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The Journal of Immunology, 2002, 168: 541-544.
Copyright © 2002 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Negative Regulation of Immune Synapse Formation by Anchoring Lipid Raft to Cytoskeleton Through Cbp-EBP50-ERM Assembly1

Katsuhiko Itoh2,*,{dagger}, Masahiro Sakakibara2,*,{dagger}, Sho Yamasaki*, Arata Takeuchi*, Hisashi Arase3,*, Masaru Miyazaki{dagger}, Nobuyuki Nakajima{dagger}, Masato Okada§ and Takashi Saito4,*,{ddagger}

* Department of Molecular Genetics and {dagger} General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan; and {ddagger} Cell Signaling Team, RIKEN Research Center of Allergy and Immunology, and § Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Immune response is initiated by direct contact between T cells and APCs. Within their interface, a specially organized cell-cell junction is formed as an immune synapse (IS)5 (1, 2, 3). Dynamic changes in the distribution of surface molecules take place that create IS at early T cell activation. In the center of IS, TCR recognizes the Ag-MHC complex and recruits various signaling molecules crucial for T cell activation into a lipid raft. Although these signaling molecules are concentrated into a lipid raft, the relationship between the aggregation of lipid raft and IS formation is not yet fully understood. The mechanism for the dynamic regulation of IS formation, particularly the aggregation of lipid rafts, is unknown.

Cbp/PAG is a recently cloned membrane adapter molecule associating with Csk (4, 5), and Cbp is exclusively localized in a lipid raft, which resembles LAT, a membrane adapter crucial for T cell activation (6). The Cbp-Csk complex increases the Csk activity through a binding and conformational mechanism (7). It has been shown that Cbp is phosphorylated in resting T cells and that TCR stimulation induces its dephosphorylation and the dissociation of Csk (4, 5). Because Csk negatively regulates src kinases, dissociation of the Cbp-Csk assembly induces activation of src kinases. Thus, T cell activation appears to be regulated by the recruitment of Csk to phosphorylated Cbp. However, the specific function of Cbp with its exclusive localization in lipid raft is unknown.

To address this question, and also to clarify the function of raft aggregation and regulation of immune synapse formation, we tried to isolate Cbp-associating proteins. We isolated NHERF/EBP50 (EBP) as a Cbp-binding protein. Because EBP is able to bind both Cbp in raft and ezrin-radixin-moesin (ERM) which then associate with cortical cytoskeleton, the assembly of Cbp and EBP functions to anchor lipid raft to the cytoskeleton. We found that T cell activation induces dissociation of this assembly and IS formation, suggesting that the anchorage through Cbp-EBP serves as negative regulation of IS formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Yeast two-hybrid screening

Yeast two-hybrid screening was performed as previously described (8) using the cytoplasmic tail of Cbp in pAS2-1 (Clontech Laboratories, Palo Alto, CA) as bait on a BALB/c mouse spleen cDNA library in pACT2 (1 x 107).

In vitro binding assay

EBP-PDZ-1 subcloned into pCITE4b (Novagen, Madison, WI) was in vitro translated using a reticulocyte lysate system (Promega, Madison, WI) with [35S]methionine. In vitro binding assay with GST fusion proteins (GFP) was performed in a binding buffer (0.05% Triton, 50 mM HEPES, 10% glycerol, 0.1% BSA, 100 mM KCl, 2 mM MgCl2, 0.05 mM DTT) as previously described (8).

Retrovirus-mediated gene transfer

pMX-IRES-GFP was kindly provided by Dr. T. Kitamura (Tokyo University, Tokyo, Japan) and used for stable transfection of FLAG (Sigma, St. Louis, MO)-tagged wild-type (WT)- and 429A-Cbp and HA-EBP into Eco-Jurkat which express the receptor for ecotropic retrovirus (9). These constructs were transiently transfected into PHOENIX (provided by Dr. G. Nolan, Stanford University, Stanford, CA) by lipofection, and Eco-Jurkat was cultured with 48-h virus supernatant. Transfectants were stained with anti-FLAG mAb and PE-anti-mouse Ig, and FLAG+ and/or GFP+ cells were sorted by FACS.

Preparation of detergent-insoluble (raft) fraction

Jurkat cells (1 x 108) were stimulated with OKT3 for 2 min, lysed in 1% Triton-X100 lysis buffer (25 mM MES (pH 6.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, and 2 mM Na3VO4) for 10 min, and homogenized 10 times; 80%, 30% and 5% sucrose were made in the same buffer. One milliliter 80% sucrose was mixed with the lysate and overlaid with 6.5 ml 30% sucrose and then 3.5 ml 5% sucrose. After centrifugation for 18 h at 200,000 x g in Beckman SW 41Ti ultracentrifuge (Beckman Coulter, Fullerton, CA), 1-ml fractions were collected from the bottom of the gradient.

Confocal microscopy and photobleaching

For fluorescence labeling of lipid raft, Jurkat transfectants were labeled with 5 µM BODDIPY-GM1 (Molecular Probes, Eugene, OR) (10), incubated at 37°C for 20 min, and placed on a poly-L-lysine-coated dish. Photobleaching was performed at room temperature with a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany) with the 488-nm line of a krypton-argon laser as previously reported (11). The ratio of the fluorescence intensity of the photobleached region to that on the opposite side was measured using the profile data of LSM software (Carl Zeiss, Oberkochen, Germany).

