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The Journal of Immunology, 2000, 164: 338-344.
Copyright © 2000 by The American Association of Immunologists

Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function

Kiyonao Sada1, Juan Zhang and Reuben P. Siraganian

Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The protein tyrosine kinase Syk plays an essential role in Fc{epsilon}RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc{epsilon}RI-mediated tyrosine phosphorylation of phospholipase C-{gamma} and a dramatic enhancement of histamine release. The in vivo Fc{epsilon}RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc{epsilon}RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc{epsilon}RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aggregation of the high affinity IgE receptor (Fc{epsilon}RI) on mast cells or basophils results in a number of biochemical events leading to the release of histamine, arachidonic acid metabolites, and cytokines. Among these biochemical changes, protein tyrosine phosphorylation is the earliest event in the signal transduction pathway from the Fc{epsilon}RI, a receptor that itself has no intrinsic tyrosine kinase activity (1, 2, 3). In this pathway the sequential activation of the nonreceptor protein tyrosine kinases (PTK)2 Lyn and Syk is critical for cell activation (3). The tyrosine phosphorylation by these kinases then results in conformational changes in proteins that may regulate enzymatic activities and allow the interaction of molecules.

The Syk family of PTK is essential for signal transduction from cell surface immune receptors (4, 5, 6). For example, the expression of Syk is critical for the Fc{epsilon}RI-mediated tyrosine phosphorylation of phospholipase C-{gamma} (PLC-{gamma}), calcium mobilization, and histamine release in rat basophilic leukemia RBL-2H3 cells (6). The importance of Syk for Fc{epsilon}RI signaling is also demonstrated by analysis of mast cells from syk-/- embryos (7). Syk has tandem SH2 domains in the N-terminal half of the molecule (8) that are involved in its association with the {gamma} subunit of Fc{epsilon}RI after receptor aggregation (9, 10). Similarly, Ab-mediated aggregation of a chimeric transmembrane protein that has the intracellular domain of the {gamma} subunit of Fc{epsilon}RI results in tyrosine phosphorylation of Syk and its activation (11). In vitro the SH2 domains of Syk bind to the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), especially of the {gamma} subunit of Fc{epsilon}RI (10, 12). Syk also associates with this {gamma} subunit in collagen-stimulated platelets, a nonimmune receptor signaling pathway in platelets (13, 14). Binding of Syk to a diphosphorylated ITAM results in a conformational change and an increase in its kinase activity (15, 16), although Src family kinases can also phosphorylate and activate Syk (17). The in vivo tyrosine phosphorylation of Syk parallels the increase in its in vitro kinase activity. Therefore, in the proposed pathway for immune receptor signaling the binding of the SH2 domains of Syk to diphosphorylated ITAM is followed by autophosphorylation and Lyn-dependent phosphorylation, both of which may contribute to the activation of Syk (5, 18).

Syk is tyrosine phosphorylated and/or activated not only by immune receptors, but also by several other cell surface molecules, such as cytokine receptors, integrins, and G protein-coupled receptors (9, 19, 20, 21, 22, 23, 24, 25, 26). Tyrosine phosphorylation of Syk in its activation loop (Tyr519 and Tyr520 in rat Syk) is essential for downstream signaling events (27, 28, 29, 30). Additional tyrosine residues have been found in Syk and ZAP-70 that are phosphorylated in vivo or in vitro (31, 32, 33). These results suggest the existence of conserved tyrosine phosphorylation sites within the linker region of Syk family PTK. For example, the yeast two-hybrid system identified Tyr341 of porcine Syk as the direct interaction site for the SH2 domain of Vav (34).

Recently, Tyr316 in porcine Syk and the homologous Tyr292 in human ZAP-70 were reported to be the binding sites for Cbl, a negative regulator of receptor signaling (35, 36). The purpose of the present study was to examine the function of this tyrosine in the linker region (Tyr317 of rat Syk) in signal transduction in mast cells. The stable expression, in a Syk-negative cell line, of Syk with this residue mutated resulted in dramatic enhancement of the Fc{epsilon}RI-induced total tyrosine phosphorylation of cellular proteins and histamine release.


