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The Journal of Immunology, 2004, 173: 1811-1818.
Copyright © 2004 by The American Association of Immunologists

Inactivation of c-Cbl or Cbl-b Differentially Affects Signaling from the High Affinity IgE Receptor

Juan Zhang1,*, Yungping J. Chiang1,{dagger}, Richard J. Hodes{dagger} and Reuben P. Siraganian2,*

* Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, and {dagger} Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Cbl family of proteins negatively regulate signaling from tyrosine kinase-coupled receptors. Among the three members of this family, only c-Cbl and Cbl-b are expressed in hemopoietic cells. To examine the role of c-Cbl and Cbl-b in Fc{epsilon}RI signaling, mast cell cultures from wild-type, c-Cbl–/–, and Cbl-b–/– mice were generated. Cell growth rates and cell surface expression of Fc{epsilon}RI were similar in the different cell populations. Compared with control cells, Cbl-b inactivation resulted in increases in Fc{epsilon}RI-induced Ca2+ response and histamine release. Fc{epsilon}RI-induced tyrosine phosphorylation of total cellular proteins, Syk, and phospholipase C-{gamma} was also enhanced by Cbl-b deficiency, whereas receptor-initiated phosphorylation of Vav, JNK, and p38 kinases was not changed in these cells. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on Fc{epsilon}RI-induced histamine release or on the phosphorylation of total cellular proteins or Syk. The absence of c-Cbl increased the phosphorylation of ERK after receptor stimulation, but resulted in slightly reduced p38 phosphorylation and Ca2+ response. These results suggest that Cbl-b and c-Cbl have divergent effects on Fc{epsilon}RI signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of Fc{epsilon}RI-induced degranulation.


   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 that includes increased protein tyrosine phosphorylation, a rise in intracellular calcium, and activation of protein kinase C. These biochemical changes eventually result in degranulation. This signal transduction pathway in mast cells has many similarities to signaling by other immune receptors, such as those on T or B cells. Like other immunoreceptors, Fc{epsilon}RI contains multiple subunits, the {alpha}-chain for binding IgE, and the {beta}- and {gamma}-chains that function to transduce signals via the paired tyrosine residues located in their ITAM. Ag stimulation leads to the phosphorylation of tyrosines in the ITAMs of the {beta}- and {gamma}-chains by the associated Src family protein tyrosine kinase (PTK)3 Lyn. The recruitment of Syk by the phosphorylated ITAMs results in the activation of Syk kinase. This leads, in turn, to the tyrosine phosphorylation of multiple downstream molecules, such as linker for activation of T cells, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, and phospholipase C{gamma} (PLC-{gamma}) (1, 2, 3).

Adapters and/or docking molecules also play a critical role in this signal transduction pathway. These proteins function by facilitating the interactions among multiple intracellular components required for signaling. Adapter molecules may provide docking sites for kinases and phosphatases to target their substrates, or to enhance or inhibit enzymatic reactions (4, 5). The Cbl family of adapter proteins plays a significant role in signal transduction as negative regulators of tyrosine kinase-coupled receptors (6, 7). The Cbl family comprises three mammalian proteins, c-Cbl, Cbl-b, and Cbl-3; among these, c-Cbl and Cbl-b are expressed in hemopoietic cells, whereas Cbl-3 is expressed only in epithelial tissues. The Cbl family proteins are tyrosine-phosphorylated in response to many stimuli, including growth factor receptors and various immune receptors. Cbl-b and c-Cbl interact with critical signaling molecules in both phosphorylation-dependent and -independent fashions, including Src family tyrosine kinases, ZAP-70/Syk family tyrosine kinases, p85 subunit of PI3K, and Vav. By associating with these proteins, Cbl-b and c-Cbl could serve as scaffolds to assemble signaling complexes and regulate their function.

Although c-Cbl and Cbl-b have a similar protein domain structure (8), gene-targeting studies demonstrate that they have distinct physiological functions. Although c-Cbl regulates receptor signaling in thymocytes, Cbl-b is involved in regulation of mature T cell signaling (9, 10). Mice deficient in Cbl-b develop spontaneous autoimmunity and exhibit hyperproliferation of the mature T and B cells. Cbl-b deficiency bypasses the requirement for CD28 costimulation in the TCR-mediated induction of T cell proliferation, IL-2 production, and phosphorylation of Vav (11, 12). Cbl-b may also regulate PI3K activation downstream of TCR (13). Characterization of B cells isolated from Cbl-b-deficient mice indicates that Cbl-b functions as a negative regulator of BCR signaling through a mechanism involving its ubiquitin ligase activity (14). However, in the DT40 chicken B cell line, Cbl-b acts as a positive regulator of BCR signaling. In these cells, Cbl-b deficiency results in attenuated activation of PLC-{gamma}2 and JNK, and reduced Ca2+ mobilization after BCR stimulation (15).

