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The Journal of Immunology, 2002, 168: 2087-2090.
Copyright © 2002 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Transmembrane Phosphoprotein Csk-Binding Protein/Phosphoprotein Associated With Glycosphingolipid-Enriched Microdomains as a Negative Feedback Regulator of Mast Cell Signaling Through the Fc{epsilon}RI

Hidenori Ohtake*, Naoki Ichikawa*, Masato Okada{dagger} and Toshiyuki Yamashita1,*

* Division of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; and {dagger} Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Tyrosine phosphorylation in the cytoplasmic domains of Fc{epsilon}RI by the Src family kinase Lyn initiates a signaling cascade leading to mast cell activation. In this study, we show that a recently identified transmembrane protein, Csk-binding protein (Cbp), also known as phospoprotein associated with glycosphingolipid-enriched microdomains (PAG), negatively regulates Fc{epsilon}RI signaling. In rat basophilic leukemia (RBL)-2H3 cells, the levels of tyrosine phosphorylation of Cbp/PAG and its association with Csk, a negative regulator for Lyn, significantly elevate immediately after aggregation of Fc{epsilon}RI. An overexpression of Cbp/PAG in RBL-2H3 cells inhibits Fc{epsilon}RI-mediated cell activation. This is accompanied with decreased levels of tyrosine phosphorylation of Fc{epsilon}RI, association of Fc{epsilon}RI with Lyn, and Fc{epsilon}RI-associated tyrosine kinase activity. These findings combined with the fact that Cbp/PAG, Lyn, and aggregated Fc{epsilon}RI are localized to lipid rafts, suggest that upon Fc{epsilon}RI aggregation Cbp/PAG down-regulates the receptor-associated Lyn activity through relocating Csk to rafts, thereby efficiently mediating feedback inhibition of Fc{epsilon}RI signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
A variety of intracellular events leading to the release of inflammatory mediators are induced when Fc{epsilon}RI on mast cells are aggregated by the binding of allergen to receptor-bound IgE (1). One of the earliest detectable events following Fc{epsilon}RI aggregation is the tyrosine phosphorylation of cellular proteins including receptor subunits. The cytoplasmic domains of the {beta}- and {gamma}-chains contain a sequence termed the immunoreceptor tyrosine-based activation motif (ITAM)2 that is also found in other multichain immune recognition receptors such as B cell receptor and TCR (2). The two tyrosine residues in each ITAM are phosphorylated upon Fc{epsilon}RI aggregation, and then the phosphorylated ITAM serves as a binding site for signaling molecules containing src homology 2 (SH2) domains such as Syk, a critical protein tyrosine kinase (PTK) responsible for cell activation (1).

It is commonly thought that the Src family kinase Lyn is responsible for the initial tyrosine phosphorylation of Fc{epsilon}RI subunits (3, 4, 5). Lyn that is constitutively associated with a small fraction of the receptors mediates the tyrosine phosphorylation of the receptors by a process of transphosphorylation when the receptors are brought into proximity upon aggregation (5, 6). A structural basis of this "transphosphorylation" model is weak interactions between Lyn and {beta} subunits of Fc{epsilon}RI (4, 7). In addition, specialized membrane domains (lipid rafts) are involved in the initial Fc{epsilon}RI signaling (8, 9, 10, 11). These rafts are enriched in a number of signaling molecules including dually acylated Src family kinases like Lyn (12). In quiescent cells, Fc{epsilon}RI is excluded from or only weakly associated with rafts, while aggregation of receptors triggers its recruitment to rafts (9, 10).

Once phosphorylated, aggregated receptors promote their association with additional Lyn, thereby the receptor-associated PTK activity is strongly enhanced (5, 13). Lyn, like other members of the Src family kinases, possesses a negative-regulatory phosphotyrosine located near the C terminus. This phosphotyrosine is able to interact with the Lyn’s N-terminal SH2 domain, thereby preventing the catalytic site of the kinase from accessing the external kinase substrates. In hematopoietic cells, CD45, a transmembrane protein tyrosine phosphatase, is implicated in the dephosphorylation of this regulatory phosphotyrosine (14). Several investigators including ourselves suggested that CD45 functions as a positive regulator in Fc{epsilon}RI signaling (13, 15, 16). In contrast, Csk, which is a ubiquitously expressed tyrosine kinase, specifically phosphorylates the regulatory tyrosine (17). Unlike Src family kinases, Csk is devoid of the amino-terminal myristylation signal required for membrane localization. With regard to the role of Csk in Fc{epsilon}RI signaling, Honda et al. (18) reported its implication in the initiation and the termination of the Fc{epsilon}RI-mediated Lyn activation.