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

Triton X-100 and protein A-agarose beads were obtained from Sigma (St. Louis, MO). Streptavidin beads were purchased from Pierce (Rockford, IL). Polyvinylidene difluoride transfer membrane was purchased from Millipore (Bedford, MA), and the enhanced chemiluminescence reagent (Renaissance) was obtained from NEN Life Science Products (Boston, MA). The plasmid for the expression of the human cytoplasmic domain of erythrocyte band 3 protein (cdb3) was provided by Dr. P. S. Law (Purdue University, West Lafayette, IN). The preparation of cdb3 was previously described (30, 37). Biotinylated diphosphorylated peptide based on the ITAM of Fc{epsilon}RI{gamma} (biotin-{gamma}PP) was previously described (38). The HRP-conjugated mouse anti-phosphotyrosine 4G10 mAb was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-Cbl mAb was purchased from Transduction Laboratories (Lexington, KY). Rabbit anti-Syk Abs, anti-Fc{epsilon}RIß mAb, anti-PLC-{gamma}1 mAb, rabbit anti-PLC-{gamma}2 Ab, and phosphoplus p44/42 mitogen-activated protein kinase (MAPK, Tyr204) Ab kit were previously described (6, 16, 30).

Cell culture

Rat basophilic leukemia RBL-2H3 cells and the Syk-negative variant of RBL-2H3 have been described previously (6). In this study the B2 subclone derived from the Syk-negative TB1A2 cells was used for stable transfection studies (39). After ionophore stimulation the B2 cells release a similar amount of histamine as the wild-type RBL-2H3 cells. RBL-2H3, B2, and various cDNA transfected cells were maintained as monolayer cultures in MEM with 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 1% antibiotic-antimycotic (100 U/ml penicillin G, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B), and 15% heat-inactivated FBS (Biofluids, Rockville, MD). The stably transfected clones were maintained with 0.4 mg/ml of active G418 (Life Technologies).

Construction of cDNAs and transfection

The rat wild-type Syk expression vector pSVL-Syk has been described previously (6). Point mutation of Tyr317 of rat Syk cDNA to Phe (Y317F) and of Lys396 to Arg (dead kinase: DK) were prepared by using either a PCR-based method or the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and were confirmed by DNA sequencing. The mutant cDNAs (Syk Y317F and Syk DK) were then subcloned into the pSVL vector (Pharmacia LKB, Piscataway, NJ). For stable transfection, 20 µg of linearized expression constructs and 2 µg of pSV2-neo vector were cotransfected into 5 x 106 Syk-negative B2 cells by electroporation (960 µF, 310 V) as described previously (6). The stably transfected clones were selected with 0.4 mg/ml of active G418 (Life Technologies). Cell lines were screened for the level of Syk protein expression by immunoblotting total cell lysates with rabbit anti-SykI Ab using blotting with anti-Fc{epsilon}RIß mAb as an internal control. Two clones transfected with each kind of cDNA that expressed Syk at a level comparable to that in the wild-type RBL-2H3 cells were selected for further analyses.

Cell activation and preparation of cell lysates

For histamine release analysis 105 cells were seeded in 24-well plates and cultured overnight with or without Ag-specific IgE. The cell monolayers were washed twice with 1 ml of MEM containing 0.1% BSA and 10 mM Tris, pH 7.4, before stimulation. The cells were stimulated with the Ag dinitrophenyl coupled to human serum albumin (DNP-HSA) or with calcium ionophore A23187 in the same medium. After incubation for 45 min at 37°C, the medium was removed for histamine analysis (1, 40). For immunoprecipitation studies cells were seeded in petri dishes and after overnight culture were stimulated with Ag as described above. At the indicated times the cell monolayers were rinsed twice with ice-cold PBS containing 1 mM Na3VO4 and 0.1 mM PMSF and solubilized in 1% Triton lysis buffer (1% Triton X-100, 50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 90 µ/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml of pepstatin A). In some experiments cells were solubilized under denaturing conditions in 1% SDS lysis buffer (1% SDS, 25 mM Tris (pH 7.4), 50 mM NaCl, 1 mM Na3VO4, and protease inhibitors). These lysates were then diluted 1/10 with immunoprecipitation buffer (25 mM Tris (pH 7.4), 50 mM NaCl, 1 mM Na3VO4, 1% Triton X-100, 0.5% Nonidet P-40, and protease inhibitors). For the preparation of total cell lysates, monolayers were rinsed twice with PBS as described above and directly lysed by the addition of 2x SDS-PAGE sample buffer.