It is unclear what roles Cbl-b and c-Cbl play in mast cell signal transduction, which involves pathways similar to those mediating TCR and BCR signaling. To evaluate the function of Cbl-b and c-Cbl in Fc{epsilon}RI signaling, mast cell cultures were generated from Cbl-b–/– and c-Cbl–/– mice and compared with cultures from wild-type (WT) mice. Ag-induced protein tyrosine phosphorylation, Ca2+ response, and mast cell degranulation were examined in the different cell populations. Our results suggest that Cbl-b and c-Cbl have divergent effects on Fc{epsilon}RI signal transduction, and that Cbl-b, but not c-Cbl, functions as a negative regulator of Fc{epsilon}RI-induced degranulation.


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

Tissue culture reagents were purchased from BioWhittaker (Walkersville, MD). Mouse IL-3 and stem cell factor were obtained from BioSource International (Camarillo, CA). A plasmid expressing the human cytoplasmic domain of erythrocyte band 3 protein (cdb3) was provided by Dr. P. S. Law (Purdue University, West Lafayette, IN). HRP-conjugated anti-phosphotyrosine Ab PY-20 was obtained from ICN Immunobiologics (Lisle, IL). HRP-conjugated anti-phosphotyrosine Ab 4G-10, anti-PLC-{gamma}1, and anti-JNK Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-c-Cbl, anti-Cbl-b, anti-PLC-{gamma}2, anti-Syk, anti-Vav, and anti-phospho-JNK Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-p38, anti-p38, anti-phospho-p44/42 MAPK, and anti-p44/42 MAPK Abs were obtained from Cell Signaling Technology (Beverly, MA). The sources of other materials not indicated were described previously (2).

Animals

Generation of c-Cbl- and Cbl-b-deficient mice was reported previously (10, 12). All animal experiments were conducted with the approval of the institutional animal care and use committee. All mice used in this study were crossed to the C57BL/6 background and maintained at Bioqual (Rockville, MD) under specific pathogen-free conditions.

Mast cell isolation and activation

Bone marrow cells from individual c-Cbl+/+, c-Cbl–/–, Cbl-b+/+, and Cbl-b–/– mice were cultured in complete medium for 4–10 wk (DMEM supplemented with 20% heat-inactivated FBS, 4 mM L-glutamine, 5 x 10–5 M 2-ME, 10% NCTC 109 medium (Sigma-Aldrich, St. Louis, MO), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, antibiotics, 25 ng/ml IL-3, and 25 ng/ml stem cell factor). IgE receptor expression level was determined by flow cytometry. For cell activation, bone marrow-derived mast cells (BMMC) were cultured with Ag-specific IgE for 36 h and stimulated with Ag at concentrations from 0.1–100 ng/ml for 45 min at 37°C. The cells were also incubated with calcium ionophore A23187 at concentrations from 0.125–1 µM. After stimulation, the supernatants were removed for histamine analysis.

Immunoprecipitation and immunoblotting

Cells were stimulated as described above for the indicated times, and reactions were stopped by the addition of a 5-fold volume of ice-cold PBS containing 5 mM EDTA, 2 mM Na3VO4, and protease inhibitors. The cells were then centrifuged at 400 x g at 4°C, and the cell pellet was used as described below for either immunoblotting or immunoprecipitation experiments. For immunoblotting, cells were washed in PBS, and the pellet was immediately lysed with hot sample buffer (75 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 1% 2-ME). For immunoprecipitation, the cell pellets were solubilized in Triton lysis buffer (1% Triton X-100, 20 mM Tris (pH 7.4), 100 mM NaCl, 50 mM NaF, plus protease inhibitors, and Na3VO4). After a 20,000 x g centrifugation, the supernatants were immunoprecipitated with Abs bound to protein A-agarose beads. Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. The blots were probed with anti-phosphotyrosine or other specific Abs. In all blots, proteins were visualized by ECL. Where indicated the blots were scanned, and the densitometric quantitation of the bands was used to calculate the ratio of the phosphorylated proteins in the Cbl-b–/– or c-Cbl–/– BMMC compared with the WT cells.