Recently, two groups have identified a novel protein that regulates the activity of Src family kinases, which is a Csk-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) (19, 20). Cbp/PAG is a ubiquitously expressed transmembrane protein that exclusively localizes in lipid rafts. When phosphorylated on a specific tyrosine residue, Cbp/PAG becomes able to bind to the SH2 domain of Csk. This binding elevates the affinity of Csk for Src family kinases (21). Thus, Cbp/PAG is involved in the negative regulation of Src family kinases not only by relocating Csk to the membrane, but also by directly activating Csk. In primary T cells, Cbp/PAG is constitutively tyrosine phosphorylated, and the level of this phosphorylation is decreased following TCR aggregation (20, 22). In addition, the overexpression of Cbp/PAG in Jurkat cells impairs TCR-induced cell activation (20, 22).

The role of Cbp/PAG in Fc{epsilon}RI-mediated signaling has not been explored. The similarities in some functional properties between Fc{epsilon}RI and TCR systems prompted us to explore the possible role of Cbp/PAG in Fc{epsilon}RI signaling and thereby further assess the functional significance of Csk in this receptor system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells, Abs, and reagents

Rat basophilic leukemia (RBL)-2H3 cells were grown as adherent monolayers and harvested following exposure to trypsin and EDTA (23). The cells were then sensitized with anti-DNP IgE mAb (H1 DNP-{epsilon}-26.82) as previously described (13, 23, 24). When the cells were fractionated by sucrose density gradient centrifugation, the cells were sensitized with biotin-conjugated IgE and stimulated with streptavidin (9, 25). Polyclonal anti-rat Cbp/PAG Ab was prepared by immunizing rabbits with a synthetic peptide (GDLQQGRDVTRL) corresponding to the C-terminal 12 residues of rat Cbp/PAG coupled with keyhole limpet hemocyanin. Anti-myc Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The other Abs and reagents we used have been described (13, 23, 25).

cDNA constructs and transfection

The full-length rat Cbp/PAG was inserted into a pCMV-Tag1 vector (Stratagene, La Jolla, CA) to generate myc-tagged Cbp/PAG. The cDNA construct was then subcloned into a pCXN2 expression vector (26) kindly provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan). RBL-2H3 cells were transfected with this vector by electroporation, and G-418-resistant clones were screened by immunoblotting with anti-myc. Among the monoclonal transfectants obtained, two cell lines stably expressing the highest levels of Cbp/PAG were used for the experiments.

Isolation of lipid rafts

Lipid rafts were isolated from cell lysates by sucrose gradient centrifugation under detergent-free conditions (27). Briefly, cells were homogenized in the homogenate buffer containing 500 mM Na2CO3 and then fractionated by a discontinuous sucrose gradient centrifugation. In some experiments, proteins in fractions were quantitatively precipitated using CH3OH and CHCl3 (28).

Immunoprecipitation, immunoblotting, and in vitro kinase assay

The conditions for immunoprecipitation and immunoblotting were described previously (13, 23, 25). The IgE-bound Fc{epsilon}RI was immunoprecipitated with anti-IgE. When we immunoprecipitated Cbp/PAG, cells were solubilized in lysis buffer containing 60 mM octyl-D-glycoside which is destructive to lipid rafts. Fc{epsilon}RI-associated PTK activities were measured using exogenous substrates (cdc-2-derived peptide; KVEKIGEGTYGVVKK) and [{gamma}-32P]ATP as described previously (6, 13).