Immunoprecipitation and immunoblotting

For immunoprecipitation, postnuclear supernatants were first precleared by mixing with protein A-agarose and then immunoprecipitated with Abs prebound to protein A-agarose. Rabbit anti-mouse IgG Ab was used to couple mouse mAb with protein A-agarose. After gentle rotation at 4°C for 1 h, the beads were washed four times, then precipitated proteins were eluted by boiling for 5 min with 2x SDS-PAGE sample buffer. Total cell lysates and immunoprecipitated proteins were separated by SDS-PAGE (10% acrylamide), then electrotransferred to polyvinylidene difluoride membranes. The membranes were incubated with 4% BSA in 10 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween-20 for 1 h. The blots were probed with individual primary Abs, then incubated with HRP-conjugated donkey anti-mouse or rabbit Ab. Proteins were visualized by the enhanced chemiluminescence reagent (Renaissance).

In vitro kinase assay

For the in vitro kinase assay of the various Syk, cell lysates were immunoprecipitated with anti-Syk Ab. Washed immunoprecipitates were incubated in kinase buffer (30 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM MnCl2, 4 µM ATP, and [{gamma}-32P]ATP) for 30 min at room temperature with cdb3 as an exogenous substrate (6, 30). The phosphorylated proteins were analyzed by autoradiography.

Other procedures

Precipitation with ITAM peptides was performed as described previously (16). Lysates from 5 x 106 cells were precipitated with biotinylated diphosphorylated synthetic peptide based on Fc{epsilon}RI{gamma} (biotin-{gamma}PP) prebound to streptavidin beads. All experiments were performed at least three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of stable cell lines expressing mutant forms of Syk

In previous experiments we found that the expression of wild-type Syk protein tyrosine kinase reconstituted cellular functions in Syk-negative RBL-2H3 cells (6, 39). To investigate the functional importance of Tyr317, the cDNAs encoding rat Syk wild type, Y317F, and dead kinase (DK) were transfected into the Syk-negative B2 cells, which do not express Syk. Cloned lines were then screened by immunoblotting using anti-Syk and anti-Fc{epsilon}RIß Abs. For further analysis at least two cloned lines transfected with each cDNA were selected in which the level of Syk expression was similar to that in the wild-type RBL-2H3 cells (Fig. 1GoA). In all the following experiments each of these lines was examined, although some figures present the results from only one representative cell line.



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  		FIGURE 1. The Y317F mutation of Syk enhances mast cell activation. A, Generation of stable cell lines expressing mutants of Syk. The Syk-negative variant of the RBL-2H3 cells was transfected with various rat Syk cDNAs and selected with 0.4 mg/ml of G418. Two positive cloned lines expressing each of the different forms of Syk were selected for further analysis. Total cell lysates from the RBL-2H3, Syk-negative B2 (Syk-), and cells expressing the wild-type (WT), Y317F, and DK Syk were immunoblotted with anti-Syk and anti-Fc{epsilon}RIß Abs, respectively. Molecular size markers are indicated at the left in kilodaltons. B, Analysis of Fc{epsilon}RI-mediated histamine release. Syk-negative cells and cells expressing the different mutant forms of Syk were cultured overnight with Ag-specific IgE and then stimulated with the indicated concentrations of the Ag DNP-HSA or with the calcium ionophore A23187. The Ag-induced release is normalized by expression as a percentage of that induced with 1 µM A23187. The results are the mean ± SE from three independent experiments. The A23187-induced average release as a percentage of the total cellular histamine content in the different cell lines was 72% (Syk- B2), 68% (DK-I4), 84% (DK-I1), 67% (WT 5), 67% (WT 1), 78% (Y317F-F17), and 72% (Y317F-F14). C, Fc{epsilon}RI-induced tyrosine phosphorylation of cellular proteins. Syk-negative (Syk-), Syk wild-type (WT)-expressing, Syk Y317F-expressing, and Syk DK-expressing cell lines were primed with IgE and stimulated with the 0.1 µg/ml of the Ag DNP-HSA for the indicated times. Total cell lysates (105 cell equivalents/lane) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb. Similar results were obtained when the other cells transfected with WT, Y317F, and DK Syk were examined in three independent experiments.

