The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (FcεRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after FcεRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing FcεRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, FcεRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-γ2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of FcεRI signaling.

The signal transduction pathways present in mast cells have many similarities to those in other cells of the immune system, such as T and B cells. The high-affinity IgE receptor (FcεRI)2 on mast cells, like the T and B cell receptors, is a multimeric protein complex consisting of ligand-binding domain(s) and signal-transducing subunits. These receptors lack intrinsic enzymatic activity, but they all contain the immunoreceptor tyrosine-based activation motif (ITAM), which is critical for cell activation (1, 2, 3, 4). Ag binding to these receptors results in phosphorylation of the tyrosines in the ITAM. Syk/ZAP-70 protein tyrosine kinases then associate with the tyrosine-phosphorylated ITAM, which results in tyrosine phosphorylation of Syk/ZAP-70 and an increase in their enzymatic activity (5, 6, 7, 8).

The importance of Syk/ZAP-70 activation in signaling through Ag and Fc receptors has been demonstrated by a number of experiments. The peripheral CD4+ T cells from ZAP-70-deficient patients are incapable of transducing the normal TCR-mediated intracellular signals (9). In Syk-deficient avian B cells, B cell receptor (BCR) engagement fails to induce intracellular calcium mobilization and does not result in the full spectrum of intracellular protein tyrosine phosphorylations (10). Similarly, in Syk-deficient mast cells, FcεRI aggregation induces no detectable increase in total cellular protein tyrosine phosphorylation and no degranulation (11, 12), defects that are reconstituted by the expression of wild-type Syk (11).

Because of the critical role of Syk/ZAP-70 in signaling, there is much interest in understanding the mechanism of the regulation of these enzymes. Most protein tyrosine kinases are substrates for phosphorylation on tyrosine residues. Phosphopeptide mapping has identified 6 tyrosine phosphorylation sites in ZAP-70 and 10 in Syk (13, 14). The tyrosine phosphorylation of Syk/ZAP-70 at some of these sites may be important in regulating the catalytic activity of these molecules and may also provide docking sites for binding of substrates and other molecules. There are two adjacent tyrosine residues in the catalytic domain of Syk (Y518, Y519) and ZAP-70 (Y492, Y493). These two tyrosines located in the “activation segment or loop” are probably important for regulating the function of kinases (15). In ZAP-70, the phosphorylation of the two tyrosines requires an Src family kinase, but they are autophosphorylated in Syk (13). In Syk-deficient B cells, the expression of Syk with both of these tyrosines mutated to phenylalanines abrogates the function of Syk in Ag signaling but not binding to ITAM (16). In 3T3E cells (NIH3T3 cells transfected with the α, β, and γ subunits of the FcεRI), Y518F- and Y519F-mutated Syk expressed by recombinant vaccinia viruses has basal activity nearly identical to that of wild-type Syk but reduced tyrosine phosphorylation and no increase in activity after FcεRI aggregation (17). Mutation of the two tyrosine residues separately in ZAP-70 has interesting effects. When the first tyrosine is mutated (Y492F), there is an increase in the intrinsic kinase activity in COS-7 cells (18) and also an increase in its capacity to reconstitute BCR-induced signaling in Syk-deficient B cells (19). When the second tyrosine is mutated (Y493F), there is no change in enzymatic activity (18), but it cannot be activated by Lck in COS-7 and Sf9 insect cells (18, 19) and fails to reconstitute BCR-mediated signaling in Syk-negative B cells (19).

Structural studies have suggested a mechanism for the regulation of protein tyrosine kinases by the activation loop tyrosines (20, 21, 22, 23). In the insulin receptor kinase there are three tyrosines (Y1158, Y1162 and Y1163) in the activation loop; Y1162 but not Y1163 lies within the catalytic center such that it prevents both ATP and substrate binding. In the activated form, phosphorylated Y1163 is the key residue that stabilizes the molecule in a different conformation and allows kinase activity. These studies suggest that activation could occur optimally only by transphosphorylation. The structure of the fibroblast growth factor receptor kinase domain suggests an alternative model in which Y653 in the activation loop interferes with substrate binding but not with ATP binding. Phosphorylation of this tyrosine is thought to induce a conformational change that would allow substrate access to the catalytic center.

