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The Journal of Immunology, 1999, 163: 2508-2516.
Copyright © 1999 by The American Association of Immunologists

CD45 Is Essential for Fc{epsilon}RI Signaling by ZAP70, But Not Syk, in Syk-Negative Mast Cells

Juan Zhang1 and Reuben P. Siraganian

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore Fc{epsilon}RI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to Fc{epsilon}RI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated Fc{epsilon}RI{gamma}-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stimulation of mast cells by the aggregation of the high affinity IgE receptor (Fc{epsilon}RI) initiates a biochemical cascade, including increased protein tyrosine phosphorylations, a rise in intracellular calcium, and activation of protein kinase C that ultimately results in degranulation. The activation of protein tyrosine kinases plays a critical role in this signal transduction pathway (1, 2). As the subunits of Fc{epsilon}RI do not have any intrinsic kinase activity, cytoplasmic protein tyrosine kinases must be involved in initiating these receptor-mediated events. The accumulated evidence suggests that Lyn and Syk are responsible for the initiation of these Fc{epsilon}RI-mediated signals.

Syk is a member of the ZAP70/Syk family of protein tyrosine kinases (3, 4). Syk is present in most hematopoietic cells, including B cells, mast cells, immature T cells, and platelets, while ZAP70 is expressed only in T cells and NK cells. Structurally, the ZAP70/Syk family of kinases has two tandem Src homology 2 (SH2)2 domains in the N-terminal half and a catalytic domain in the COOH-terminal half. The Ag and Fc receptors in hematopoietic cells all contain the immunoreceptor tyrosine-based activation motif (ITAM). Stimulation of these receptors results in tyrosine phosphorylation of the ITAM by a Src family protein tyrosine kinase. ZAP70/Syk protein tyrosine kinases then associate by their SH2 domains with the tyrosine-phosphorylated ITAM. This results in tyrosine phosphorylation of ZAP70/Syk, conformational changes, and an increase in enzymatic activity (5, 6, 7, 8, 9).

The Syk/ZAP70 family of protein tyrosine kinases is essential for signal transduction from cell surface immune receptors (6, 10, 11, 12). ZAP70-deficient T cells are incapable of receptor-mediated signal transduction, while Syk-deficient B cells exhibit a marked decrease in Ag receptor-induced mobilization of intracellular calcium. Similarly, Fc{epsilon}RI aggregation in Syk-deficient mast cells does not induce changes in intracellular calcium or degranulation.

The structural similarity of Syk and ZAP70 suggests that there may also be functional overlap between these two kinases. In Syk-deficient mice, thymocyte maturation is not grossly impaired, suggesting that ZAP70 coexpressed in these cells compensates for the absence of Syk (13, 14). Furthermore, in a Syk-negative B cell line, expression of either ZAP70 or Syk reconstitutes B cell receptor signaling (15). However, other experiments suggest that there are significant differences in activation requirements between these two kinases. Phosphorylated ITAM induces the activation of Syk, while ZAP70 requires additional stimulatory input from Lck (16, 17, 18, 19). In Jurkat T cell lines, Syk, but not ZAP70, can transduce TCR signaling independently of CD45 and of Lck (20). Also, in a Syk- and ZAP70-negative Jurkat T cell line, the expression of Syk, but not ZAP70, results in a significant degree of cellular activation in the absence of TCR stimulation (21).

CD45 is a transmembrane protein tyrosine phosphatase expressed on most hematopoietic cells (22, 23). Accumulated data suggest that this enzyme dephosphorylates the negative regulatory tyrosine of Src family kinases, which then permits these kinases to become activated (24, 25, 26). In CD45-deficient T cells, TCR stimulation fails to couple to the phosphatidylinositol pathway (27, 28). CD45 also plays an important role in the proliferative response of murine T cell clones to specific Ag (29). In CD45 knockout mice, most thymocytes are arrested in their development, and the T cells that do develop are unable to response to TCR stimulation (30).

CD45 expression is detected on basophils and mast cells. Several experiments suggest that CD45 is important for signal transduction leading to degranulation. First, incubation of human basophils with monoclonal anti-CD45 Ab blocks IgE-mediated histamine release (31). Second, bone marrow-derived mast cells from mice in which the CD45 is genetically inactivated fail to secrete after Fc{epsilon}RI aggregation (32). Third, in CD45-deficient Jurkat cells transfected with Fc{epsilon}RI, there is aggregation-induced tyrosine phosphorylation of the receptor subunits without any calcium influx, a defect that is reconstituted by the expression of CD45 (33). However, there are other experiments that suggest that CD45 may not be important for signal transduction in mast cells. The Fc{epsilon}RI-induced histamine release of CD45-deficient and positive RBL-2H3 cell variants are similar (Refs. 34, 35 , and our unpublished observations). Therefore, the function of CD45 in mast cells is not clear.