Degranulation and Ca2+ mobilization assay

The assay conditions for degranulation were described previously (23). Measurements of intracellular free Ca2+ concentration were performed using Fura-PE3 as a Ca2+ indicator (13).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
When lysates of resting RBL-2H3 cells were subjected to centrifugation on sucrose density gradient and subsequently to immunoblotting with anti-Cbp/PAG Abs, we found that Cbp/PAG is exclusively localized to the low density sucrose region containing lipid rafts (Fig. 1Go). The Cbp/PAG in RBL-2H3 cells are constitutively tyrosine phosphorylated, and aggregation of Fc{epsilon}RI results in an enhanced tyrosine phosphorylation of Cbp/PAG (see Fig. 4GoA). The time-course study demonstrated that within 30 s after Ag addition, Cbp/PAG became heavily tyrosine-phosphorylated. This stimulated tyrosine phosphorylation, then gradually declined.



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  		FIGURE 1. The exclusive localization of Cbp/PAG in the lipid raft fractions in RBL-2H3 cells. The cells were lysed under detergent-free conditions and fractionated by sucrose density gradient centrifugation. Equal aliquots of each fraction were analyzed by immunoblotting for the presence of Cbp/PAG.

 


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  		FIGURE 4. Tyrosine phosphorylation of Cbp/PAG followed by the recruitment of Csk to lipid rafts upon Fc{epsilon}RI aggregation. A, Tyrosine phosphorylation of Cbp/PAG. IgE-sensitized cells were stimulated with 1 µg/ml DNP-BSA for indicated times. Cbp/PAG were then immunoprecipitated and subjected to immunoblotting with antiphosphotyrosine. The same blot was stripped and reprobed with anti-Cbp/PAG. B, Recruitment of Csk to lipid rafts. The cells were sensitized with biotinylated IgE, and then unstimulated or stimulated with 0.5 µg/ml streptavidin for 30 s, and fractionated by sucrose density gradient centrifugation. Low density fractions were pooled, concentrated by CH3OH/CHCl3, and subjected to immunoblotting with anti-Csk. The same blot was reprobed with anti-Cbp/PAG. The Csk blot was scanned and the absorbance of the Csk bands was measured. Data shown are the mean ± SD of three separate experiments.

 
Cbp/PAG has been known to interact with Csk. This interaction is dependent on the phosphorylation of a specific tyrosine residue (Tyr314 of rat Cbp/PAG), which allows Csk to bind to Cbp/PAG through the SH2 domain of Csk (19). This interaction is detectable in the resting RBL-2H3 cells when Cbp/PAG is immunoprecipitated and analyzed by immunoblotting with anti-Csk (Fig. 2Go). Moreover, following Fc{epsilon}RI aggregation, the association of Cbp/PAG with Csk is enhanced. These results indicate that more Csk are recruited to lipid rafts upon Fc{epsilon}RI aggregation, which may result in the down-regulation of the receptor-mediated signaling.



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  		FIGURE 2. Association of Cbp/PAG with Csk in RBL-2H3 cells. RBL-2H3 cells that had been sensitized with anti-DNP IgE were unstimulated or stimulated with 1 µg/ml DNP-BSA for 2 min at 37°C. Cbp/PAG was then immunoprecipitated and analyzed by immunoblotting with anti-Csk. The same blot was stripped and reprobed with anti-Cbp/PAG.

 
As an attempt to analyze the role of Cbp/PAG in Fc{epsilon}RI-mediated signaling, we overexpressed a wild-type (WT) form of Cbp/PAG in RBL-2H3 cells. Two established clones, Cbp/WT-1 and Cbp/WT-2, were used for detailed analysis. The detection of Cbp/PAG by immunoblotting with anti-Cbp/PAG Abs demonstrates that the levels of expression of Cbp/PAG in Cbp/WT-1 and Cbp/WT-2 are ~6- to 7-fold higher than that in the parental RBL-2H3 cells (Fig. 3GoA). These clones and parental cells express comparable amounts of Lyn and Csk (Fig. 3GoB). In addition, FACS analysis shows that these cells also express comparable amounts of Fc{epsilon}RI and CD45 (data not shown). As compared with that in RBL-2H3 cells, the overall intensity of tyrosine phosphorylation of Cbp/PAG both at quiescence and after aggregation of Fc{epsilon}RI was increased in cells overexpressing Cbp/PAG (Fig. 4GoA). This, in turn, led to an increase in the amount of Csk relocated to the low density lipid raft fractions (Fig. 4GoB).