The purpose of the present studies was to characterize the role of the activation loop tyrosines in the function of Syk in FcεRI signaling. Therefore the tyrosines were mutated either singly or in combination, and these mutant forms were expressed by stable transfection in a Syk-deficient variant of the RBL-2H3 (rat basophilic leukemia 2H3) cells. The use of these cells allowed the function of Syk to be examined in a background completely deficient of this kinase and without any modification such as tags. Our results suggested that phosphorylation of Y519 or Y520 (the double tyrosines in rat Syk that are equivalent to Y518, Y519 in chicken Syk) are not critical for the in vitro kinase activity of Syk but are essential for propagating the FcεRI signaling in vivo.

Triton X-100, Nonidet P-40, ATP, protein A agarose beads, and protease inhibitors were obtained from Sigma (St. Louis, MO). The materials for electrophoresis were purchased from Novex (San Diego, CA), and polyvinylidene difluoride transfer membrane was purchased from Millipore (Bedford, MA). The plasmid expressing the human cytoplasmic domain of erythrocyte band 3 protein (cdb3) was kindly provided by Dr. P. S. Low (Purdue University, West Lafayette, IN). The cdb3 protein was purified as described previously (24). The rabbit anti-rat Syk Abs have been described previously; the anti-SykI is to a sequence between the second Syk Src homology region 2 (SH2) and kinase domains, and the anti-SykC is to a peptide corresponding to the carboxyl-terminal amino acids (6). Mouse monoclonal anti-FcεRIα (mAb BC4) and the anti-trinitrophenol-specific IgE have been described previously (25, 26). The horseradish peroxidase-conjugated anti-phosphotyrosine Ab PY-20 was from ICN Immunobiologics (Lisle, IL). Phosphoplus p44/42 mitogen-activated protein kinase (MAPK) (Tyr204) Ab kit was from New England BioLabs (Beverly, MA). The sources of other materials not indicated were as described previously (6).

Y519 and/or Y520 of rat Syk were mutated to phenylalanine using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s directions. Following is the nomenclature used to refer to the different mutations in Syk: Y519F (FY), Y520F (YF), and FY plus YF (double mutation; FF). The identities of all mutations were verified by standard DNA sequencing. Mutant Syk cDNAs were cloned into the SacI site of the pSVL expression vector (Pharmacia LKB, Piscataway, NJ). The expression constructs were cotransfected with pSV2-neo vector into Syk-negative TB1A2 cells by electroporation, and stable transfected clones were selected with 400 μg/ml of active G418 (Life Technologies, Grand Island, NY) as described previously (11). The cell lines were screened for the expression of mutated Syk by immunoblotting with anti-Syk Abs, and four clones with each mutation were selected for further study.

TB1A2 cells and their transfectants were cultured as monolayers in Eagle’s MEM supplemented with 15% heat-inactivated FBS, penicillin, streptomycin, and amphotericin (27). For activation, cell monolayers after overnight culture were stimulated as described previously (11). When cells were cultured with Ag-specific IgE, activation was with the Ag DNP coupled to human serum albumin at concentrations from 0.01 to 1.0 μg/ml or the calcium ionophore A23187 at 0.25 to 2 μM. Cells were also stimulated with 0.3 μg/ml anti-FcεRIα Abs (mAb BC4) or 40 nM PMA. After the indicated times, the supernatants were removed for histamine analysis.

After stimulation for the indicated times, the cell monolayers were rinsed with ice-cold PBS containing 1 mM Na3VO4 and protease inhibitors (2 mM PMSF, 90 mU/ml aprotinin, 50 μg/ml leupeptin, and 50 μg/ml pepstatin) and solubilized in Brij lysis buffer (3% Brij-96, 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM Na3VO4, and 10 mM 2-ME plus protease inhibitors) or with modified RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, in addition to protease inhibitors and Na3VO4). The postnuclear supernatants were immunoprecipitated with Abs bound to protein A-agarose beads. After rotation at 4°C for 1 h, the beads were washed four times with ice-cold lysis buffer, and the proteins were eluted by boiling for 5 min with sample buffer as described previously (7). Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. The blots were probed with 40 ng/ml anti-phosphotyrosine mAb PY-20 conjugated to horseradish peroxidase and, after stripping, were reblotted with other Abs. In all blots, proteins were visualized by enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL).

Syk immunoprecipitated with rabbit anti-SykI Abs as described above was further washed with kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl2, and 2 mM MnCl2) and resuspended in 50 μl of kinase buffer. The reactions were started by the addition of 3 μCi of [γ-32P]ATP and 4 μM ATP with or without cdb3 as substrate. After the indicated incubation time at room temperature, the reactions were stopped by the addition of 50 μl of 2× Laemmli sample buffer and boiling for 10 min. The eluted proteins were separated under reducing conditions by SDS-PAGE (10% gels), electrotransferred to membranes, and visualized by autoradiography. The same membranes were immunoblotted with anti-Syk Abs as described above.