Flow cytometric analysis of IgE receptor expression

Cells were cultured without or with IgE for 36 h, washed, and incubated with PE-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL). Analysis was performed by flow cytometry using a FACScan (BD Bioscience, San Jose, CA).

Measurement of calcium influx

Cells were cultured with IgE for 36 h, washed with loading medium (medium 199 supplemented with 2 mM CaCl2, 0.1% BSA, and 250 µM sulfinpyrazone), and loaded with 2 µM fura 2-AM for 1 h at 37°C. Then cells were washed with working medium (medium 199 containing 2 mM CaCl2, 10 mM Tris (pH 7.4), 0.01% BSA, and 250 µM sulfinpyrazone), plated in 48-well plates and spun at 1100 rpm for 10 min. Fura-2 fluorescence in single cells was measured using a Tillvision System (Till Photononic, Grafelfing, Germany). A pair of fluorescence images was acquired every 2 s at excitation wavelengths of 340 and 380 nm. For data analysis, 100 cells were chosen at random, and the ratio of the background-subtracted fluorescence intensity excited at the two wavelengths was calculated and plotted for individual cells.

In vitro kinase assay

Syk 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 40 µl of the same buffer. The kinase reactions were performed for 18 min at room temperature with 3 µCi of [{gamma}-32P]ATP and 4 µM ATP. The reactions were stopped by the addition of 40 µl of 2x Laemmli sample buffer and boiling for 10 min. The eluted proteins were separated under reducing conditions by SDS-PAGE, electrotransferred to membranes, and visualized by autoradiography. The membranes were then also immunoblotted with anti-Syk Ab as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cbl-b is phosphorylated after Fc{epsilon}RI aggregation

Ag receptor stimulation results in the phosphorylation of both c-Cbl and Cbl-b in T and B cells. In mast cells, Fc{epsilon}RI engagement induces tyrosine phosphorylation of c-Cbl (16, 17), but phosphorylation of Cbl-b has not been characterized. We therefore used mast cells from WT mice to test the receptor-induced phosphorylation of Cbl-b. BMMC were incubated with monoclonal IgE specific for DNP and stimulated by 10 ng/ml DNP-HSA for the indicated times. The proteins immunoprecipitated with anti-Cbl-b Ab were analyzed by immunoblotting with anti-phosphotyrosine and anti-Cbl-b. As shown in Fig. 1A, Fc{epsilon}RI aggregation resulted in rapid tyrosine phosphorylation of Cbl-b, which was detectable at 1 min after stimulation, remained strong for up to 5 min, and then decreased slowly during the next 15 min. The Ag dose response of Cbl-b tyrosine phosphorylation was also tested by stimulating the cells with different concentrations of Ag for 2 min, and the immunoprecipitated Cbl-b was immunoblotted with anti-phosphotyrosine Ab. The optimal concentration of Ag for stimulating tyrosine phosphorylation of Cbl-b was 10 ng/ml (Fig. 1B). These results indicate that, as in B cells and Jurkat T cells, immune receptor stimulation of mast cells results in tyrosine phosphorylation of Cbl-b as well as c-Cbl.



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  		FIGURE 1. Fc{epsilon}RI aggregation resulted in the tyrosine phosphorylation of Cbl-b. A, Time course of Ag-induced Cbl-b phosphorylation. BMMC from WT mice were cultured with Ag-specific IgE, washed, and stimulated by 10 ng/ml Ag for the indicated times. Cbl-b was immunoprecipitated with goat anti-Cbl-b and analyzed by immunoblotting with anti-phosphotyrosine or anti-Cbl-b Abs. B, Ag dose response of Cbl-b tyrosine phosphorylation. WT BMMC were stimulated with the indicated concentrations of Ag for 2 min. Cbl-b was immunoprecipitated and analyzed as described in A. The results shown are representative of at least three different experiments.

 
Cbl-b negatively regulates Fc{epsilon}RI-induced mast cell degranulation

To investigate the function of Cbl-b in mast cell signal transduction, BMMC were isolated from Cbl-b knockout mice and WT animals. Bone marrow from each mouse was cultured individually in medium containing both IL-3 and stem cell factor, and Cbl-b phenotypes were confirmed by PCR and immunoblotting with anti-Cbl-b Ab (data not shown and Fig. 2A). The rate of cell growth was similar in WT and Cbl-b knockout BMMC cultures, and cell surface expression of Fc{epsilon}RI was comparable on WT and Cbl-b–/– cells (data not shown).



