase Pathway and Release of Arachidonic Acid

In studies with RBL-TT and RBL-TT, cross-linking of TT with biotinylated anti-Tac antibody and avidin did not result in release of secretory granules or arachidonic acid (Fig. 1 A). Both responses were evoked by cross-linking of TT (Fig. 1 B) or of IgE/FcR1 with antigen (Fig. 1, A and B). Anti-Tac and avidin failed to evoke responses in either clone to indicate that the combination of anti-Tac and avidin were required for stimulation of RBL-TT and that dimerization of TT was an insufficient stimulus. Similar results were obtained with permeabilized cells (Fig. 1, C and D).


Figure 1: Aggregation of TT but not of TT chimeras induces release of arachidonic acid as well as secretion of hexosaminidase. RBL-TT and RBL-TT were incubated for 2 h with optimal concentrations of biotinylated anti-Tac (anti-Tac) or DNP-specific IgE. Cells were left intact or permeabilized with streptolysin O in 1 µM [Ca] and stimulated with 15 µg/ml avidin ( Avid.) or 20 ng/ml (intact cells) and 100 ng/ml (permeabilized cells) DNP-BSA ( Ag.) for 10 min. Cells were also treated with biotinylated anti-Tac or avidin alone for controls as indicated. Values (mean ± S.E. for three cultures for this and subsequent figures) were corrected for basal release and were from one of two similar experiments with different clones of RBL-TT cells.



Release of arachidonic acid was transient when compared to release of secretory granules. In this series of experiments, release of arachidonic acid ceased within 3 to 5 min of addition of stimulants (Fig. 2). Secretion of granules had essentially ceased by 10 min (Fig. 2), although in other experiments secretion continued for as long as 15 to 20 min (e.g. Ref. 39). The only difference between anti-Tac/avidin and antigen was that the responses to antigen were more prompt than those to the anti-Tac/avidin (compare A with B in Fig. 2 ).


Figure 2: Time course of release of arachidonic acid and secretion of hexosaminidase following aggregation of FcR1 or TT chimera. RBL-TT were incubated for 2 h with optimal concentrations of DNP-specific IgE ( A) or biotinylated anti-Tac ( B) and then stimulated with 20 ng/ml DNP-BSA ( A) or avidin ( B) for the indicated times. Values were not corrected for spontaneous release of mediators.



Cross-linking of TT also resulted in the tyrosine phosphorylation of various proteins, including p72 (25) , previously identified as Syk (40) and p42 (Fig. 3 A). The phosphorylation of p42 was associated with a shift in electrophoretic migration of p42, such that a second retarded band of p42 was apparent (Fig. 3 A), and an increase in MAP-kinase activity in cell extracts (Fig. 3 B). Two additional proteins, possibly p44 and p46, were detected by the antibody (21, 20) . These proteins also exhibited a shift in migration but, as in previous studies (21) , they exhibited no detectable increase in tyrosine phosphorylation (Fig. 3 A). Cross-linking of TT or use of anti-Tac or avidin failed to evoke these responses ( e.g.Fig. 3 ). Consistent with previous studies (25) , however, cross-linking of TT resulted in tyrosine phosphorylation of a p56 protein, most probably Lyn. This and concurrent experiments indicated that both chimeras were consistently expressed in approximately equivalent amounts (data not shown). These studies indicated collectively that the MAP kinase/phospholipase A pathway can be activated by cross-linking of the chain alone.


Figure 3: Aggregation of TT but not of TT chimeras induces activation of MAP kinase. RBL-TT and RBL-TT were incubated for 2 h with optimal concentrations of biotinylated anti-Tac (anti-Tac) or DNP-specific IgE and then stimulated with avidin or 20 ng/ml DNP-BSA ( Ag.) for 5 min. Cells were also treated with biotinylated anti-Tac alone for controls as indicated. For A, tyrosine-phosphorylated proteins in whole cell extracts were detected by anti-phosphotyrosine antibody and the ECL-detection technique ( Anti-PY). The same membrane was used for detection of p42 by immunoblotting with antibody to MAP kinase ( Anti-MAPK). Note that the protein p40 was previously identified as unphosphorylated p42; the identity of the additional proteins is discussed in the text. For B, cells were permeabilized with streptolysin O before stimulation in 1 µM [Ca] and then assayed for MAP kinase activity. Values indicate percent of the activity in unstimulated cells.