These studies were essentially as described previously (28). The phosphorylated synthetic peptide used was based on the γ subunit of FcεRI and immunoprecipitation was from lysates of the different transfected cell lines with anti-SykC in the presence or absence of this peptide. For the effect of the ITAM on autophosphorylation, Syk was immunoprecipitated with anti-SykI Abs, and the reaction was as described previously (28).

To analyze the potential role of the activation loop tyrosines in Syk on FcεRI signaling, Y519 and Y520 of Syk were singly or in combination changed to phenylalanines by site-directed mutagenesis. The mutated Syk cDNA was transfected into a Syk-negative variant of RBL-2H3 cells and clones were selected with G418 containing media. Expression of Syk was assessed by anti-Syk immunoblotting. Clones were selected with the level of expression of Syk comparable with or higher than that expressed in cells transfected with wild-type Syk (Fig. 1). In general, for every mutant at least two clones with Syk expressed at levels similar to wild-type and two with levels that were higher were used for further characterization.

FIGURE 1.

Immunoblot analysis of Syk expression in transfected cell lines. The Syk-negative TB1A2 cells were transfected with wild-type or the different mutant Syk and stable cloned lines selected with G418. Positive clones expressing the indicated constructs were analyzed by immunoblotting of total cell lysates with an affinity-purified polyclonal anti-SykI Ab. N indicates cells in which the expression level of mutant Syk was comparable with that in cells transfected with wild-type Syk, whereas O indicates overexpression. Shown are immunoblots of cells expressing Syk and Syk mutant FY (A), Syk YF (B), and Syk FF (C).

FIGURE 1.

Immunoblot analysis of Syk expression in transfected cell lines. The Syk-negative TB1A2 cells were transfected with wild-type or the different mutant Syk and stable cloned lines selected with G418. Positive clones expressing the indicated constructs were analyzed by immunoblotting of total cell lysates with an affinity-purified polyclonal anti-SykI Ab. N indicates cells in which the expression level of mutant Syk was comparable with that in cells transfected with wild-type Syk, whereas O indicates overexpression. Shown are immunoblots of cells expressing Syk and Syk mutant FY (A), Syk YF (B), and Syk FF (C).

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FcεRI aggregation results in tyrosine phosphorylation and increased kinase activity of Syk (6, 29). To test whether receptor stimulation still induced tyrosine phosphorylation of the different mutant Syk, cells were activated with anti-FcεRIα mAb. Repeated experiments showed that FcεRI aggregation induced tyrosine phosphorylation of the different mutant Syk (Fig. 2). There were, however, differences in the extent of this phosphorylation. The tyrosine phosphorylation of FY mutant was comparable with that of wild-type Syk, whereas the level of the tyrosine phosphorylation of the YF mutant was weaker than that of wild-type Syk, although the difference was not dramatic. Unlike the single mutants, the FF double mutant was tyrosine phosphorylated less than the wild-type Syk after receptor stimulation. This decreased phosphorylation of the FF mutated Syk was also apparent in time course experiments (data not shown). The level of Syk expression in cells had no effect on the extent of the receptor-induced tyrosine phosphorylations. Therefore, FcεRI aggregation results in tyrosine phosphorylation of Syk even when both of the activation loop tyrosines are mutated. This phosphorylation must be on sites other than the activation loop.

FIGURE 2.

Tyrosine phosphorylation of the different Syk proteins in response to FcεRI aggregation. Cells (3 × 106/lane) from indicated clones were either not stimulated (−) or stimulated (+) with the anti-FcεRIα mAb BC4 for 15 min and then solubilized with 3% Brij buffer. Syk was immunoprecipitated with polyclonal anti-Syk Ab conjugated to protein A beads. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine (anti-PY), stripped, and then reblotted with anti-Syk Abs (anti-Syk). Arrows indicate Syk.

FIGURE 2.

Tyrosine phosphorylation of the different Syk proteins in response to FcεRI aggregation. Cells (3 × 106/lane) from indicated clones were either not stimulated (−) or stimulated (+) with the anti-FcεRIα mAb BC4 for 15 min and then solubilized with 3% Brij buffer. Syk was immunoprecipitated with polyclonal anti-Syk Ab conjugated to protein A beads. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine (anti-PY), stripped, and then reblotted with anti-Syk Abs (anti-Syk). Arrows indicate Syk.