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  		FIGURE 2. The absence of Cbl-b enhanced Ag-initiated mast cell degranulation. A, Expression of Cbl-b. Cell lysates prepared from WT and Cbl-b–/– BMMC were analyzed by anti-Cbl-b Ab immunoblotting. B, Fc{epsilon}RI-induced histamine release. Cells were stimulated with the indicated doses of Ag for 45 min at 37°C. The results shown are the percentage of total histamine released in the supernatant and are the mean (±SE) of at least 12 different assays. **, p ≤ 0.01 for Cbl-b–/– cells compared with Cbl-b+/+ cells at that Ag concentration, as determined by paired t test. C, Calcium ionophore-induced histamine release. Cells were incubated with the indicated dose of A23187, and histamine was assayed and calculated as described in B. {square}, WT cells; {blacksquare}, Cbl-b–/– cells.

 
Degranulation is one of the major functional responses of mast cells to Ag stimulation. Therefore, we compared the Fc{epsilon}RI-induced histamine release of WT and Cbl-b–/– BMMC to determine whether Cbl-b inactivation has any effect on Fc{epsilon}RI signal transduction. As shown in Fig. 2B, the absence of Cbl-b increased receptor-mediated histamine release at all concentrations of Ag tested. Calcium ionophore A23187 was used as a control in the same experiments for nonreceptor-induced degranulation, and the results showed that the A23187-induced histamine release was not enhanced by Cbl-b deficiency (Fig. 2C). These experiments suggest that Cbl-b plays a specific role in negatively regulating Fc{epsilon}RI signal transduction.

Effects of Cbl-b deficiency on cellular protein phosphorylation

Because the absence of Cbl-b enhanced Fc{epsilon}RI-induced mast cell degranulation, we investigated the effect of Cbl-b inactivation on early parameters of signal transduction. The earliest event detected after Fc{epsilon}RI aggregation is protein phosphorylation on tyrosine residues, which is critical for downstream signal propagation. To detect whether Cbl-b deficiency had any effect on the phosphorylation of cellular proteins, BMMC from Cbl-b+/+ and Cbl-b–/– mice were stimulated with IgE plus different concentrations of Ag, and total cell lysates were blotted with anti-phosphotyrosine Ab. As shown in Fig. 3A, 10 ng/ml Ag was the optimal concentration for inducing total cellular protein tyrosine phosphorylation of WT mast cells, which is the same concentration that resulted in the maximum Cbl-b phosphorylation and induced the highest histamine release. Compared with WT cells, the absence of Cbl-b enhanced Ag-initiated cellular protein tyrosine phosphorylation. In contrast, basal phosphorylation in nonstimulated cells was slightly reduced by Cbl-b deficiency. Time-course experiments with the optimal concentration of Ag (10 ng/ml; Fig. 3B) indicated that in Cbl-b+/+ BMMC, cellular protein phosphorylation peaks at 2 min after receptor stimulation, whereas the signal in Cbl-b–/– BMMC was more sustained and peaks at 8 min.



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  		FIGURE 3. Cbl-b deficiency enhanced multiple steps of the Fc{epsilon}RI signaling pathway. A and B, Total cellular protein tyrosine phosphorylation. Cells were stimulated as indicated with different Ag concentrations (A) or for different times (B), and total cell lysates were analyzed by immunoblotting with anti-phosphotyrosine Ab. Total cell lysates were analyzed by immunoblotting with anti-phospho-p44/42 MAPK (I), anti-phospho-JNK (J), anti-phospho-p38 (K), or the specific Abs. Lysates were also immunoprecipitated with anti-Fc{epsilon}RI-{beta} (C), anti-Syk (D), anti-PLC-{gamma}1 (F), anti-PLC-{gamma}2 (G), and anti-Vav (H) Abs and were analyzed by immunoblotting with anti-phosphotyrosine or other specific Abs. E, In vitro kinase assay of Syk. Syk was immunoprecipitated, then incubated with [{gamma}-32P]ATP and cdb3 in a protein kinase assay. Stimulation was performed with 10 ng/ml Ag for 2 min unless indicated otherwise. The results shown are representative of two to four different experiments. The changes in phosphorylation in Cbl-b–/– BMMC were compared with those in WT cells by densitometry after normalization for protein loading. The numbers are the average from the different experiments.