Overexpression of Syk Enhances Antigen-induced Activation of MAP Kinase and Expression of Truncated Syk, Syk(T), Blocks This Activation

Because of the association of Syk with the chain of FcR1 and TT and the indications that it may transduce early signals for activation of RBL-2H3 cells (25) , the effects of overexpression of Syk and Syk(T) were examined by use of a transient expression system. Expression of Syk resulted in enhanced activation of MAP kinase when cells were stimulated with antigen, as indicated by the increase in enzyme activity (Fig. 4 A) and in the amount of protein in the second retarded band of p42(Fig. 4 B). This was most evident with concentrations of antigen that were suboptimal for activation of MAP kinase (Fig. 4 A). Expression of Syk by itself did not result in constitutive activation of MAP kinase ( i.e. ``0 ng/ml DNP-BSA'' in Fig. 4, A and B).


Figure 4: Expression of porcine Syk enhances antigen-induced activation of MAP kinase. Cultures of RBL-2H3 cells were incubated for 16 h with 5 plaque-forming units/RBL-2H3 cell of either wild type (vaccinia/control) or Syk recombinant vaccinia (vaccinia/Syk). The cultures were permeabilized with streptolysin O ( A) or left intact ( B) before stimulation with the indicated concentration of DNP-BSA ( Ag.) for 2.5 min. For A, extracts of the permeabilized cells were assayed for MAP kinase activity. Values indicate percent of activity of unstimulated cells that were infected with the vaccinia/Syk virus ( solid line) or vaccinia/control virus ( dashed line). Similar results were obtained in another experiment. For B, immunoblots of intact cells, vaccinia/control (-) or vaccinia/Syk (+), were probed with antibody to MAP kinase ( Anti-MAPK) as described in the legend of Fig. 3.



Expression of Syk(T), in contrast, blocked activation of MAP kinase in antigen-stimulated cells, as indicated by measurement of enzyme activity (Fig. 5 A), tyrosine phosphorylation of p42 (Fig. 5 B), and the appearance of the second retarded band of p42 (Fig. 5 C). The expression of Syk(T) was also associated with marked diminution of all antigen-induced tyrosine phosphorylated proteins with the notable exception of a p46 protein (Fig. 5 B). The size of this protein matched exactly that expected of truncated Syk (estimated size, 46 kDa).


Figure 5: Expression of truncated Syk (Syk(T)) supresses antigen-induced activation of MAP kinase. Cultures of RBL-2H3 cells were incubated for 16 h with vaccinia/control or Syk(T) recombinant vaccinia virus ( A, as indicated; B and C, 5 plaque-forming units/RBL-2H3 cell). The cultures were permeabilized with streptolysin O ( A) or left intact ( B) before stimulation with the antigen, DNP-BSA (100 ng/ml in A and 20 ng/ml in B), for 5 min. For A, extracts of the permeabilized cells were assayed for MAP kinase activity, and values indicate percent of activity of unstimulated cells that were infected with the vaccinia/Syk(T) virus ( solid line) or vaccinia/control virus ( dashed line). Similar data were obtained in two separate experiments. For B and C, immunoblots of intact cells were probed with anti-phosphotyrosine antibody ( Anti-PY in Fig. 3) and antibody to MAP kinase ( Anti-MAPK in Fig. 3): Key: wt, vaccinia/control; Syk(T), vaccinia/Syk(T); (-), unstimulated; and (+), antigen ( Ag.)-stimulated cells.



Antigen-induced release of arachidonic acid (data not shown, but see later experiments) and secretion (data not shown) were also blocked in Syk(T)-transfected cells and were enhanced in Syk-transfected cells. These studies indicated, therefore, that Syk transduced necessary signals for the activation of MAP kinase and release of arachidonic acid as well as secretion.