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  		FIGURE 3. Characterization of Cbp/PAG overexpressing cell lines. A, Expression of Cbp/PAG. RBL-2H3 cells and two Cbp/PAG overexpressing cell lines, Cbp/WT-1 and Cbp/WT-2, were analyzed by immunoblotting with anti-Cbp/PAG and anti-myc Ab. B, Immunoblot analysis of Lyn and Csk expression.

 
We tested for the ability of Cbp/PAG overexpressing cells to degranulate in response to Fc{epsilon}RI aggregation. The cells were also stimulated simultaneously with PMA and calcium ionophore A23187 to estimate their maximal ability to degranulate. Although the levels of degranulation in response to PMA and A23187 were comparable, the Fc{epsilon}RI-mediated responses of Cbp/WT-1 and Cbp/WT-2 cells were significantly reduced compared with that of the parental RBL-2H3 cells (Fig. 5GoA). We also found that Ca2+ mobilization induced by Fc{epsilon}RI aggregation was impaired in these Cbp/PAG overexpressing cells (Fig. 5GoB). Thus, these results indicate that Cbp/PAG could play a role in negatively regulating mast cell signaling through Fc{epsilon}RI.



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  		FIGURE 5. Effect of overexpression of Cbp/PAG on Fc{epsilon}RI-induced mast cell activation. A, Degranulation responses. IgE-sensitized cells were incubated with various amounts of DNP-BSA for 40 min at 37°C. The cells were also incubated with 50 nM PMA and 5 µM calcium ionophore A23187. {beta}-Hexosaminidase in the supernatant was measured as a percentage of total {beta}-hexosaminidase. Each point represents the mean ± SD of three independent experiments. B, Fc{epsilon}RI-stimulated Ca2+ mobilization. The cells were sensitized with IgE and loaded with Fura-PE3. The fluorescence of the stirred cell suspension was continuously monitored with a fluorescence spectrophotometer. The cells were stimulated with 1 µg/ml DNP-BSA at time 100 s.

 
It is likely that the overexpression of Cbp/PAG affects the early events following Fc{epsilon}RI aggregation. Immunoprecipitation of Fc{epsilon}RI following immunoblotting with antiphosphotyrosine Ab showed that the overexpression of Cbp/PAG significantly decreased the aggregation-induced tyrosine phosphorylation of both the {beta}- and {gamma}-chains of the receptors (Fig. 6GoA). This strongly suggests that Cbp/PAG is involved in the regulation of the initial tyrosine phosphorylation of Fc{epsilon}RI induced by receptor aggregation.



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  		FIGURE 6. Effect of overexpression of Cbp/PAG on early events following Fc{epsilon}RI aggregation. A, Tyrosine phosphorylation of Fc{epsilon}RI. Cells were stimulated with 1 µg/ml DNP-BSA for indicated times and Fc{epsilon}RI were then immunoprecipitated and analyzed by immunoblotting with antiphosphotyrosine mAb. B, Association of Fc{epsilon}RI with Lyn. Cells were unstimulated or stimulated with 1 µg/ml DNP-BSA for 2 min, and Fc{epsilon}RI was then immunoprecipitated and analyzed by immunoblotting with anti-Lyn. C, Fc{epsilon}RI-associated PTK activity toward exogenous substrates. Fc{epsilon}RI immunoprecipitates were prepared as above and their in vitro kinase activities were assessed with the cdc-2 peptide and [{gamma}-32P]ATP. Incorporation of radioactivity into the substrate peptides was visualized by SDS-PAGE on 16% tricine gels followed by autoradiography.

 
When we analyzed coimmunoprecipitation of Fc{epsilon}RI with Lyn, we found that the level of preassociation was similar between parental RBL-2H3 cells and two Cbp/PAG overexpressing cell lines (Fig. 6GoB). However, the receptor aggregation in Cbp/PAG overexpressing cells did not induce an increase in association of Lyn with the receptors, which was seen in parental cells. In Cbp/PAG overexpressing cells, the Lyn’s regulatory tyrosine may not be dephosphorylated due to the increasing amounts of Csk in the lipid rafts where aggregated Fc{epsilon}RI and Lyn reside, which prevents the recruitment of any additional Lyn proteins toward phosphorylated receptors.