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The functional activity of the mutant Syk was tested in an immune-complex kinase assay using the cdb3 band protein as exogenous substrate. Surprisingly, cdb3 band was phosphorylated by all of these mutated Syk (Fig. 3), and none of the different Syk showed decreased enzymatic activity compared with wild-type Syk, even though the in vivo tyrosine phosphorylation of the double mutant (FF) was clearly weaker than that of wild-type Syk (see above). The extent of the autophosphorylation of the different mutants of Syk was also similar. Since no receptor-induced increase in Syk kinase activity was detected in these experiments, shorter incubation times were used for the in vitro kinase reaction. Under these conditions, there was no phosphorylation of the exogenous substrate by wild-type or the different Syk mutants, but all showed FcεRI aggregation-induced increase in autophosphorylation (Fig. 4). These results indicate that mutation of the activation loop tyrosines does not affect the in vitro kinase activity of Syk and that FcεRI aggregation still induces an increase in the autophosphorylation activity of Syk.

FIGURE 3.

In vitro enzymatic activity of the different Syk mutants. Syk was immunoprecipitated from lysates of nonstimulated (BC4−) and stimulated (BC4+) cell lines and incubated with [γ-32P]ATP and the substrate cdb3. The proteins were separated by SDS-PAGE in 10% gels and electrotransferred, and radiolabeled proteins were detected by autoradiography. The same membranes were then blotted with a polyclonal anti-Syk Ab. Here the incubation time was 30 min. The results are representative of three independent experiments. Cells were transfected with Syk FY (A), YF (B), and FF (C) mutants.

FIGURE 3.

In vitro enzymatic activity of the different Syk mutants. Syk was immunoprecipitated from lysates of nonstimulated (BC4−) and stimulated (BC4+) cell lines and incubated with [γ-32P]ATP and the substrate cdb3. The proteins were separated by SDS-PAGE in 10% gels and electrotransferred, and radiolabeled proteins were detected by autoradiography. The same membranes were then blotted with a polyclonal anti-Syk Ab. Here the incubation time was 30 min. The results are representative of three independent experiments. Cells were transfected with Syk FY (A), YF (B), and FF (C) mutants.

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FIGURE 4.

In vitro enzymatic activity of the different Syk mutants tested with a shorter in vitro incubation time (8 min). There was no phosphorylation of cdb3 under these conditions. The results are representative of two independent experiments. Arrows indicate Syk.

FIGURE 4.

In vitro enzymatic activity of the different Syk mutants tested with a shorter in vitro incubation time (8 min). There was no phosphorylation of cdb3 under these conditions. The results are representative of two independent experiments. Arrows indicate Syk.

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Receptor-induced tyrosine phosphorylation of cellular proteins is one of the earliest events after FcεRI aggregation (30). We therefore examined the FcεRI-induced tyrosine phosphorylation of total cellular proteins in the different transfected cell lines (Fig. 5). Transfection of wild-type Syk restored receptor-mediated cellular protein tyrosine phosphorylations in the Syk-negative TB1A2 cells. In the cells expressing mutant Syk at levels comparable with those of cells transfected with the wild type, receptor aggregation failed to induce an obvious increase in cellular protein tyrosine phosphorylations. FcεRI-induced protein tyrosine phosphorylation was partially reconstituted in cells that were overexpressing either the FY or the YF mutant forms of Syk. However, these phosphorylations were weaker in the cells with the YF compared with the FY mutant Syk. There were minimal receptor-mediated protein tyrosine phosphorylations in the cells expressing double-mutated (FF) Syk. Therefore, although the different Syk mutants have in vitro kinase activity, they are defective in propagating intracellular signals.

FIGURE 5.

FcεRI-induced tyrosine phosphorylation of cellular proteins. Cells (105) expressing the indicated Syk constructs were either not stimulated (−) or stimulated (+) with anti-FcεRIα (BC4) for 20 min. Total cellular lysates were analyzed by immunoblotting with anti-phosphotyrosine Ab PY-20.

FIGURE 5.

FcεRI-induced tyrosine phosphorylation of cellular proteins. Cells (105) expressing the indicated Syk constructs were either not stimulated (−) or stimulated (+) with anti-FcεRIα (BC4) for 20 min. Total cellular lysates were analyzed by immunoblotting with anti-phosphotyrosine Ab PY-20.