In the present study, a variant of the RBL-2H3 cell line that was deficient in Syk and CD45 was characterized and transfected with ZAP70, Syk, or CD45. Stable cell lines were isolated that expressed one or more of these different molecules. It was found that CD45 was essential for reconstitution of Fc{epsilon}RI-mediated signaling by ZAP70. In contrast, Syk was able to function in a CD45-independent manner, and coexpression of CD45 with Syk did not result in significant changes in intracellular responses to receptor stimulation. The expression of CD45 itself in the double-deficient cell did not reconstitute Fc{epsilon}RI-mediated signaling. Furthermore, we also investigated the possible mechanisms of the different functional effects of ZAP70 and Syk in mast cell signaling.


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

Triton X-100 and protein A-agarose beads were obtained from Sigma (St. Louis, MO). The materials for electrophoresis were from Novex (San Diego, CA). The plasmid that expresses the human cytoplasmic domain of erythrocyte band 3 protein (cdb3) was kindly provided by Dr. P. S. Law (Purdue University, West Lafayette, IN). The cdb3 protein was purified as described previously (36, 37). The sources of other materials not indicated were as described previously (7).

Antibodies

The mouse anti-ZAP70 Ab was from Transduction Laboratories (Lexington, KY). The rabbit anti-ZAP70 and anti-mouse CD45 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat CD45 mAb OX-1 was from Serotec (Serotec, U.K.). Phosphoplus p44/42 microtubule-associated protein kinase (MAPK) Ab kit was from New England BioLabs (Beverly, MA). The anti-phospholipase C-{gamma} (PLC) 1 Ab was from Upstate Biotechnology (Lake Placid, NY). The HRP-conjugated anti-phosphotyrosine Ab PY-20 was from ICN Immunobiologics (Lisle, IL). Anti-v-src residues 2 to 17 Ab, LA074, was from Quality Biotech (Camden, NJ). Abs to Fc{epsilon}RIß, Fc{epsilon}RI{gamma}, Lyn, and Syk were as described previously (38, 39).

Construction of cDNAs and transfections

A 1.9-kb fragment containing the open reading frame for human ZAP70 was excised from a pBluescript vector encoding the full-length cDNA of human ZAP70 (kindly provided by Dr. A. Weiss, University of California, San Francisco, CA), and ligated into the pSVL expression vector (Pharmacia LKB, Piscataway, NJ). The Syk expression construct was as described previously (12). For stable transfection, 20 µg of linearized ZAP70 or Syk expression constructs and 2 µg pSV2-neo vector were cotransfected into 5 x 106 Syk-negative B2 cells by electroporation (310 V, 960 µF), as described previously (12). The stable transfected clones were selected with 300 µg/ml of active G418 (Life Technologies, Gaithersburg, MD). The expression of ZAP70 or Syk was confirmed by immunoblotting using anti-ZAP70 or anti-Syk Abs.

For the expression of CD45, a chimeric membrane-targeted molecule was used that has the first 15 amino acids of Src joined to the enzymatically active portion of murine CD45 intracellular domain (kindly provided by Dr. J. D. Ashwell, National Cancer Institute, National Institutes of Health, Bethesda, MD) (40). The DNA encoding this fragment was subcloned into the pSVL expression vector and with the pSV2-hph vector was coelectroporated into the Syk-negative B2 cells, stable ZAP70, or Syk-transfected cloned lines. Stable transfected clones were selected with 1–1.5 mg/ml hygromycin B. CD45-expressed clones were identified by immunoprecipitation with an anti-v-Src (residues 2 to 17) Ab followed by immunoblotting with anti-mouse CD45 Ab.

Cell culture and activation

Rat basophilic leukemia RBL-2H3 cells and the Syk-negative variant of RBL-2H3 have been described previously (12). In this study, the B2 subclone derived from the Syk-negative TB1A2 cells was used for stable transfection studies (41). The stable transfected clones were maintained with 300 µg/ml of active G418 (Life Technologies).

For cell activation, the monolayers were cultured overnight either with or without Ag-specific IgE. The cells cultured with IgE were stimulated with the Ag DNP coupled to human serum albumin at concentrations from 0.01 to 1.0 µg/ml. Cells were also stimulated with calcium ionophore A23187 at 0.25–2 µM or with 70 nM of PMA. After stimulation for the indicated times, the supernatants were removed for histamine analysis.