 
To further study the effect of Cbl-b deficiency on Fc{epsilon}RI-induced cell activation, we examined the Ag-induced tyrosine phosphorylation of several important signaling molecules. Ag stimulation results in rapid tyrosine phosphorylation of the {beta} and {gamma} subunits of Fc{epsilon}RI. As shown in Fig. 3C, compared with Cbl-b+/+ BMMC, the absence of Cbl-b resulted in stronger Ag-induced tyrosine phosphorylation of both {beta} and {gamma} subunits of Fc{epsilon}RI.

Syk plays a critical role in transducing signals from Fc{epsilon}RI to downstream events, such as protein tyrosine phosphorylation and degranulation. As shown in Fig. 3D, Fc{epsilon}RI aggregation induced a rapid increase in Syk tyrosine phosphorylation in both WT and Cbl-b–/– cells. Compared with BMMC isolated from Cbl-b+/+ mice, Cbl-b deficiency resulted in increased tyrosine phosphorylation of Syk. Syk kinase activity was determined by an immune complex kinase assay using cdb3 band protein as exogenous substrate (Fig. 3E). Although Cbl-b deficiency obviously enhanced Syk phosphorylation, there was only a slight increase in Syk kinase activity in Cbl-b–/– cells compared with Cbl-b+/+ BMMC.

Receptor stimulation induces the activation of PLC-{gamma}, which is downstream of Syk. Therefore, tyrosine phosphorylation of PLC-{gamma}1 and PLC-{gamma}2 was measured for WT and Cbl-b–/– BMMC (Fig. 3, F and G). Fc{epsilon}RI aggregation induced rapid tyrosine phosphorylation of PLC-{gamma} in both Cbl-b+/+ and Cbl-b–/– cells. Compared with WT cells, the absence of Cbl-b resulted in only a slight increase (1.5-fold) in PLC-{gamma}1 phosphorylation, whereas the tyrosine phosphorylation of PLC-{gamma}2 was enhanced 4.4-fold by Cbl-b deficiency.

Vav is a guanine nucleotide exchange factor of the Rho family of GTPases. Because Vav has been reported to play an important role in Cbl-b function in T cells, we examined whether the absence of Cbl-b had any effect on Vav tyrosine phosphorylation in mast cells. As shown in Fig. 3H, Ag-induced Vav tyrosine phosphorylation was similar in Cbl-b+/+ and Cbl-b–/– BMMC.

Engagement of Fc{epsilon}RI also led to the activation of MAPK ERK, JNK, and p38 pathways. Compared with Cbl-b+/+ BMMC, Cbl-b deficiency resulted in a slightly reduced ERK phosphorylation after receptor aggregation (Fig. 3I), whereas Ag-induced phosphorylation of JNK and p38 was similar in Cbl-b+/+ and Cbl-b–/– cells (Fig. 3, J and K).

Cbl-b negatively regulates Fc{epsilon}RI-induced calcium response

Ag stimulation results in the generation of inositol 1,4,5-triphosphate, which initiates the release of calcium from intracellular stores, and calcium influx through calcium release-activated calcium channels in the plasma membrane. Because receptor-induced phosphorylation of Fc{epsilon}RI{beta} and {gamma} subunits, Syk, and PLC-{gamma} were all enhanced in Cbl-b–/– BMMC, we measured the changes in intracellular calcium in individual cells to monitor the effects of Cbl-b deficiency on receptor-mediated changes in the intracellular free Ca2+ concentration ([Ca2+]i). Both the rapid and the sustained calcium response to Ag stimulation were enhanced in Cbl-b–/– cells (Fig. 4). In contrast, calcium mobilization induced by thrombin stimulation was similar in the two populations (data not shown).



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  		FIGURE 4. Cbl-b deficiency enhanced the Ag-induced [Ca2+]i response. BMMC were cultured with IgE and loaded with fura 2. After washing, pairs of fluorescence images were acquired at excitation wavelengths of 340 and 380 nm. Ag (10 ng/ml) was added at the time indicated by the arrow. For analysis, 100 cells were chosen at random, and the ratio of the background-subtracted fluorescence intensity excited at the two wavelengths was calculated and is presented as the mean 340:380 ratio. The solid line is for WT cells, and the dashed line is for Cbl-b–/– BMMC. The results shown are representative of three different BMMC preparations tested at multiple Ag concentrations (1, 10, and 100 ng/ml).

 
c-Cbl is not critical for regulating mast cell degranulation

The c-Cbl and Cbl-b proteins share overall domain structure, and overexpression of c-Cbl in RBL-2H3 cells inhibits Fc{epsilon}RI-induced cellular protein phosphorylation and degranulation (18). To test the effect of c-Cbl deficiency on BMMC signal transduction, bone marrow from c-Cbl+/+ and c-Cbl–/– mice was isolated and cultured individually in medium containing both IL-3 and stem cell factor. Western blot analysis with anti-c-Cbl Ab confirmed that there was no c-Cbl protein expression in c-Cbl–/– BMMC (Fig. 5A). The cell surface expression of Fc{epsilon}RI was similar in c-Cbl+/+ and c-Cbl–/– cells (data not shown).