 
Tyr317 in Syk negatively regulated Fc{epsilon}RI-mediated histamine release and tyrosine phosphorylation of cellular proteins

To examine the importance of Tyr317 in the function of Syk, we first analyzed Fc{epsilon}RI-mediated histamine release (Fig. 1GoB). The Syk-negative cells and cells transfected with wild-type or mutated Syk were stimulated either by Fc{epsilon}RI aggregation or with the calcium ionophore A23187. The histamine release induced by the calcium ionophore was similar among all these clones and was therefore used as an internal control. There was no Fc{epsilon}RI-mediated release with the DK-expressing cells (clones I1 and I4) or Syk-negative cells (B2). However, the release in cells expressing Syk Y317F (clones F14 and F17) was dramatically enhanced compared with that in cells with the wild-type Syk (clones 1 and 5). Similar enhancement of Fc{epsilon}RI-induced release was seen in all five cloned lines that expressed Y317F Syk, although in one of these lines Y317F was expressed at lower levels than in the RBL-2H3 cells or in cells with wild-type Syk (data not shown). These results indicate that in vivo Tyr317 functions to negatively regulate Fc{epsilon}RI-mediated histamine release.

Receptor-induced tyrosine phosphorylation is a very early event after Fc{epsilon}RI aggregation. Therefore, we tested the tyrosine phosphorylation of total cellular proteins induced by Fc{epsilon}RI aggregation in the different transfected cell lines. Cells were stimulated at optimum concentration of Ag, and total cell lysates were analyzed by anti-phosphotyrosine immunoblotting. As we had observed previously (6), expression of wild-type Syk reconstituted Fc{epsilon}RI-mediated tyrosine phosphorylation of cellular proteins (Fig. 1GoC, lanes 1–5 and 16–20). Moreover, the expression of Syk Y317F resulted in enhanced tyrosine phosphorylation of cellular proteins (Fig. 1GoC, lanes 1–10). In contrast, tyrosine phosphorylation of cellular proteins in Syk DK-expressing cells was similar to that in the nontransfected Syk-negative cells (Fig. 1GoC, lanes 11–20). The results in Fig. 1GoC are from one cell line expressing each of the different Syk mutants; similar results were obtained with the other cloned lines. These results indicated that mutation of Syk Tyr317 resulted in enhancement of receptor-mediated tyrosine phosphorylation of cellular proteins and histamine release.

Negative role of Syk Tyr317 in tyrosine phosphorylation of PLC-{gamma}

The increase in intracellular calcium is critical for receptor-mediated histamine release. Reconstitution studies of B cells and Syk-negative mast cells demonstrate that expression of Syk is necessary for receptor-triggered tyrosine phosphorylation of PLC-{gamma}, generation of inositol 1,4,5-trisphosphate, and calcium mobilization (5, 6). The Ab-mediated clustering of chimeric transmembrane proteins with Syk as the intracellular domain triggers PLC-{gamma} phosphorylation and calcium mobilization (41). We therefore tested the effects of the different mutations of Syk on the tyrosine phosphorylation of PLC-{gamma}. The Fc{epsilon}RI-induced tyrosine phosphorylation of PLC-{gamma}2 was dependent upon Syk kinase activity and was enhanced by the Y317F mutation (Fig. 2GoA). Similarly, the Fc{epsilon}RI-mediated tyrosine phosphorylation of PLC-{gamma}1 was enhanced in cells expressing Y317F Syk (Fig. 2GoB). These data suggest that Tyr317 in Syk functions as a negative regulator of the tyrosine phosphorylation of PLC-{gamma} and therefore in the signals that result in the increase in intracellular calcium.