Immunoprecipitation and immunoblotting

After stimulation, the cell monolayers were rinsed with ice-cold PBS containing 2 mM Na3VO4 and protease inhibitors (2 mM PMSF, 90 mU/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml pepstatin) and solubilized in Triton lysis buffer (1% Triton, 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, plus protease inhibitors and Na3VO4), or with Brij lysis buffer (3% Brij-96, 20 mM Tris (pH 7.5), 100 mM NaCl, 2 mM Na3VO4, 10 mM 2-ME plus protease inhibitors) or with modified RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, plus 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 eluted by boiling for 5 min with SDS-PAGE sample buffer, as described previously (8). Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The blots were probed with anti-phosphotyrosine Ab or other Abs. In all blots, proteins were visualized by enhanced chemiluminescence (NEN Life Science, Boston, MA).

Precipitation with ITAM peptides

Lysates from different cell lines were precipitated with immobilized, nonphosphorylated, or phosphorylated Fc{epsilon}RIß- or {gamma}-ITAM peptides, as described previously (42).

Immunocytometry

Single cell suspensions were prepared from different cell lines by detaching the cell monolayers with EDTA and washing with cold PBS. The cells were incubated with the following mAb: anti-Fc{epsilon}RI (mAb BC4) or anti-CD45 (mAb OX-1). The mAb were visualized with FITC-conjugated rat anti-mouse IgG and analyzed using a FACScan (Becton Dickinson, Mountain View, CA).

In vitro kinase assay

Syk or ZAP-70, immunoprecipitated 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 kinase reactions were for 10 min at room temperature with 3 µCi of [{gamma}-32P]ATP and 4 µM ATP, with or without cdb3 as substrate. The reactions were stopped by the addition of 50 µl of 2x 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-ZAP70 or anti-Syk Abs, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD45 is essential for ZAP70 to reconstitute Fc{epsilon}RI-induced degranulation in Syk-deficient cells

Syk is essential for Fc{epsilon}RI-mediated degranulation (12, 43). To investigate whether ZAP70 could reconstitute this function of Syk in mast cells, a Syk-deficient variant of RBL-2H3 cells was transfected with wild-type ZAP70. Eight cloned lines were selected based on their expression of ZAP70 at levels approximating to or higher than that found in Jurkat cells (Fig. 1GoA). Histamine release induced by Fc{epsilon}RI or calcium ionophore stimulation was tested in these lines (Fig. 2Go). Incubation with calcium ionophore resulted in >70% of the total cellular histamine release. However, Fc{epsilon}RI aggregation failed to induce degranulation in six of the eight clones (release not different from the parental Syk-negative B2 line). The other two lines had some minimal release. The release in these two ZAP70-transfected cell lines was still much less than when the cells were reconstituted with Syk. Therefore, unlike Syk, transfection of ZAP70 did not reconstitute significant Fc{epsilon}RI-mediated degranulation.



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  		FIGURE 1. Generation of stable mast cell lines expressing ZAP70. The cloned Syk-deficient variant line (B2) was transfected with cDNA encoding human ZAP70 and selected with G418. Cell lysates of cloned lines were screened by immunoblotting with anti-ZAP70 Ab, and eight positive clones were selected for further study. Lysates from these cells were immunoblotted with anti-ZAP70 Ab (A) and with anti-Fc{epsilon}RIß or anti-Fc{epsilon}RI{gamma} Abs (B). For comparison, lysates from an equivalent number of Jurkat cells were blotted for expression of ZAP70.

 


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  		FIGURE 2. Fc{epsilon}RI-induced histamine release in different ZAP70-expressing lines. Cells were cultured overnight with Ag-specific IgE, washed, and either stimulated or not with Ag or with calcium ionophore A23187 for 45 min at 37°C. Supernatants were assayed for histamine. The maximum Ag-induced histamine release (which was at 0.2 µg/ml) is presented as the percentage of that with calcium ionophore (1 µM) and is the mean from three independent experiments. The results with two cloned lines obtained after Syk transfection are shown for comparison.

 
To investigate why the expression of ZAP70 failed to reconstitute the Ag-stimulated degranulation, we examined several of the molecules that are important in the early steps of the Fc{epsilon}RI signaling pathway. By immunoblotting and/or FACS analysis, the expression level of Fc{epsilon}RI was similar among all of these cloned lines (Fig. 1GoB, and data not shown). By in vitro kinase assay, the activity of Lyn was also similar in all of these transfected cell lines (data not shown). However, there were differences in the expression of protein tyrosine phosphatase CD45 among these clones. By FACS analysis, the parental B2 Syk-negative cells had minimal expression of CD45 (Fig. 3Go). The six clones that failed to release histamine after receptor stimulation had minimal or no expression of CD45, whereas the other two cloned lines that did release some histamine were CD45-positive. In contrast, two lines obtained by the transfection with wild-type Syk (Syk-1 and Syk-5), which released histamine after IgE-Ag stimulation, were also negative for CD45 by FACS analysis (data not shown). These results suggested that the difference in reconstitution by Syk, compared with ZAP70, could be due to CD45.