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In mast cells, FcεRI activation results in degranulation by a pathway in which Syk is essential (11, 12). Therefore, we investigated the effects of the mutation of these tyrosines on FcεRI-stimulated histamine release. Syk-negative cells and cells transfected with wild-type or mutated Syk were activated by either FcεRI aggregation or by the calcium ionophore A23187 (Fig. 6). The histamine release stimulated by the calcium ionophore A23187 was similar among all of these clones and was therefore used as an internal control. As we have observed previously, the transfection of wild-type Syk reconstituted histamine release. In cells with the mutated forms of Syk, there was no FcεRI-induced histamine release when the expression level was the same as in the cells with wild-type Syk. However, overexpression of either the FY or the YF mutant form of Syk partially reconstituted secretion. In cells expressing the double mutant form of Syk (FF), there was no FcεRI-induced secretion even in the lines in which there was overexpression of the protein. Therefore, Y519 and Y520 in Syk are essential for propagation of the intracellular signals that result in degranulation.

FIGURE 6.

Histamine release. Cells were cultured for 16 h with Ag-specific IgE and then washed and either not stimulated or stimulated for 45 min at 37°C with either the Ag (DNP-HSA) or the calcium ionophore A23187. Supernatants were assayed for histamine. The Ag-induced histamine release is presented as the percentage of that with calcium ionophore for the optimal release, which was with 0.2 μg/ml of Ag and 1 μM calcium ionophore. The results are the means from three independent experiments.

FIGURE 6.

Histamine release. Cells were cultured for 16 h with Ag-specific IgE and then washed and either not stimulated or stimulated for 45 min at 37°C with either the Ag (DNP-HSA) or the calcium ionophore A23187. Supernatants were assayed for histamine. The Ag-induced histamine release is presented as the percentage of that with calcium ionophore for the optimal release, which was with 0.2 μg/ml of Ag and 1 μM calcium ionophore. The results are the means from three independent experiments.

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Previous studies have shown that FcεRI-induced tyrosine phosphorylation of PLC-γ2 is dependent on Syk (11). In cells in which the expression of mutant forms of Syk were comparable with the level of wild-type Syk, there was no detectable tyrosine phosphorylation of PLC-γ2 (data not shown). Therefore, we examined whether overexpression of FY, YF, or the double mutant (FF) forms of Syk could support the FcεRI-mediated tyrosine phosphorylation of PLC-γ2. In cells overexpressing FY Syk, aggregation of FcεRI induced some tyrosine phosphorylation of PLC-γ2, although this increase was much weaker than that of wild-type Syk (Fig. 7). Overexpression of the YF Syk resulted in only a very slight increase in PLC-γ2 tyrosine phosphorylation, while the double mutant Syk failed to reconstitute the FcεRI-mediated PLC-γ2 tyrosine phosphorylation.

FIGURE 7.

FcεRI aggregation-induced tyrosine phosphorylation of PLC-γ2. The parental Syk-negative cells and cells expressing the indicated forms of Syk were either not stimulated (BC4−) or stimulated by mAb BC4 (0.3 μg/ml) (BC4+) for 10 min. Cell lysates were immunoprecipitated with anti-PLC-γ2 Ab. The immunoprecipitated proteins were analyzed by immunoblotting with anti-phosphotyrosine Ab (4G10) and anti-PLC-γ2 Ab. Arrows indicate PLC-γ2.

FIGURE 7.

FcεRI aggregation-induced tyrosine phosphorylation of PLC-γ2. The parental Syk-negative cells and cells expressing the indicated forms of Syk were either not stimulated (BC4−) or stimulated by mAb BC4 (0.3 μg/ml) (BC4+) for 10 min. Cell lysates were immunoprecipitated with anti-PLC-γ2 Ab. The immunoprecipitated proteins were analyzed by immunoblotting with anti-phosphotyrosine Ab (4G10) and anti-PLC-γ2 Ab. Arrows indicate PLC-γ2.

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The FcεRI-induced activation of MAPK is also downstream of Syk (31). Therefore, we investigated the role of the activation loop tyrosines on regulating activation of MAPK in these transfected cells (Fig. 8). FcεRI aggregation did not induce activation of MAPK in the Syk-negative cells. Transfection of wild-type Syk reconstituted FcεRI-induced tyrosine phosphorylation. In the cells expressing the different mutant Syk at levels comparable with those of wild-type, receptor stimulation did not result in phosphorylation of MAPK. The overexpression of Syk FY or of YF partially reconstituted this MAPK activation, which was greater with the FY than with the YF mutation. In contrast, FcεRI aggregation did not induce any tyrosine phosphorylation of MAPK in cells overexpressing the double mutant (FF) Syk. As an internal control, MAPK became phosphorylated to the same extent when all of the different cell lines were stimulated with PMA to directly activate protein kinase C.