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  		FIGURE 2. The Y317F mutation of Syk enhances Fc{epsilon}RI-induced tyrosine phosphorylation of PLC-{gamma}. Cells expressing the wild-type (WT), Y317F, and DK forms of Syk were stimulated with Ag for the indicated times, and cell lysates were then immunoprecipitated with anti-PLC-{gamma}2 (A) or PLC-{gamma}1 (B) Abs. The immunoprecipitated proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb. The membrane was then stripped and reprobed with the respective anti-PLC-{gamma} Abs.

 
Effect of Y317F mutation on Syk function

Fc{epsilon}RI aggregation results in tyrosine phosphorylation of Syk. Therefore, we examined the effect of mutation of Tyr317 on Fc{epsilon}RI-mediated phosphorylation of Syk. In unstimulated cells, the different mutant forms of Syk were not tyrosine phosphorylated (Fig. 3Go, lanes 1, 4, and 7). Fc{epsilon}RI aggregation induced similar tyrosine phosphorylation of wild-type and Y317F Syk (Fig. 3Go, lanes 1–6). In contrast, there was minimal tyrosine phosphorylation of the DK Syk after receptor aggregation, presumably due to phosphorylation by Lyn (Fig. 3Go, lanes 7–9). Therefore, although there was enhanced Fc{epsilon}RI-induced histamine release and tyrosine phosphorylation of cellular proteins in cells expressing Y317F Syk, this was not accompanied by an increase in the phosphorylation of Syk itself.



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  		FIGURE 3. Fc{epsilon}RI-induced tyrosine phosphorylation of Y317F Syk is unchanged. The cell lines expressing wild-type (WT), Y317F, and DK forms of Syk were stimulated with Ag for the indicated times, and cell lysates were then immunoprecipitated with anti-Syk Ab. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine (upper panel) and anti-Syk Abs (lower panel).

 
The binding of Syk to phosphorylated ITAM peptides or the tyrosine phosphorylation of Syk results in a conformational change that is recognized by anti-Syk Ab that reacts with carboxyl-terminal amino acids of Syk (16). This anti-Syk Ab precipitated similar amounts of wild-type and Y317F Syk from stimulated cells (data not shown). Therefore, Y317F Syk undergoes this same conformational change as wild-type Syk after cell activation. Because Ag-induced phosphorylation of the ß and {gamma} subunits of Fc{epsilon}RI were similar in the various cell lines expressing the mutant forms of Syk (data not shown), we tested the binding of Syk to diphosphorylated ITAM based on Fc{epsilon}RI{gamma} (Fig. 4Go). There was similar precipitation of wild-type and Y317F Syk with different amounts of biotin-{gamma}PP, suggesting that mutation of Tyr317 did not affect the interaction of the SH2 domains with diphosphorylated {gamma}-ITAM.



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  		FIGURE 4. The Y317F mutation does not affect the binding of Syk to diphosphorylated peptide based on the ITAM of Fc{epsilon}RI{gamma}. Streptavidin beads were preincubated with the indicated amounts of biotinylated diphosphorylated {gamma}-ITAM peptide and then added to lysates from 5 x 106 unstimulated cells expressing wild-type (WT) or Y317F Syk. The precipitates were analyzed by immunoblotting with anti-Syk Ab.

 
The functional activity of the mutant Syk was tested in an in vitro kinase assay using cdb3 band protein as an exogenous substrate. Even though the Y317F mutant form was not tyrosine phosphorylated in unstimulated cells (see Fig. 3Go), it had increased kinase activity compared with that of wild-type Syk (Fig. 5Go, lane 1 vs lane 4). Fc{epsilon}RI aggregation also resulted in the activation of Syk. The tyrosine phosphorylations of wild-type and Y317F Syk were similar after Fc{epsilon}RI aggregation (see Fig. 3Go), but the Tyr317 mutant had increased kinase activity (Fig. 5Go). This was apparent by the increased level of both autophosphorylation and phosphorylation of the cdb3 substrate. As would be expected, Syk DK had no kinase activity (Fig. 5Go, lanes 7 and 8), which strongly indicates that Syk and cdb3 are tyrosine phosphorylated not by other coprecipitated kinases, but by Syk itself. These results suggest that mutation of Tyr317 in Syk results in enhanced catalytic activity.