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  		FIGURE 3. FACS analysis of the cell surface expression of CD45 on the various cell lines. Negative control cells incubated with only FITC anti-mouse IgG are shown by broken lines.

 
CD45 is a transmembrane tyrosine phosphatase that is normally present on many hematopoietic cells. To investigate the role of CD45 in ZAP70’s function, the CD45-deficient, ZAP70 stable transfected line (clone 1) was retransfected with a membrane-targeted form of CD45. Stable transfected clones were screened for the expression of CD45 and several CD45-positive lines (Fig. 4Go, and data not shown) were tested for histamine release. Surprisingly, in contrast to the parental line that failed to degranulate with Ag stimulation, all these doubly transfected clones released histamine after Fc{epsilon}RI aggregation (Fig. 5Go, and data not shown). These results suggest that ZAP70 can restore Fc{epsilon}RI-initiated degranulation in mast cells only when CD45 is present.



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  		FIGURE 4. Expression of mouse CD45 by transfection in the different cell lines. The ZAP70-transfected cloned line (clone 1 in the previous figures) and Syk-transfected cloned line (clone 5) were retransfected with a chimeric cDNA encoding the intracellular enzymatically active portion of mouse CD45 preceded by the membrane-targeting amino-terminal sequence from p60c-src. The stable transfected clones were selected by hygromycin B, and mouse CD45-expressing cells were identified by immunoprecipitation with an anti-v-src Ab and immunoblotting with an anti-mouse CD45 Ab. As a control, the parental Syk-negative B2 cells were also transfected with CD45 (marked as CD45).

 


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  		FIGURE 5. Reconstitution of Fc{epsilon}RI-mediated histamine release by transfection with ZAP70 or Syk. Cells were cultured overnight with Ag-specific IgE, washed, and either stimulated or not with Ag or with the calcium ionophore A23187 at 37°C for 45 min. Supernatants were assayed for histamine. The Ag-induced histamine release (0.01 µg/ml) is presented as the percentage of that with calcium ionophore (1 µM). The results are the mean from three experiments.

 
ZAP70 and Syk have different requirements for CD45 in Fc{epsilon}RI signaling pathway

ZAP70 and Syk belong to the same tyrosine kinase family. The results with ZAP70 suggested a role for CD45 in signal transduction in mast cells. However, in mast cells, Syk is the major, if not only, protein tyrosine kinase of this family. Therefore, Syk-negative cells that had been reconstituted by transfection with wild-type Syk were retransfected with CD45. As a control, a CD45-transfected, Syk-deficient cell line was also included. The Syk/CD45 double-negative cells that, after transfection, had stable expression of the following proteins were used for analysis: CD45, ZAP70, Syk, CD45 plus ZAP70, or CD45 plus Syk (Fig. 4Go, and Fig. 6GoA). The Fc{epsilon}RI-induced histamine release was determined in the different cell lines (Fig. 5Go). There was no receptor-induced release in the Syk-negative cells transfected with CD45. In the cells reconstituted with Syk, CD45 cotransfection did not result in any significant increase in Fc{epsilon}RI-mediated degranulation. Therefore, there appeared to be a clear difference between ZAP70 and Syk in CD45 requirement for histamine release.



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  		FIGURE 6. Analysis of ZAP70 or Syk functional status in the different cell lines. A, Expression of ZAP70 or Syk. Lysates from the indicated cell lines were analyzed by immunoblotting with anti-ZAP70 or anti-Syk Ab. The cell lines used were the following: the parental Syk-negative line (B2), Syk-negative cells transfected with CD45, ZAP70, or Syk, with or without CD45 as indicated. For comparison, lysates from an equivalent number of Jurkat cells were blotted for expression of ZAP70. B, Fc{epsilon}RI-induced tyrosine phosphorylation of ZAP70 or Syk. Cells were cultured overnight with Ag-specific IgE and either nonstimulated or stimulated with 0.1 µg/ml Ag (Ag, - or +) for 20 min. Lysates from 4 x 106 cells were immunoprecipitated with either rabbit anti-ZAP70 or anti-Syk Abs. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine (4G10) and mouse anti-ZAP70 or anti-Syk Abs. C, In vitro kinase assay of ZAP70 or Syk immunoprecipitated from the different cell lines. ZAP70 or Syk was immunoprecipitated from the different cell lines before and after cell stimulation, as in B, and then incubated with [{gamma}-32P]ATP and the substrate cdb3 in a protein kinase assay. The proteins were separated by SDS-PAGE, electrotransferred, and visualized by autoradiography. The same membranes were then blotted with anti-ZAP70 or anti-Syk Abs.