FIGURE 8.

FcεRI-mediated tyrosine phosphorylation of MAPK. The parental Syk-negative cells and cells expressing the indicated forms of Syk were either not stimulated (BC4 −) or stimulated by mAb BC4 (0.3 μg/ml; BC4+) or by PMA for 4 min. Cell lysates were analyzed by immunoblotting with anti-phospho-p44/42 MAPK or anti-p44/42 MAPK Abs.

FIGURE 8.

FcεRI-mediated tyrosine phosphorylation of MAPK. The parental Syk-negative cells and cells expressing the indicated forms of Syk were either not stimulated (BC4 −) or stimulated by mAb BC4 (0.3 μg/ml; BC4+) or by PMA for 4 min. Cell lysates were analyzed by immunoblotting with anti-phospho-p44/42 MAPK or anti-p44/42 MAPK Abs.

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The interaction of Syk with tyrosine-phosphorylated ITAM peptides or the tyrosine phosphorylation of Syk results in a conformational change recognized by a polyclonal anti-peptide Ab (anti-SykC) raised to the carboxyl-terminal sequence of Syk (28). Anti-SykC also immunoprecipitates Syk that is tyrosine phosphorylated in vivo after cell activation (28). For example, addition of synthetic diphosphorylated ITAM peptide corresponding to the γ subunits of FcεRI results in a conformational change that allows the immunoprecipitation of nonphosphorylated Syk by anti-SykC (28). Therefore, anti-SykC was used to detect conformational changes. There was enhanced precipitation of Syk with the anti-SykC Abs when diphosphorylated γ ITAM peptide was added to the lysates of cells expressing the different mutant forms of Syk (Fig. 9). There was no detectable difference in the extent of this precipitation in mutants that had either one or both of the tyrosines mutated. The amount of Syk immunoprecipitated with anti-SykC increased when lysates were from stimulated cells, and this correlated with the extent of its in vivo tyrosine phosphorylation (data not shown). FcεRI was also coprecipitated with Syk in amounts that correlated with the extent of Syk tyrosine phosphorylation (data not shown). Therefore, the mutation of the tyrosines in the activation loop does not interfere in the conformational changes induced in Syk by either tyrosine phosphorylation or by interaction with diphosphorylated ITAM peptides.

FIGURE 9.

Changes in the conformation of Syk induced by binding of tyrosine-phosphorylated ITAM peptide. Lysates from nonactivated cells expressing the different mutated Syk were incubated with tyrosine-phosphorylated FcεRIγ ITAM peptide (at 1 μM) and then immunoprecipitated with anti-SykC. The immunoprecipitates were analyzed by immunoblotting with anti-SykI Abs.

FIGURE 9.

Changes in the conformation of Syk induced by binding of tyrosine-phosphorylated ITAM peptide. Lysates from nonactivated cells expressing the different mutated Syk were incubated with tyrosine-phosphorylated FcεRIγ ITAM peptide (at 1 μM) and then immunoprecipitated with anti-SykC. The immunoprecipitates were analyzed by immunoblotting with anti-SykI Abs.

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The addition of diphosphorylated ITAM peptides enhances the kinase activity of Syk (28, 32, 33). We therefore tested whether the binding of the tyrosine-diphosphorylated γ ITAM peptide induced any change in the kinase activity of the Syk mutants. Syk was immunoprecipitated with anti-SykI Abs and subjected to immune complex kinase assays with or without added ITAM peptide (Fig. 10). The slow autophosphorylation of the different Syk mutants was increased by the addition of the diphosphorylated ITAM peptides. This effect was less when both Y519 and Y520 were mutated. These results indicate that the conformational changes induced by the ITAM peptides still result in enhanced kinase activity when these tyrosines are mutated.

FIGURE 10.

Phosphorylated FcεRIγ ITAM peptide enhances the in vitro autophosphorylation of mutated Syk. Syk was immunoprecipitated with anti-SykI Abs from nonactivated cells and then used for immune complex kinase assay in the presence or absence of the γ tyrosine-phosphorylated ITAM peptide. The precipitates were analyzed by immunoblotting with anti-phosphotyrosine and anti-SykI Abs. The diphosphorylated ITAM peptide based on the γ subunit of FcεRI was at a final concentration of 1 μM.

FIGURE 10.