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  		FIGURE 5. c-Cbl deficiency had minimal effects on Ag-induced mast cell degranulation and [Ca2+]i response. A, Expression of c-Cbl. Cell lysates prepared from WT and c-Cbl–/– BMMC were analyzed by immunoblotting with anti-c-Cbl Ab. B and C, Histamine release. Cells were stimulated with the indicated concentrations of Ag (B) or with calcium ionophore A23187 (C) for 45 min at 37°C. The results shown are the percentage of total histamine released in the supernatant and are the mean (±SE) of at least six different experiments. {square}, WT cells; {blacksquare}, c-Cbl–/– cells. D, Ag-induced calcium response. The c-Cbl+/+ (solid line) or c-Cbl–/– BMMC (dashed line) cultured with IgE were loaded with fura 2, and fluorescence in single cells was measured as described in Fig. 4. Ag (10 ng/ml) was added at the time indicated by the arrow.

 
The effect of c-Cbl deficiency on mast cell degranulation was determined by histamine assay. Even though the overexpression of c-Cbl blocks Fc{epsilon}RI-initiated degranulation in RBL-2H3 cells, in BMMC the absence of c-Cbl had no detectable effect on Ag-induced histamine release with a wide range of Ag concentrations (Fig. 5B). Histamine release initiated by calcium ionophore A23187 was also comparable in c-Cbl+/+ and c-Cbl–/– cells (Fig. 5C). The role of c-Cbl in calcium response was investigated with c-Cbl+/+ and c-Cbl–/– cells using the calcium-sensitive dye fura 2. The absence of c-Cbl slightly reduced the IgE-Ag-initiated calcium mobilization (Fig. 5D). In contrast, thrombin stimulated similar calcium responses in WT and c-Cbl-deficient cells (data not shown).

The overexpression of c-Cbl in RBL-2H3 cells inhibits receptor-induced cellular protein phosphorylation (18). We therefore investigated whether the absence of c-Cbl in BMMC has any effect on Fc{epsilon}RI-induced phosphorylation of proteins. The tyrosine phosphorylation of total cellular proteins before and after Ag stimulation was compared in WT and c-Cbl–/– cells (Fig. 6A). Again, c-Cbl deficiency had no discernible effect on total cellular protein phosphorylation in BMMC.



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  		FIGURE 6. Comparisons of Ag-induced protein phosphorylation. WT and c-Cbl–/– BMMC were cultured with IgE and stimulated by Ag at 10 ng/ml. Lysates were analyzed by immunoblotting with anti-phosphotyrosine (A), anti-phospho-p44/42 MAPK (D), anti-phospho-JNK (E), anti-phospho-p38 (F), or other specific Abs. Lysates were also immunoprecipitated with anti-Syk (B) and anti-PLC-{gamma}2 (C) Abs and analyzed by immunoblotting with anti-phosphotyrosine and the specific Abs. Stimulation was performed for the indicated times, except for 2 min for B and C. The results shown are representative of at least two independent experiments.

 
Previous reports have also suggested that c-Cbl can regulate the activation of Syk. Therefore, Syk was immunoprecipitated from WT and c-Cbl–/– BMMC before and after Ag stimulation. The results shown in Fig. 6B suggested that c-Cbl deficiency had no effect on Fc{epsilon}RI-induced tyrosine phosphorylation of Syk. PLC-{gamma} is downstream of Syk and critical for Ag-induced calcium response in mast cells. As shown in Fig. 6C, the lack of c-Cbl resulted in a slightly reduced tyrosine phosphorylation of PLC-{gamma}2 after receptor stimulation, which correlated with the effect of c-Cbl deficiency on the Fc{epsilon}RI-induced calcium response.