 
Ag receptor stimulation results in tyrosine phosphorylation and increased kinase activity of ZAP70/Syk kinases. Therefore, we examined the functional status of the ZAP70/Syk expressed in these cloned lines. Fc{epsilon}RI aggregation induced the tyrosine phosphorylation of ZAP70 and Syk (Fig. 6GoB). This tyrosine phosphorylation of Syk or ZAP70 was slightly increased in the cells that coexpressed CD45. Both ZAP70 and Syk precipitated from nonstimulated cells phosphorylated cdb3 in the in vitro kinase reaction (Fig. 6GoC). However, the presence of CD45 resulted in differences in the receptor-induced change in kinase activity. Syk had increased kinase activity when immunoprecipitated from Fc{epsilon}RI-stimulated cells, irrespective of the presence or absence of CD45 in the cells. In contrast, the receptor aggregation-induced increase in kinase activity of ZAP70 was only observed with the CD45-positive cells.

One of the earliest events following Fc{epsilon}RI stimulation is tyrosine phosphorylation of cellular proteins. The pattern of protein tyrosine phosphorylation was similar in lysates of the different nonstimulated cells, suggesting that the expression of ZAP70/Syk with or without CD45 did not result in any unregulated kinase activity (Fig. 7Go). In the Syk-deficient parental B2 cells, as reported previously, receptor stimulation induced only a slight increase in cellular protein tyrosine phosphorylation. Syk transfection of these B2 cells reconstituted Fc{epsilon}RI-aggregation-induced cellular protein tyrosine phosphorylation. Cotransfection of CD45 with Syk slightly increased this response. Surprisingly, in the cells expressing ZAP70, there was only a minimal increase in receptor-induced cellular protein tyrosine phosphorylation. Furthermore, coexpression of CD45 with ZAP70 had no dramatic effect on this function. Therefore, although the coexpression of CD45 and ZAP70 reconstituted secretion in these cells, it only resulted in minimal, if any, increase in total cellular tyrosine phosphorylations.



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  		FIGURE 7. Fc{epsilon}RI-induced tyrosine phosphorylation of cellular proteins. Cells were cultured overnight with Ag-specific IgE and were then either not stimulated (Ag -) or stimulated (Ag +) with 0.01 µg/ml Ag for 20 min. Lysates prepared from 1.5 x 105 cell equivalents per lane were analyzed by immunoblotting with anti-phosphotyrosine Ab. The cell lines are the following: Syk-negative parental cells (B2), Syk-negative cells transfected with only CD45 (CD45), ZAP70 (clones 1 and 3), Syk (clones 1 and 5) with or without CD45, as indicated.

 
Fc{epsilon}RI stimulation induces the activation of PLC, which results in the formation of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. These secondary messengers are responsible for releasing Ca2+ and activating protein kinase C. The receptor-initiated tyrosine phosphorylation of PLC-{gamma} is downstream of Syk (12). As has been observed previously, Fc{epsilon}RI stimulation did not induce an increase in tyrosine phosphorylation of PLC-{gamma}1 in the parental Syk-negative cells (Fig. 8Go). This phosphorylation was restored by the expression of Syk but not ZAP70. The coexpression of CD45 with ZAP70 reconstituted the receptor-aggregation-induced tyrosine phosphorylation of PLC-{gamma}1, though this was still weaker than that in the Syk-reconstituted cells. In contrast, CD45 coexpression had minimal, if any, effect on tyrosine phosphorylation of PLC-{gamma}1 in the Syk-transfected cells.



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  		FIGURE 8. Fc{epsilon}RI-induced tyrosine phosphorylation of PLC-{gamma}1. Cells expressing the indicated proteins were stimulated with 0.1 µg/ml Ag for 10 min, and cell lysates from 6 x 106 cells were then immunoprecipitated with anti-PLC-{gamma}1 Ab. The immunoprecipitated proteins were analyzed by immunoblotting with anti-phosphotyrosine and anti-PLC-{gamma}1 Abs.

 
Aggregation of Fc{epsilon}RI results in the activation of MAPK, which is downstream of Syk. Therefore, we examined whether ZAP70 could substitute for Syk in this pathway. The different transfected cell lines were stimulated with Ag, and total cell lysates were analyzed with an anti-phospho-MAPK Ab (Fig. 9Go). As previously observed, receptor stimulation induced minimal phosphorylation of MAPK in the Syk-negative cells. This defect was corrected by transfection with Syk. In ZAP70-expressing cells, Fc{epsilon}RI-induced tyrosine phosphorylation of MAPK was similar to that in Syk-deficient parental cells. Only when CD45 was coexpressed did ZAP70 restore the receptor-induced tyrosine phosphorylation of MAPK. The expression of CD45 also slightly increased MAPK tyrosine phosphorylation in the Syk-reconstituted cells. Therefore, ZAP70, unlike Syk, requires CD45 for the activation of downstream molecules, such as PLC-{gamma} and MAPK.