Phosphorylated FcεRIγ ITAM peptide enhances the in vitro autophosphorylation of mutated Syk. Syk was immunoprecipitated with anti-SykI Abs from nonactivated cells and then used for immune complex kinase assay in the presence or absence of the γ tyrosine-phosphorylated ITAM peptide. The precipitates were analyzed by immunoblotting with anti-phosphotyrosine and anti-SykI Abs. The diphosphorylated ITAM peptide based on the γ subunit of FcεRI was at a final concentration of 1 μM.

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Although Syk plays a critical role in FcεRI-induced signaling in mast cells, the mechanism of its regulation is not fully understood. One mechanism for regulation is by binding to tyrosine-phosphorylated ITAM peptides, which results in a conformational change and an increase in the kinase activity of Syk (28). Here we demonstrate another mechanism for regulating the in vivo function of Syk that requires the activation loop tyrosines. When Syk mutated in these tyrosines was transfected into Syk-negative TB1A2 cells, FcεRI-aggregation failed to induce cellular protein tyrosine phosphorylation and phosphorylation of PLC-γ2 or MAPK and did not result in histamine release. However, receptor cross-linking still induced increased tyrosine phosphorylation and increased enzymatic activity of the mutant Syk. In vitro binding of a phosphorylated peptide based on the ITAM of FcεRIγ induced increased kinase activity of the mutant Syk. Furthermore, the mutated Syk also showed similar conformational changes as wild-type Syk after cell stimulation. These results suggest that these activation loop tyrosines are not essential for the in vitro enzymatic activity of Syk, but they are necessary for in vivo propagation of FcεRI signaling.

The expression level of the mutant Syk was an important parameter for determining the functional impairment in signal transduction. There was no reconstitution of signal transduction when the FY or the YF mutants were expressed at the same level as the wild-type Syk. However, overexpression of these two mutant forms exhibited some of the wild-type responses in activation of signaling pathways and in degranulation. Therefore, overexpression of these mutant forms could mask defects induced by these mutations and/or induce alternative signaling pathways that are not active in the native state. In contrast, the overexpression of the doubly mutated form of Syk still did not activate any of the intracellular signaling pathways.

Mutation of either of the two tyrosines had effects on the in vivo tyrosine phosphorylation of Syk. There was minimal if any change when the first tyrosine was mutated but some decrease after mutation of the second tyrosine. However, when both tyrosines were mutated, there was a more dramatic decrease, although there was still in vivo tyrosine phosphorylation of Syk. However, all of the different mutants autophosphorylated to an equal extent in an in vitro kinase reaction, suggesting that the in vivo differences are not due to changes in autophosphorylating activity but to interactions of Syk with other molecules. Such interactions of Syk could be with other kinases that can potentially phosphorylate Syk and other downstream molecules. For example, several Src family kinases interact by their SH2 domain with tyrosine-phosphorylated Syk (7, 34, 35, 36, 37, 38), and the in vitro binding of Lck requires the phosphorylation of the two activation loop tyrosines (36). The activation loop tyrosines of Syk are autophosphorylated in vitro probably by transphosphorylation, which then can bind other molecules including kinases (such as Lyn) that are critical for downstream signal transduction (14).

The strong tyrosine phosphorylation of Syk, which has the two tyrosines in the activation loop mutated suggests that other tyrosines in the molecule are potential sites both for autophosphorylation (transphosphorylation) and for phosphorylation in vivo by other kinases. This is not surprising, as Syk has multiple tyrosines that are phosphorylated in vitro. The difference between in vitro and in vivo results could suggest that these alternative sites are not as good for transphosphorylation in vivo as they are in vitro.

The surprising observation was that the mutation of either or both of the activation loop tyrosines in Syk did not significantly reduce its in vitro kinase activity. Thus, by both autophosphorylation and by the ability to tyrosine phosphorylate a substrate in vitro, there was no change in the activity of Syk. However, there was a dramatic difference in the capacity of Syk to phosphorylate in vivo substrates, such as PLC-γ2, when these tyrosines were mutated. Therefore, the effect of these mutations of Syk on signaling could be due either to subtle changes in the catalytic activity of Syk or to effects on the interaction of Syk with substrates.