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  		FIGURE 5. The Y317F mutation increases the in vitro kinase activity of Syk. Cells expressing the wild-type (WT), Y317F, and DK forms of Syk were stimulated with Ag for the indicated times, and lysates were then immunoprecipitated with anti-Syk Ab. The immunoprecipitates were incubated for 30 min at room temperature in an in vitro kinase reaction buffer containing [{gamma}-32P]ATP and 2.5 µg of the substrate cdb3. Radiolabeled proteins were detected by autoradiography (upper panel), and the precipitates were also immunoblotted with the anti-Syk Ab (lower panel). The numbers at the bottom of the figure are the normalized densitometric analysis of Syk and cdb3.

 
Role of Tyr317 in Syk in tyrosine phosphorylation of Cbl

Recent studies indicate that Cbl interacts with Syk and can down-regulate the function of Syk (42, 43, 44). Studies with the yeast two-hybrid system suggest that Tyr317 is a candidate binding site for the truncated transforming form of Cbl (36). In transfection studies in COS-1 cells, there is decreased coprecipitation of this two molecules and also decreased tyrosine phosphorylation of Cbl when Syk is mutated at this tyrosine. Therefore, we examined the tyrosine phosphorylation of Cbl in the cells expressing the different mutated forms of Syk. The Fc{epsilon}RI-induced tyrosine phosphorylation of Cbl was downstream of Syk kinase activity (Fig. 6Go, lanes 1–3 vs lanes 7–9). Interestingly, the tyrosine phosphorylation of Cbl was not affected by the mutation of Tyr317 in Syk (Fig. 6Go, lanes 1–6). Therefore, the tyrosine phosphorylation of Cbl did not correlate with the enhancement of histamine release, and Cbl was not one of the proteins whose Fc{epsilon}RI-mediated tyrosine phosphorylation was enhanced by the expression of Syk Y317F (see Fig. 1GoC). The results also suggest that mutation of Tyr317 in Syk selectively affected the increase in tyrosine phosphorylation of cellular proteins that are downstream of Syk activation.



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  		FIGURE 6. The Y317F mutation of Syk does not affect Fc{epsilon}RI-induced tyrosine phosphorylation of Cbl. Cells expressing wild-type (WT), Y317F, and DK forms of Syk were stimulated with Ag for the indicated times, and lysates were then immunoprecipitated with anti-Cbl mAb. The immunoprecipitated proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with an anti-phosphotyrosine mAb. The membrane was stripped and then reprobed with anti-Cbl mAb.

 
Effect on MAPK-activating pathway

Fc{epsilon}RI-mediated tyrosine phosphorylation of Vav and activation of MAP kinase are downstream of Syk (7, 45). We previously observed that mutations of the activation loop tyrosines in Syk abrogate tyrosine phosphorylation of MAPK (30). We therefore examined whether MAPK activation was affected by the mutation of Tyr317 in Syk. Immunoblotting of cell lysates using anti-phospho-MAPK Ab showed that tyrosine phosphorylation of MAPK was not affected by the Y317F mutation, although it was downstream of Syk kinase activity (Fig. 7Go). As an internal control, MAPK in these cells was equally tyrosine phosphorylated when they were stimulated with PMA to directly activate protein kinase C (Fig. 7Go, lanes 3, 6, and 9). In addition, tyrosine phosphorylation of Vav and the ß and {gamma} subunits of Fc{epsilon}RI was not enhanced by the Y317F mutation (data not shown). The results indicated that Y317F mutation in Syk selectively enhanced the Fc{epsilon}RI-induced tyrosine phosphorylation of only some signaling molecules.