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  		FIGURE 9. Fc{epsilon}RI-induced tyrosine phosphorylation of MAPK. The indicated cell lines were either not stimulated (Ag -) or stimulated for 4 min with 0.1 µg/ml Ag (Ag +). 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.

 
Expression of CD45 did not restore Fc{epsilon}RI signal pathway in the Syk-deficient cells

To better understand the function of CD45 in Fc{epsilon}RI signaling, a cell line was used that expressed CD45 in the absence of the ZAP70/Syk kinase. Receptor aggregation did not induce detectable degranulation in these cells, even though these cells released ~70% of total histamine with calcium ionophore incubation (Fig. 5Go, and data not shown). CD45 reconstitution also failed to restore Fc{epsilon}RI-induced total cellular protein tyrosine phosphorylation (Fig. 7Go) and tyrosine phosphorylation of PLC-{gamma}1 and MAPK in the Syk-deficient cells (Figs. 8Go and 9Go). Therefore, CD45, in the absence of Syk or ZAP70, could not reconstitute these late steps in mast cell signaling.

As previous experiments suggest that CD45 plays a role in the early steps of Ag receptor signal pathway, we then examined the status of the tyrosine phosphorylation of Fc{epsilon}RI in the different transfected cell lines (Fig. 10GoA). Fc{epsilon}RI aggregation did induce tyrosine phosphorylation of both the ß- and {gamma}-subunits of the receptor in all cell lines, including the parental double-deficient cells. Surprisingly, the extent of this phosphorylation was weaker in the CD45-positive than in the CD45-negative lines. To confirm this difference in receptor tyrosine phosphorylation, we examined the parental Syk-negative and the CD45-transfected cells (that were lacking in ZAP70/Syk). In time course experiments, at every time point, the tyrosine phosphorylation of the ß- and {gamma}-subunits was weaker in the CD45-transfected cells than that in CD45-negative cells (Fig. 10GoB). Although tyrosine phosphorylation of the receptor subunits was weaker in the CD45-transfected cells, there still was signal transduction that led to degranulation. In the cells expressing ZAP70, there was degranulation only when there was coexpression of CD45, conditions under which there was a decrease in tyrosine phosphorylation of the receptor subunits.



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  		FIGURE 10. Ag-induced tyrosine phosphorylation of the ß- and {gamma}- subunits of Fc{epsilon}RI. A, Cells were either nonstimulated or stimulated with 0.1 µg/ml Ag for 30 min. The Fc{epsilon}RI was immunoprecipitated with anti-Fc{epsilon}RIß Ab from 4 x 106 cells and analyzed by immunoblotting with anti-phosphotyrosine Ab (4G10) and anti-Fc{epsilon}RIß. The cells used are the following: Syk-negative cells (B2), Syk-negative cells transfected with CD45 (CD45), ZAP70, or Syk, as indicated. B, Time course of Ag-induced tyrosine phosphorylation of the ß- and {gamma}- subunits in Syk-negative CD45- (B2) or CD45+ (CD45) cells. The cells were stimulated with Ag for the indicated times and lysates from 4 x 106 cells were immunoprecipitated with anti-Fc{epsilon}RIß Ab and analyzed by immunoblotting with anti-phosphotyrosine Ab.

 
Stronger binding of Syk than ZAP70 to the phosphorylated ITAM of Fc{epsilon}RI

Both ZAP70 and Syk have two tandem SH2 domains, which associate with the phosphorylated Ag receptors. To investigate the possible mechanism of why ZAP70 could not reconstitute the function of Syk in CD45-deficient mast cells, synthetic nonphosphorylated and phosphorylated peptides based on the ITAMs of Fc{epsilon}RI ß- or {gamma}- subunits were used to compare the binding ability of ZAP70 and Syk with Fc{epsilon}RI. As reported previously, the nonphosphorylated ITAM peptide was unable to precipitate ZAP70 or Syk (42, 44). Phosphorylated ß-ITAM peptide only precipitated a minimum amount of Syk, not ZAP70, while the two kinases associated much more strongly with the phosphorylated {gamma}-ITAM (Fig. 11Go, and data not shown). Interestingly, there was a dramatic difference in the binding of ZAP70 and Syk with the phosphorylated Fc{epsilon}RI{gamma}-peptide. The immobilized, phosphorylated {gamma}-ITAM precipitated ~15% of the expressed Syk from nonstimulated cell lysates, while it only bound ~1.5% of expressed ZAP70 under the same conditions. Experiments with stimulated cells gave similar results (data not shown). Furthermore, this difference in binding was not due to the variation in the expression level of the two kinases, since the precipitation with different concentrations of cellular proteins demonstrated similar result (Fig. 11Go). Therefore, even though ZAP70 and Syk have similar structures, Syk binds much better than ZAP70 to the ITAM of Fc{epsilon}RI{gamma}.