Syk Also Regulates Tyrosine Phosphorylation of Vav via FcR1 and TT

We next examined the effects of Syk on the tyrosine phosphorylation of Vav because it is one of several proteins that are tyrosine-phosphorylated in antigen-stimulated RBL-2H3 cells (41) and it is known to regulate GTP/GDP exchange activity of Ras in B cells and T cells (42, 43, 44) . It may serve, therefore, as a potential link between Syk and the MAP kinase pathway via the interaction of Ras with Raf (28, 29, 30) . Both Syk (Fig. 6, line 1) and Vav (Fig. 6, line 2) showed a marked increase in tyrosine phosphorylation in antigen-stimulated cells. The tyrosine phosphorylation of Vav was a specific response in that it was observed only in cells stimulated via TT (Fig. 6, line 2) or via FcR1 (Fig. 6, line 3). Such phosphorylation was not observed in cells stimulated with carbachol or thapsigargin (Fig. 6, line 3). The latter two stimulants were known to activate the MAP kinase pathway in RBL-2H3 cells, but probably not through the activation of Syk (20) . Indeed, tyrosine phosphorylation of Syk itself was induced by antigen but not by carbachol (Fig. 6, line 1).


Figure 6: Vav is tyrosine-phosphorylated when cells are stimulated via FcR1 and Syk but not when stimulated via carbachol or thapsigargin. RBL-TT, RBL-TT, and RBL-2H3(m1) cells, as well as vaccinia-infected cells that expressed the porcine Syk or Syk(T) gene, were exposed to the indicated stimulants for 5 min. Immunoblots of immunoprecipitated Syk ( blot 1) and Vav ( blots 2-5) were probed with anti-phosphotyrosine antibody. Key: (-), no stimulant; Ag., 20 ng/ml DNP-BSA; CBC, 1 mM carbachol; Thaps., 150 nM thapsigargin; T/Av., cells treated with biotinylated anti-Tac and avidin as described Fig. 1; Syk(T), Syk, or (-), cells infected with vaccinia/Syk(T), vaccinia/Syk, or vaccinia/control virus, respectively, as described in Figs. 4 and 5.



Expression of Syk(T) blocked antigen-induced tyrosine phosphorylation of Vav (Fig. 6, line 4). Conversely, expression of Syk enhanced this phosphorylation (Fig. 6, line 5). The lack of response of Vav to carbachol was not affected by expression of Syk(T) or Syk (Fig. 6, lines 4 and 5). An incidental observation was that basal phosphorylation of Vav in unstimulated cells was reduced, but not blocked, in Syk(T)-transfected cells (Fig. 6, line 4). Collectively, the results indicated that Syk and Vav were selectively and specifically tyrosine-phosphorylated via cross-linking of the chain of FcR1 and that phosphorylation of Vav was downstream to that of Syk.

A Requirement for Syk for Activation of MAP Kinase and Release of Arachidonic Acid Is Apparent in Antigen-stimulated Cells but Not in Carbachol-stimulated Cells

The above studies established that activation of MAP kinase was dependent on Syk in antigen-stimulated cells and that Syk was not required for such activation in carbachol-stimulated cells. Because MAP kinases transduce necessary signals for release of arachidonic acid through activation of cytosolic phospholipase A(20) , we next examined the effect of expression of Syk and Syk(T) on this release.

As previously observed (20) , antigen and carbachol caused an appreciable increase in MAP kinase activity in intact cells (data not shown). Further studies, however, were undertaken with permeabilized cells to avoid the potential interactions between the phospholipase C/Ca/protein kinase C and MAP kinase/phospholipase A cascades, both of which appear to be activated via Syk in antigen-stimulated cells. For example, we knew that MAP kinases were activated by an increase in [Ca]alone and that an increase in [Ca]was necessary for release of arachidonic acid (20) . Therefore, primary and secondary ( i.e. via phospholipase C) effects of Syk(T) could not be readily distinguished in the intact cell.

The effects of expression of Syk and Syk(T) on release of arachidonic acid in permeabilized cells are shown in Fig. 7. As was observed with the activation of MAP kinase, expression of Syk enhanced antigen-induced release of arachidonic acid, and expression of Syk(T) blocked this release.() In contrast, carbachol-induced release was unaffected by these expressions to indicate that only FcR1 was dependent on Syk for activation of the MAP kinase/phospholipase A pathway.


Figure 7: Expression of porcine Syk or Syk(T) results in, respectively, enhancement or suppression of antigen-induced release of arachidonic acid without affecting carbachol-induced release of arachidonic acid. Cultures of RBL-2H3(m1) cells were incubated for 16 h with 5 plaque-forming units/RBL-2H3 cell of wild type Syk or Syk(T) recombinant vaccinia. The cultures were permeabilized with streptolysin O in the presence of 1 µM [Ca] and 100 ng/ml DNP-BSA ( Ag.) or 1 mM carbachol ( CBC). Release of arachidonic acid was determined 10 min thereafter.