Structural studies indicate that activation loop tyrosines are important in regulating the function of protein kinases. In protein tyrosine kinases that are receptors, the binding of ligand results in a conformational change, which allows transphosphorylation of the activation loop tyrosines. Phosphorylation with the resulting change in the charge induces a conformational change in the activation loop and allows the binding of substrates. This results in further transphosphorylation of the molecule and the phosphorylation of substrates. Comparison of the crystal structure of insulin and of the fibroblast growth factor receptor suggests that there are different mechanisms by which the activation loop inhibits transphosphorylation of protein tyrosine kinases. The inhibitory effect in the insulin receptor is present because the activation loop inhibits binding of both ATP and of the substrate, whereas in the case of the fibroblast growth factor receptor there is inhibition of only the substrate. These differences would suggest that inhibition by the activation loop is stronger in insulin than in the fibroblast growth factor receptor. Furthermore, depending on the concentration of substrate, it is possible that there could be activation of the kinase in the absence of phosphorylation of the activation loop residues in the case of fibroblast growth factor. The capacity of Syk to phosphorylate substrate even when it is not tyrosine phosphorylated suggests that there is weak inhibition by the activation loop.

There are several studies of the effect of mutations in the activation loop tyrosines on the function of Syk and the related ZAP-70. Although they are structurally similar, there are differences between these two kinases in their signaling capacity (39). For example, activation of ZAP-70 but not of Syk requires an Src family kinase, and the enzymatic activity of Syk is at least 100-fold greater than that of ZAP-70 (40, 41, 42, 43). In vitro the activation loop tyrosines of ZAP-70 but not of Syk require Lck to become tyrosine phosphorylated (13, 14). With ZAP-70, mutation of the first activation loop tyrosine increases, whereas mutation of the second or both tyrosines has minimally or no effect on enzymatic activity. When such ZAP-70 mutants are transfected into a Syk-negative avian B cell line, the ZAP-70 mutated at the first activation loop tyrosine (FY) results in increased activation signals compared with the wild-type ZAP-70, while the kinase with mutation of the second (YF) or both tyrosines (FF) does not function (19, 44, 45). In transient transfection in COS cells, the FY mutation results in a fourfold increase in ZAP-70 basal kinase activity, while the YF mutant has the same basal kinase activity as wild-type ZAP-70. Incubation with pervanadate, a tyrosine phosphatase inhibitor, results in an increase in the tyrosine phosphorylation in cells transfected with wild-type and FY mutant, but no obvious change with the YF mutant (18). Also in transfected COS cells, a chimeric Syk with both of the activation loop tyrosines mutated does not induce tyrosine phosphorylation of endogenous PLC-γ1, and it is only minimally precipitated by the SH2 domain of PLC-γ1, even though the mutated Syk protein is tyrosine phosphorylated (46). In Jurkat T cells, overexpression of ZAP-70 with both of these mutated tyrosines inhibits TCR-induced activation of NFAT (a nuclear factor enhancing IL-2 gene transcription), intracellular calcium increase, and activation of extracellular signal-regulated kinases. However, this FF mutant is tyrosine phosphorylated; binds to the TCR ζ subunit; associates with Lck; and, by in vitro kinase reaction, has increased basal kinase activity compared with wild-type ZAP-70 (47). These results suggest that there are differences in the regulation of Syk compared with ZAP-70 by the two tyrosines in the activation loop. However, altogether the data strongly indicate that the activation loop tyrosines are critical in signaling by both of these kinases.

The loss of signaling by Syk mutated at these tyrosines could also imply that the activation loop tyrosines once phosphorylated are potential binding sites for both intramolecular and intermolecular interactions. The change in the charge of these tyrosines once phosphorylated could allow intramolecular interactions that stabilize a conformation that results in interactions with potential substrates. Alternatively, the phosphorylated tyrosines in the activation loop could be potential binding sites for interaction with other molecules. The structure of insulin crystallized in the active form suggests that the two activation loop tyrosines do not interact with other residues in the kinase and therefore could potentially be docking sites for downstream signaling proteins (22) such as insulin receptor substrate 2 (48, 49), Grb-2 (50, 51), or SHP-1 (52). For Syk there is evidence that the two tyrosines once phosphorylated are involved in interaction with Lck (36). Therefore, further work is required to define the interactions of Syk with other molecules that may be disrupted by mutation of the activation loop tyrosines that would explain the in vivo dramatic changes in downstream signaling.

We thank Hitoshi Okazaki and Elsa Berenstein for reviewing the manuscript and for helpful suggestions. We also thank Greta Bader for histamine analysis.

2

Abbreviations used in this paper: FcεRI, receptor with high affinity for IgE; ITAM, immunoreceptor tyrosine-based activation motif; BCR, B cell receptor; SH2, Src homology region 2; MAPK, mitogen-activated protein kinase; FY, Syk Y519F mutation; YF, Syk Y520F mutation; FF, Syk FY plus YF mutation; PLC, phospholipase C.

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