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  		FIGURE 11. Binding of ZAP70 and Syk to phosphorylated Fc{epsilon}RI{gamma}-ITAM peptide. Lysates from the indicated number of nonstimulated, ZAP70- or Syk-transfected cells were precipitated with the phosphorylated {gamma}-ITAM peptide that had been prebound to streptavidin beads. The ZAP70 and Syk in lysates from 2.5 x 104 cells are used for comparison. The precipitated proteins were analyzed by immunoblotting with anti-ZAP70 or anti-Syk Abs. Incubation was for 80 min at 4°C with 1 nmol of biotinylated and phosphorylated {gamma}-ITAM peptide bound to 25 µl of streptavidin beads.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we characterized a mast cell line deficient in both Syk and CD45. Fc{epsilon}RI aggregation on these cells did result in tyrosine phosphorylation of the receptor subunits, but it failed to initiate intracellular responses, such as cellular protein tyrosine phosphorylation and histamine release. The expression of ZAP70 in these double-deficient cells did not restore Ag-induced receptor signaling. Cotransfection of CD45 with ZAP70 reconstituted Fc{epsilon}RI-mediated degranulation and tyrosine phosphorylation of signaling molecules. This reconstitution required both molecules, as expression of only CD45 in these cells did not restore the signaling pathway. Syk, despite being a member of the same kinase family as ZAP70, was capable of functioning independently of CD45 in these double-deficient mast cells. It reconstitutes both the receptor-induced histamine release and cellular protein tyrosine phosphorylation. Furthermore, coexpression of CD45 had no dramatic effects on the function of Syk.

Both ZAP70 and Syk belong to the same protein tyrosine kinase family with ~55% amino acid homology. The structure similarity of ZAP70 and Syk suggests that these two kinases may play overlapping and/or similar functions in Ag receptor signaling. Indeed, in ZAP70-/- mice, Syk can replace ZAP70 function in thymocyte development, in TCR-mediated Ca2+ mobilization, CD69 expression, and in the proliferative response of thymocytes (45). In Jurkat cells that over-expressed wild-type ZAP70 or Syk, receptor activation induces similar levels of IL-2 promoter activity (46). Similarly, in Syk-negative chicken B cells, expression of ZAP70 reconstitutes B cell receptor signaling (15). However, there have also been reports of differences in regulation and signaling by these two kinases. TCRs have been expressed as chimeras with ZAP70 or Syk in a variant of a murine T cell hybridoma that lacks Ag receptors (47). When exposed to Ag, Syk is capable of transmitting signals for T cell activation, while ZAP70 is not, although both kinases autophosphorylate in immune kinase assays. Similarly, ZAP70 or Syk can restore the TCR signaling in a ZAP70- and Syk-negative variant of the Jurkat cell line, but only Syk induces a significant degree of cellular activation in the absence of receptor aggregation (21). Furthermore, in an Lck-negative variant of the Jurkat cells, Syk, but not ZAP70, augments both basal and TCR-stimulated protein tyrosine phosphorylation (20, 21). In the present experiments, we found that, despite effective expression, ZAP70 did not restore Fc{epsilon}RI signaling in mast cells unless CD45 was also present in the cells. Syk, on the other hand, was fully functional in the CD45-negative cells. These results are consistent with the study of CD45-negative Jurkat cells, in which Syk, but not ZAP70, will transmit TCR signals (20). Altogether, these studies strongly suggest that ZAP70 and Syk have different regulatory requirements.

Both ZAP70 and Syk have tandem SH2 domains, which are important for the association of these kinases with phosphorylated Ag receptors in stimulated cells. GST fusion proteins of the SH2 domains of Syk bind to the different ITAM motifs of the TCR with affinities comparable to the SH2 domains of ZAP70 (48). However, there are differences in the crystal structure of the tandem SH2 domains of ZAP70 and Syk. The C-terminal phosphotyrosine-binding site of Syk is different from that of ZAP70, which suggests that, in contrast to ZAP70, the two SH2 domains of Syk can function as independent units. Furthermore, the relative orientation of Syk SH2 domains displays remarkable conformational flexibility (49, 50). In the present experiments using whole molecules, we found that there was 10 times greater binding of Syk than ZAP70 to phosphorylated Fc{epsilon}RI{gamma}-ITAM peptide. These differences in binding were not due to the different expression level of the two kinases. This decrease in the binding of ZAP70 to Fc{epsilon}RI{gamma}-ITAM likely provides one of the reasons for its poor capacity to substitute for the function of Syk in CD45-deficient cells.