A further indication of the differences between FcR1 and the muscarinic m1 receptor was that activation of MAP kinase, as indicated by increased activity (Fig. 8) and shift in migration of p42 (data not shown), by antigen was not dependent on [Ca], whereas the activation by carbachol was highly dependent on [Ca]. In these experiments, [Ca]was buffered at concentrations that mimicked those in unstimulated (75 nM free Ca) and stimulated (1000 nM Ca) intact cells. Although as noted previously (20) , elevation of [Ca]by itself induced a modest activation of MAP kinase activity, the data clearly demonstrated that transduction of signals via FcR1 occurred at basal [Ca].


Figure 8: Activation of MAP kinase activity by antigen, in contrast to activation by carbachol, is not dependent on Ca. Washed-permeabilized cells were stimulated with 100 ng/ml DNP-BSA ( Ag.) or 1 mM carbachol ( CBC) in the presence of 75 nM or 1 µM [Ca] for 5 min. Extracts of the cells were assayed for MAP kinase. Values indicate percent of activity in the presence of 1 µM [Ca] alone and are representative of two similar experiments.




DISCUSSION

As shown here, Syk not only provides a necessary link between FcR1 and phospholipase C 1 (25) , but also between FcR1 and the MAP kinase/cytosolic phospholipase A pathway. Thus, Syk appears to activate two divergent signaling pathways; one that leads to the generation of a Ca signal and release of secretory granules (25) and another that results in the activation of MAP kinase and phospholipase A with release of arachidonic acid (this study).

Previous studies have indicated that elevation of Ca and activation of protein kinase C are sufficient signals for secretion and that secretion is blocked by inhibition or removal of protein kinase C (22) . Phospholipase A-mediated release of arachidonic acid, in contrast, is not dependent on protein kinase C but rather on the activation of MAP kinase and elevation of [Ca](20) . Increased [Ca]is not required, however, for the activation of MAP kinase (this study) and phospholipase A(20, 45) when cells are stimulated via FcR1. Rather, the increase in [Ca]is necessary for the association of phospholipase A with cell membrane and the release of arachidonic acid from membrane substrates (20) (see schema in Fig. 9).


Figure 9: Pathways for mediating degranulation, via protein kinase C and Ca, and release of arachidonic acid, via MAP kinase/phospholipase A, in RBL-2H3(m1) cells. The schema is based on present and previous (20, 22) work. It depicts the simultaneous activation of the phospholipase C ( PLC)/protein kinase C ( PKC) as well as the MAP kinase ( MAPK)/phospholipase A ( PLA) pathways through the tyrosine kinase, Syk, when cells are stimulated via FcR1. Also indicated is the activation of the MAP kinase/phospholipase A pathway via the muscarinic m1 receptor by a Syk-independent, Ca-dependent mechanism. The stimulatory ligands, namely, antigen and carbachol, are thought to activate the same pathway: Ras, Raf1, MEK-1, MAP kinase, and phospholipase A as shown (20). The connection between Syk and Ras/Raf1 is undetermined, but the possible participation of Vav and other components is discussed in the text.



The effects of overexpression of Syk(T) and porcine Syk demonstrate that Syk is not only essential for communication between the -subunit of FcR1 and the MAP kinase pathway but also for FcR1-mediated tyrosine phosphorylation of Vav which may participate in this communication. Our data, therefore, do not exclude the possible activation of the MAP kinase cascade via phospholipase D. Diglycerides are generated through the activation of the phospholipases C and D in antigen-stimulated RBL-2H3 cells (46) and diglycerides are known to activate Vav (42) which, as previously noted, is thought to regulate Ras in B cells and T cells (43, 44) . Nevertheless, if these reactions occur in RBL-2H3 cells, they must depend on the prior activation of Syk.