The Fc{epsilon}RI-induced activation of the MAPK ERK, JNK, and p38 pathways was also evaluated in c-Cbl+/+ and c-Cbl–/– cells. Compared with control cells, c-Cbl–/– BMMC showed an increase in ERK phosphorylation (Fig. 6D). The phosphorylation of JNK was comparable in WT cells and c-Cbl–/– BMMC (Fig. 6E), whereas the phosphorylation of p38 was somewhat reduced by c-Cbl deficiency (Fig. 6F). These results suggested that in BMMC, c-Cbl does not play a critical role in mast cell degranulation, but may affect other Ag receptor signaling pathways.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To evaluate the function of c-Cbl and Cbl-b in mast cells, BMMC were cultured from WT, c-Cbl–/–, and Cbl-b–/– mice, and Ag-induced protein tyrosine phosphorylation, Ca2+ response, and degranulation were compared in the different cell populations (Table I). In WT cells, Fc{epsilon}RI aggregation induced rapid tyrosine phosphorylation of Cbl-b, suggesting a role for this protein in mast cell signal transduction. This was confirmed by the observations that the absence of Cbl-b enhanced receptor-induced histamine release, calcium mobilization, and tyrosine phosphorylation of cellular proteins, including the {beta} and {gamma} subunits of Fc{epsilon}RI, Syk, and PLC-{gamma}, whereas the phosphorylation of Vav, JNK, and p38 was essentially unchanged. In contrast to Cbl-b, c-Cbl deficiency did not affect histamine release and had different and more limited effects on other parameters of receptor-induced cellular responses.


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  		Table I. Fc{epsilon}RI signaling in Cbl-b–/– or c-Cbl–/– BMMCsa

 
c-Cbl is the product of the proto-oncogene c-cbl (19). After receptor stimulation, c-Cbl becomes phosphorylated and binds to several critical signaling molecules, including Src family PTK, Syk/ZAP-70, Btk, Grb2, Crk, Nck, PI3K, and Vav (16, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Mice with c-Cbl deficiency show enhanced thymic positive selection, higher expression of cell surface TCR, and increased TCR-induced tyrosine phosphorylation of cellular proteins in thymocytes (9, 10). In contrast, there are no abnormalities in the peripheral T cells of these c-Cbl knockout mice (9, 10, 30). In the DT40 B cell line, lack of c-Cbl has no effect on receptor-induced phosphorylation of Syk and Btk, but enhances BCR-mediated PLC-{gamma}2 activation, which leads to increased apoptosis (31). Coinfecting RBL-2H3 cells with recombinant vaccinia Syk and c-Cbl inhibits IgE-induced Syk phosphorylation, kinase activity, and Syk association with Fc{epsilon}RI. The 10-fold overexpression of c-Cbl reduces receptor-initiated mast cell degranulation. However, a 3-fold overexpression of c-Cbl in the same experiments failed to inhibit receptor-mediated degranulation (18). Similarly, we observed that an up to 4-fold overexpression of hemagglutinin-tagged c-Cbl in RBL-2H3 cells did not inhibit receptor-stimulated histamine release (unpublished observations). In the present study c-Cbl deficiency in BMMC did not affect Fc{epsilon}RI-induced degranulation and had minimal effects on Fc{epsilon}RI signal transduction.

Cbl-b is the second member of mammalian Cbl family proteins. In COS-1 cells, cotransfection of Cbl-b with several PTKs results in the tyrosine phosphorylation of Cbl-b, with Syk inducing the most prominent effect (32). In T and B cells, receptor stimulation results in the tyrosine phosphorylation of Cbl-b (14, 32, 33). In the present experiments we found that in mast cells, aggregation of Fc{epsilon}RI induced rapid tyrosine phosphorylation of Cbl-b. Furthermore, our results indicated that the optimal Ag concentration for inducing Cbl-b phosphorylation was the same as that for cellular protein phosphorylation and mast cell degranulation. Taken together, these results suggest that Cbl-b is involved in Fc{epsilon}RI-mediated intracellular signaling.