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  		FIGURE 7. The Y317F mutation does not increase Fc{epsilon}RI-induced tyrosine phosphorylation of MAPK. Cell lines expressing wild-type (WT), Y317F and the DK forms of Syk were either nonstimulated (-) or stimulated for 4 min with Ag (DNP) or PMA. Total cell lysates from 1.5 x 105 cells were analyzed by immunoblotting with anti-phospho-p44/42 MAPK or anti-p44/42 MAPK Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrate that mutation of Tyr317 in Syk resulted in an enhancement of Syk kinase activity, an increase in Fc{epsilon}RI-induced tyrosine phosphorylation of phospholipase C-{gamma}, and an enhancement of histamine release. Mutation of Tyr292 in human ZAP-70, which corresponds to Tyr317 in rat Syk, results in increased production of inositol trisphosphate, early gene transcription, and a hyperactive phenotype in T and B cell receptor signaling (46, 47). Similarly, deletion of the linker region of ZAP-70 results in a gain of function, suggesting that this is a negative regulatory site (47). Because mutation of Tyr292 did not affect the intrinsic kinase activity of ZAP-70, it was proposed that there is an inhibitory protein that binds to this site after the tyrosine is phosphorylated (46, 47). The PTB domain in Cbl binds the D(N/D)XpY motif, a sequence that is at Tyr292 in ZAP-70 and is phosphorylated after T cell activation (35, 48). Furthermore, the recent evidence from Cbl-negative mice supports the concept that Cbl acts as a negative regulator of ZAP-70 in T cells (49). These results with ZAP-70 suggest that the corresponding Tyr317 in rat Syk, which is likewise located within a NXpY motif, may be a similar negative regulatory Cbl binding site. In the yeast two-hybrid system, this tyrosine was found to be critical for the interaction of Syk with v-Cbl, the truncated oncogenic form of Cbl (36). Therefore, these results suggest that Tyr317 in Syk, by recruiting Cbl, may function as one mechanism to negatively regulate histamine release.

Direct regulation of the kinase activity of Syk by Tyr317 may be another mechanism for the enhancement of signal transduction and receptor-induced secretion. The Y317F mutated Syk from unstimulated cells had increased intrinsic kinase activity (Fig. 5Go). Similarly, although the in vivo Fc{epsilon}RI-induced tyrosine phosphorylations of wild-type and Y317F mutated Syk were similar (Fig. 3Go), the in vitro kinase activity of Y317F from stimulated cells was greater than that of the wild-type Syk (Fig. 5Go). This suggests that there may be conformational changes resulting from the Y317F mutation that results in an increase in intrinsic kinase activity. We previously reported that both tyrosine phosphorylation of Syk and binding of Syk with diphosphorylated ITAM peptides induce a conformational change that is recognized by Abs that react with the carboxyl-terminal amino acids of Syk (16). However, these same Abs did not detect a difference between wild-type and Y317F Syk, suggesting that there may be other structural changes due to the Y317F mutation. Interestingly, mutation of Tyr292 in human ZAP-70 (equivalent to Tyr317 in rat Syk) did not result in a change in intrinsic kinase activity (46, 47). The linker region of Syk is longer than that of ZAP-70 and can regulate the capacity of Syk to bind to ITAM and to function in signaling from immune receptors such as Fc{epsilon}RI (39). For example, an alternatively spliced form of Syk that is 23 aa shorter in the linker region is inefficient in coupling Fc{epsilon}RI aggregation to protein tyrosine phosphorylations and degranulation. Collectively, our data suggest that Syk could have a unique self-inhibitory regulatory site(s) within the linker region.

The results also indicate that the Y317F mutation affected only some of the events that are downstream of Syk. Among these, the Fc{epsilon}RI-induced tyrosine phosphorylation of PLC-{gamma} and the subsequent histamine release were enhanced by the Y317F mutation (Figs. 1GoB and 2). In contrast, tyrosine phosphorylation of Cbl, Vav, and MAPK was not affected (Figs. 6Go and 7Go and data not shown). Therefore, besides the enhanced enzymatic activity of Syk, other mechanisms must control the cellular events that result in the augmented release of histamine. Recently, several substrates of nonreceptor PTK have been identified that may function as possible adapter molecules. Among these are Vav, SLP-76, BLNK, LAT, DAP-12, TRIM, and Cbl, which are present either in many hemopoietic cells or in only a limited lineage of cells (34, 50, 51, 52, 53, 54, 55). Phosphorylation of Tyr315 in human ZAP-70 or of Tyr341 in porcine Syk is critical for the interaction of these two PTK with Vav and for downstream tyrosine phosphorylation of molecules such as SLP-76 (34, 54). Therefore, it is possible that there could be adapter proteins that interact with Tyr317 of Syk to regulate downstream signal transduction. It is also possible that proteins directly downstream of Syk are phosphorylated to a greater extent than molecules that are more distal in the signaling cascade.