If Syk is the initiator of two divergent signaling pathways, what can be concluded about events preceding the tyrosine phosphorylation and activation of Syk? The studies with the TT and TT chimeric receptors revealed that aggregation of the chain of FcR1 is sufficient, and almost as efficient as the aggregation of intact FcR1, for tyrosine phosphorylation of Syk (25 and this paper), activation of MAP kinase, and release of arachidonic acid. Expression of the porcine Syk facilitated coupling of FcR1 to the MAP kinase pathway, especially at low suboptimal concentrations of antigen, whereas expression of Syk(T), which lacks the kinase domain, abrogated this coupling. These findings collectively suggest that the SH2 domains of Syk(T) effectively compete with endogenous Syk for binding to ITAM domains on the FcR1 chains. Interestingly, Syk(T) is a substrate for tyrosine phosphorylation when cells are stimulated through FcR1. Presumably, this phosphorylation is catalyzed by Lyn which is known to be associated with FcR1 (23) , specifically with the subunit (25) , in RBL-2H3 cells.

The mechanisms described here for FcR1 might apply to other immunologically responsive cells. Activation of the MAP kinase pathway via FcR1 or via the multimeric antigen receptors in T cells and B cells has a common requirement for cytosolic tyrosine kinases and other signaling components. The pathway for RBL-2H3 cells appears to be a G protein (most probably Ras) Raf-1 MEK-1 p42(20) as appears to be the case in activated T cells (47, 48) and B cells (49) . The FcR1 -subunit is functionally analogous to the -subunit of the antigen T cell receptor in that the subunits consist of and homodimers, and each contain one or more ITAM domains which when phosphorylated permit association with Syk-related tyrosine kinases, i.e. Syk with and ZAP-70 with (4, 5) . It is unknown whether ZAP-70 is linked to the MAP kinase pathway in T cells, as Syk appears to be in RBL-2H3 cells.

It is apparent from the differences between antigen and carbachol that convergent pathways exist for the activation of the MAP kinase/phospholipase A in RBL-2H3 cells. For example, stimulation of the pathway by carbachol is not dependent on Syk but is dependent on Ca, whereas the converse is true for stimulation of the pathway by antigen (see Fig. 9). Our previous studies (20) have indicated that all stimulants, including antigen, carbachol, thapsigargin, and Ca ionophore, utilize the same pathway namely; a G protein (Ras?) Raf-1 MEK-1 MAP kinase cytosolic phospholipase A. If they do so, the point of convergence is likely to be at the level of Ras for which multiple activation mechanisms have been shown to exist (30) .

In conclusion, the data in this and previous papers indicate that for FcR1-mediated signals, Syk is the point of divergence for the phospholipase C/protein kinase C and the MAP kinase signaling pathways. Both pathways, however, have additional stimulatory inputs from other types of receptors. For the phospholipase C-dependent cascade of signals, convergent inputs are accommodated by the existence of different isoforms of phospholipase C to allow activation via trimeric G proteins, primarily G, and tyrosine kinases (50) . For the MAP kinase pathway, it is apparent that Syk-dependent and Syk-independent mechanisms exist for the activation of this pathway.


FOOTNOTES

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

§
To whom correspondence and reprint requests should be addressed: Bldg. 10/Rm. 8N114, National Insitutes of Health, Bethesda, MD 20892-1760. Tel.: 301-496-6188; Fax: 301-402-0171; E-mail: beaven@helix.nih.gov.

The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif as a proposed consensus for nomenclature of an 26-amino acid residue which was originally recognized by M. Reth (3) in receptors of immunological importance and since referred to as ARAM, TAM, or Reth motif (4, 5) among other designations (J. Cambier, personal communication); [Ca], concentration of free cytosolic calcium; IgE, immunoglobulin E; DNP, 2,4-dinitrophenol; DNP-BSA, antigen consisting of 24 molecules of dinitrophenol conjugated with 1 molecule of bovine serum albumin; PIPES, 1,4-piperazinediethanesulfonic; MAP kinase, mitogen-activated protein kinase; Syk(T), the gene for Syk that lacks the kinase domain; RBL-TT and RBL-TT, RBL-2H3 cells that have been stably transfected and thereby express, respectively, the Tac-Tac- and Tac-Tac- chimeric constructs as discussed in the text.

S. Chakrabarti and B. Moss, personal communication.

Release of hexosaminidase (a granule marker) is similarly enhanced by the expression of porcine syk and is blocked by expression of Syk(T).


ACKNOWLEDGEMENTS

We thank Drs. S. Chakrabarti and B. Moss, NIAID, for kindly supplying the pSC-65 vaccinia recombination plasmid.


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