Overexpression of Cbl-b in different cell systems suggests that this molecule may play either a positive or a negative role in signal transduction (34, 35). However, the studies of Cbl-b-deficient mice suggest a negative regulatory function for this molecule. In T cells, Cbl-b deficiency enhances receptor-induced cell proliferation and Vav activation and uncouples TCR-initiated receptor clustering, T cell proliferation, and IL-2 production from the requirement for CD28 costimulation. Furthermore, the Cbl-b-null mutation restores proliferation and in vivo immune responses in Vav1–/– T cells and fully restores T cell-dependent Ab responses in CD28–/– mice (11, 12, 36). In B cells, lack of Cbl-b results in the sustained phosphorylation of Ig{alpha}, Syk, and PLC-{gamma}2; prolonged Ca2+ mobilization; and increased phosphorylation of ERK and JNK after BCR stimulation (14). Our finding that Cbl-b deficiency enhanced Fc{epsilon}RI-induced protein tyrosine phosphorylation, Ca2+ response, and histamine release suggests that the function of Cbl-b in mast cell is similar to the role of this molecule in mouse B and T cells. A recent report that the membrane-targeted expression of Cbl-b inhibited degranulation of RBL-2H3 cells supports the present findings (37).

Several signaling molecules associate with Cbl-b, including Vav, Grb2, and PI3K (11, 34, 38, 39). By associating with these proteins, Cbl-b could serve as a scaffold to assemble signaling complexes and regulate their function. In T cells, the lack of Cbl-b enhances basal and TCR-induced Vav tyrosine phosphorylation and Vav guanine nucleotide exchange factor activity (11, 12). However, in the present study the absence of Cbl-b did not have an obvious effect on receptor-induced Vav tyrosine phosphorylation. Therefore, it is unlikely that Vav is the major cause of the enhanced signal transduction in Cbl-b–/– BMMC. Cbl-b constitutively interacts with the p85 subunit of PI3K and negatively regulates PI3K activity in T cells. As PI3K is important for the sustained calcium influx after receptor activation, the changes in the regulation of PI3K could contribute to the enhanced tyrosine phosphorylation of PLC-{gamma} and calcium influx in mast cells.

The negative regulatory function of Cbl proteins may be partly dependent on the activity of Cbl as an ubiquitin protein ligase. Overexpression of Cbl-b in breast cancer cells enhances epidermal growth factor-induced ubiquitination and degradation of the epidermal growth factor receptor (40). In T cells, Cbl-b deficiency moderately decreases ligand-induced TCR down-modulation (41). Conversely, we found that in BMMC, the absence of Cbl-b did not inhibit Ag-induced Fc{epsilon}RI internalization, nor did it have an effect on receptor degradation after 6-h Ag stimulation (data not shown). Furthermore, in WT mast cells, 6-h Ag incubation resulted in even a slight increase in Cbl-b protein expression (data not shown) similar to that observed after TCR stimulation of primary T cells (42). Fc{epsilon}RI stimulation induces the ubiquitination of Syk (43). Cbl-b deficiency increases BCR-induced Syk ubiquitination in B cells (14). Even though Cbl-b-deficient mast cells showed enhanced Ag receptor signaling, similar to what was observed in B cells, we failed to detect any increased Syk ubiquitination in WT BMMC compared with that in Cbl-b–/– cells (data not shown). Clearly, additional experiments will be necessary to fully define the mechanism by which Cbl-b negatively regulates Fc{epsilon}RI signal transduction.

In summary, the functional roles of c-Cbl and Cbl-b in the Fc{epsilon}RI signal pathway were compared. Ag stimulation induces tyrosine phosphorylation of Cbl-b as well as c-Cbl in mast cells. The absence of Cbl-b, but not c-Cbl, increases receptor-mediated protein phosphorylation, Ca2+ mobilization, and histamine release. In contrast, the knockout of c-Cbl results in changes in the phosphorylation of ERK and p38. These results suggest that Cbl-b and c-Cbl differ in their effects on Fc{epsilon}RI signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of Fc{epsilon}RI-induced degranulation.


   Acknowledgments
 
We are grateful to Drs. Tomohiro Hitomi and Kyungduk Moon for reviewing this manuscript. We thank Lynda Weedon and Greta Bader for excellent technical help.


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

1 J.Z. and Y.J.C. contributed equally to this work. Back

2 Address correspondence and reprint requests to Dr. Reuben Siraganian, RAST Section, OIIB, Building 10/Room 1N106, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. E-mail address: rs53x{at}nih.gov Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; BMMC, bone marrow-derived mast cell; Btk, Bruton’s tyrosine kinase; [Ca2+]i, intracellular free Ca2+ concentration; cdb3, cytoplasmic domain of erythrocyte band 3 protein; Fc{epsilon}RI, high affinity IgE receptor; PLC-{gamma}, phospholipase C-{gamma}; WT, wild type. Back

Received for publication February 11, 2004. Accepted for publication May 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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