To determine whether Cbp/PAG affects the Fc{epsilon}RI-associated PTK activity, Fc{epsilon}RI was immunoprecipitated and subjected to in vitro kinase assay. As reported previously (13), the PTK activity of receptors from the parental RBL-2H3 cells was markedly enhanced by receptor aggregation, as judged by the PTK activity toward exogenous substrates that is known to be a good substrate for the Src family kinases (Fig. 6GoC). In contrast, although the basal activity was indistinguishable, the enhanced PTK activity following Fc{epsilon}RI aggregation was hardly seen in both Cbp/PAG overexpressing cell lines. These data clearly indicate that Cbp/PAG negatively regulates the enhancement of Fc{epsilon}RI-associated PTK activity following receptor aggregation.

Upon the triggering of TCR, Cbp/PAG in T cells is rapidly dephosphorylated by unknown phosphatases, leading to the dissociation of Csk from rafts (20, 22). Shortly thereafter, Cbp/PAG is rephosphorylated, thereby recruiting Csk back into rafts, which results in the termination of TCR signaling. Thus, Cbp/PAG in T cells functions to keep resting T cells in a quiescent state. In contrast, we demonstrate that in mast cells the level of tyrosine phosphorylation of Cbp/PAG and its association with Csk increases following the aggregation of Fc{epsilon}RI. Therefore, the important role of Cbp/PAG in Fc{epsilon}RI signaling appears to suppress the excessive responses once the receptors are aggregated, rather than to repress the Lyn activity in resting cells. The tyrosine phosphorylation of Cbp/PAG is thought to be mediated by the Src family kinases (19). Indeed, both constitutive and inducible tyrosine phosphorylation of Cbp/PAG was suppressed by the treatment of cells with PP2, a selective inhibitor for Src family kinases (data not shown). Therefore, we speculate that in lipid rafts the up-regulated Fc{epsilon}RI-associated Lyn kinase upon receptor aggregation phosphorylates Cbp/PAG followed by recruitment of Csk to the rafts and suppression of the receptor-associated Lyn. Thus, in mast cells, Cbp/PAG is likely to function as a negative feedback regulator for cell activation upon aggregation of Fc{epsilon}RI.

Our results combined with a previous report (13) strongly support the notion that mast cell signaling through Fc{epsilon}RI is dynamically regulated by CD45 and Csk. Because Fc{epsilon}RI rapidly translocates into lipid rafts upon aggregation, the exclusive localization of Cbp/PAG could allow an efficient inhibition of aggregated Fc{epsilon}RI-associated Lyn activity by Csk. However, the precise mechanism by which CD45 positively regulates tyrosine phosphorylation of Fc{epsilon}RI upon receptor aggregation is unclear. Whether the functions of CD45 in Fc{epsilon}RI signaling relate to lipid rafts awaits further investigation. Nevertheless, lipid rafts are likely to play key roles in the regulation of Fc{epsilon}RI signaling.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Toshiyuki Yamashita, Division of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, HokkaidoUniversity, Kita-ku, Sapporo 060-0812, Japan. E-mail address: yamashit{at}pharm.hokudai.ac.jp Back

2 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; Cbp, Csk-binding protein; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; PTK, protein tyrosine kinase; RBL, rat basophilic leukemia; SH2, src homology 2; WT, wild type. Back

Received for publication December 3, 2001. Accepted for publication January 9, 2002.


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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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N. Kato, M. Nakanishi, and N. Hirashima
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Requirement of the tyrosines at residues 258 and 270 of MAIR-I in inhibitory effect on degranulation from basophilic leukemia RBL-2H3
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S. Odom, G. Gomez, M. Kovarova, Y. Furumoto, J. J. Ryan, H. V. Wright, C. Gonzalez-Espinosa, M. L. Hibbs, K. W. Harder, and J. Rivera
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R. M. Young, D. Holowka, and B. Baird
A Lipid Raft Environment Enhances Lyn Kinase Activity by Protecting the Active Site Tyrosine from Dephosphorylation
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