The Syk family PTK has two members, Syk and ZAP-70 (8, 56). Although structurally similar, there are functional differences between these two kinases. The earliest results were with Ab-mediated clustering of chimeric transmembrane proteins that had a PTK as the intracellular domain. In such experiments Syk alone is able to initiate the intracellular signals for the increase in intracellular calcium, whereas ZAP-70 requires the presence of an Src family kinase (41). Similar results were observed when PTK were expressed in nonhemopoietic cells (55, 57). The swapping of domains between Syk and ZAP-70 suggests that these differences are due to the greater catalytic activity of the kinase domain of Syk (57, 58). Moreover, analysis of the crystal structure demonstrates that the conformational flexibility and structural independence of the SH2 domains of Syk are different from those of ZAP-70 (59, 60). Mutation of Tyr292 in ZAP-70 enhanced T or B cell receptor signaling without affecting the kinase activity of ZAP-70 (46, 47). In contrast to these results with ZAP-70, we observed that in Syk the mutation of Tyr317 increased kinase activity. This indicates that phosphorylation of homologous tyrosine residues in Syk and ZAP-70 can have distinct functions. It further implies that mutation of this tyrosine in the linker region of Syk vs ZAP-70 could have different effects on Cbl.

In B cell receptor signaling, Cbl is constitutively associated with and phosphorylated by Lyn, but not by Syk (61, 62). In contrast, our results indicate that in Fc{epsilon}RI-mediated signaling, the tyrosine phosphorylation of Cbl was downstream of Syk kinase activity. The overexpression of Cbl in RBL-2H3 cells inhibits Syk tyrosine phosphorylation, activation, and secretion (44). In the present experiments, the Fc{epsilon}RI-induced tyrosine phosphorylation of Cbl was similar in cells expressing the Y317F and wild-type Syk. This suggests that the extent of the tyrosine phosphorylation of Cbl does not regulate histamine release. The tyrosine phosphorylation of Cbl in the Syk Y317F-expressing cells could be due to Cbl interacting with Syk at sites besides Tyr317 (36, 43, 63). The Y317F Syk could still be coimmunoprecipitated with Cbl from COS-1 cells (data not shown) (63). However, we did not detect Cbl-Syk complexes by coprecipitation experiments in the stable transfected mast cell lines, suggesting that such interactions may be transient and involve a small fraction of the molecules. Therefore, in mast cells the tyrosine phosphorylation of Cbl is downstream of Syk, but is not regulated by Tyr317.

In summary, the results presented in this report demonstrate that a mutation of a tyrosine in the linker region of Syk results in an increase in catalytic activity and an enhancement of Fc{epsilon}RI-induced histamine release. As Syk is critical in propagating the Fc{epsilon}RI-induced signals that result in calcium influx and degranulation, it is not surprising that there are multiple mechanisms to regulate its function. Therefore, the activity of Syk can be down-regulated not only by dephosphorylation, as seen in nonstimulated cells, but also by the phosphorylation of Tyr317. Because this tyrosine is one of several sites that are autophosphorylated in vitro, further investigation will be necessary to fully understand the mechanism of the regulation of Syk.


   Acknowledgments
 
We thank Elsa Berenstein and Hitoshi Okazaki for helpful discussion and criticism of the manuscript. We also thank Greta Bader for histamine analysis, and Diana Rector for secretarial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Kiyonao Sada, Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, Building 10/1N106, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-1188. E-mail address: Back

2 Abbreviations used in this paper: PTK, protein tyrosine kinases; PLC-{gamma}, phospholipase C-{gamma}; SH2, Src homology 2 region; ITAM, immunoreceptor tyrosine-based activation motif; MAPK, mitogen-activated protein kinase; DK, dead kinase; HSA, human serum albumin. Back

Received for publication August 13, 1999. Accepted for publication October 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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