Previous studies have reported that ZAP70 and Syk have different intrinsic kinase activities. When these kinases are precipitated from transfected cells, the capacity of Syk to undergo autophosphorylation and to phosphorylate cdb3 is at least 100-fold greater than that of ZAP70 (51). In the present experiments, to avoid alterations of kinase structure, we used nontagged wild-type ZAP70 and Syk. Therefore, it is difficult to directly compare the activities of the two kinases. However, total cellular phosphorylations were less in ZAP70 than that in the Syk-expressing cells, irrespective of the presence or absence of CD45. This suggests that Syk is stronger than ZAP70 as a positive regulator for Fc{epsilon}RI-mediated tyrosine phosphorylation signals in mast cells. However, the situation was different for Fc{epsilon}RI-induced histamine release in these cells, the extent of receptor-mediated histamine release was similar in ZAP70- or Syk- transfected cells, as long as CD45 was also present in the cells. Therefore, in the cells transfected with ZAP70, there is a discrepancy between Fc{epsilon}RI-induced cellular protein tyrosine phosphorylation and degranulation, which suggests that the level of histamine release is not always related to the extent of cellular protein tyrosine phosphorylation.

CD45 is a transmembrane protein tyrosine phosphatase expressed in hematopoietic cells (23). Alternative splicing results in the expression of various isoforms with different extracellular domains. The mAb OX-1 used for FACS analysis recognizes all the different isoforms; therefore, the absence of binding in the present experiments suggests that this cell line is negative for CD45. Studies on both T and B cells suggest that CD45 may affect Ag-receptor signaling by modulating the activity of Src family kinases. These kinases contain a conserved tyrosine residue in the C-terminal region that negatively regulates enzymatic activity. CD45 can dephosphorylate this C-terminal tyrosine in vitro and in vivo and activate these tyrosine kinases (24, 52, 53). Loss of CD45 expression in T or B cells abrogates Ag receptor-dependent activation of Src family protein tyrosine kinases (54, 55). Previous studies have indicated that activation of ZAP70, but not Syk, requires an Src family kinase. In transfection studies in COS cells, a chimeric transmembrane protein with the cytoplasmic domain of the {gamma} subunit of Fc{epsilon}RI activates Syk, whereas ZAP70 requires the cotransfection of Lyn (56). Therefore, CD45, by activating Src kinases, may regulate ZAP70. In the present experiments, ZAP70 displayed a receptor-induced increase in kinase activity only when CD45 was coexpressed, suggesting that CD45 is essential for the activation of ZAP70.

In contrast to this positive function, a negative regulatory role of CD45 is observed in CD45-negative T cell lines and in thymocytes from CD45-/- mice. Lck in these cells is hyperphosphorylated on tyrosine residues, and its kinase activity is substantially increased (57, 58). Similarly, we found that, after Fc{epsilon}RI aggregation, there was stronger tyrosine phosphorylation of the receptor subunits in CD45-deficient cells than that in CD45-transfected cells. This suggests that the extent of receptor tyrosine phosphorylation is regulated by CD45. However, the extent of the histamine release did not correlate with this tyrosine phosphorylation.

In summary, we found that, in mast cells, CD45 was not essential for Fc{epsilon}RI-induced signal transduction by Syk. However, as in T cells, CD45 was required for ZAP70 to mediate Fc{epsilon}RI signaling in mast cells. The mechanism for these functional differences between the two kinases is probably the much stronger binding of Syk than ZAP70 with Fc{epsilon}RI{gamma}-ITAM and the requirement for CD45 by ZAP70, but not Syk, for receptor-stimulated enzymatic activation. Furthermore, we also found that the level of tyrosine phosphorylation of several signaling molecules did not correlate with the eventual degranulation. In cells expressing either Syk or ZAP70, the expression of CD45 decreased the tyrosine phosphorylation of the receptor subunits but increased that of PLC-{gamma}1.


   Acknowledgments
 
We thank Dr. Arthur Weiss and Dr. Jonathan D. Ashwell for the plasmids containing ZAP-70 and CD45, respectively; Dr. Kiyonao Sada and Dr. Katsuhiro Suzuki for reviewing this manuscript; Greta Bader and Neil Hardegen for excellent technical help; and Diana Rector for secretarial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Juan Zhang, RAST Section, OIIB, Building 10, Room 1N106, NIDCR, National Institutes of Health, Bethesda, MD 20892. E-mail address: Back

2 Abbreviations used in this paper: SH2, Src homology 2; PLC, phospholipase C; MAPK, microtubule-associated protein kinase; ITAM, immunoreceptor tyrosine-based activation motif. Back

Received for publication April 9, 1999. Accepted for publication June 14, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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