Superantigen (sAg)-induced IS formation

Jurkat cells were mixed with Raji B cell line as APC together with Staphylococcus enterotoxin E (SEE) and placed on a poly-L-lysine-coated coverslip. Jurkat cells were stained for talin with anti-talin Ab, and Raji cells were stained with anti-CD19 Ab to discriminate from T cells. The IS formation was analyzed by confocal microscopy.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
We screened Cbp-binding proteins with the yeast two-hybrid system (8) using the cytoplasmic tail of murine Cbp as bait and a spleen cDNA library. To this end, we isolated NHERF/EBP as a specific binding molecule to Cbp, which is also known as Na+-H+ exchanger-regulatory factor (NHE-RF). EBP consists of 429 aa and contains two PDZ domains and a C-terminal domain, which binds to the N terminus of ERM (12). EBP is expressed in spleen (12) and also in T cells (not shown). EBP also interacts through the first PDZ (PDZ-1) with the C terminus of several receptors such as {beta}2-adrenergic receptor (13) and cystic fibrosis transmembrane conductance regulator (CFTR) (14). The isolated clone contained only PDZ-1. Because we found that Cbp contains the consensus motif VTRL at the C terminus capable of binding to PDZ (15), two mutant Cbp constructs, a deletion mutant of the C-terminal four amino acids, {Delta}C, and a single substitution mutant, 429A, were prepared. The specific association between Cbp and EBP in yeast (data not shown) was confirmed by in vitro binding and in vivo transfection. EBP-PDZ-1 was translated in vitro, and 35S-labeled PDZ-1 probe was assessed for in vitro binding with GST fused to WT, {Delta}C, and 429A of Cbp. GST-WT specifically bound to EBP-PDZ-1, but 429A and {Delta}C of Cbp did not (Fig. 1GoA). The binding was observed with the PDZ-1 but not the PDZ-2 domain of EBP (data not shown). The in vivo association between Cbp and EBP was confirmed by transfection of FLAG-tagged WT and 429A-Cbp with HA-EBP into 293T cells (Fig. 1GoB). Only WT-Cbp, not 429A or {Delta}C (not shown), associated with EBP.



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  		FIGURE 1. Specific association of Cbp with the PDZ-1 domain of EBP. A, In vitro binding assay. GFPs of WT, {Delta}C (DC), and 429A of Cbp and GST alone were prepared, and the aliquots used were analyzed by staining with CBB (lanes 1–4). 35S-Labeled EBP50-PDZ-1 (lanes 10–14) and luciferase as a control (lanes 5–9) were in vitro translated and interacted with these GFPs. One one-hundredth of the product was applied (lanes 5 and 10). B, Cbp-EBP association in transfected COS cells. HA-EBP was cotransfected with FLAG-WT or 429A of Cbp. Cell lysates in a digitonin buffer were immunoprecipitated (I.P.) and/or blotted (I.B.) as indicated. {blacktriangleleft}, Cbp; {triangleleft}, EBP. Note that EBP has two bands and that the high molecular mass band was a phosphorylated form. The smaller EBP appeared with a nonspecific band. {alpha}, Anti.

 
To analyze the function of the Cbp-EBP association, the localization and phosphorylation of Cbp mutants were analyzed. Both FLAG-WT and 429A were stably transfected with HA-EBP into Jurkat cells, and similar amounts of WT and 429A-Cbp were expressed (Fig. 2GoA). Cbp was highly phosphorylated without stimulation but dramatically dephosphorylated on stimulation with TCR cross-linking (5). This regulation was similarly observed in 429A-Jurkat in whole cell lysates (Fig. 2GoA, top) as well as by immunoprecipitation (Fig. 2GoA, bottom). WT- but not 429A-Cbp bound to EBP. We found that the WT-Cbp-EBP association was severely diminished on stimulation. Analyzing raft fractions separated by sucrose gradient, Cbp was localized predominantly in the raft as indicated by cholera toxin staining for GM1 (Fig. 2GoB). The majority of Cbp in the lipid raft was phosphorylated, but it was dramatically dephosphorylated after TCR stimulation (Fig. 2GoB, top). The EBP-Cbp association was only detected in unstimulated cells while EBP was not detected in raft fractions upon stimulation (Fig. 2GoB, bottom).



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  		FIGURE 2. Assembly of Cbp-EBP-ERM and its dissociation upon TCR stimulation. A, TCR stimulation induces dephosphorylation of Cbp and dissociation of EBP from Cbp. HA-EBP was transfected into Eco-Jurkat with FLAG-WT or 429A. Cells were left unstimulated (unstim., -) or stimulated (stim., +) for 2 min by cross-linking with anti ({alpha})-CD3 mAb (OKT3). Total cell lysate was blotted with anti-PY mAb (4G10) (top). For the lower three panels, lysates were immunoprecipitated (I.P.) and/or blotted (I.B.) as indicated. {blacktriangleleft}, position of Cbp; {triangleleft}, position of EBP. B, Dephosphorylation of Cbp and dissociation from EBP in lipid raft. Stimulated or unstimulated Jurkat cells (1 x 108) were lysed in 1% Triton X-100 buffer and fractionated by sucrose gradient separation. Upper panels, Equal amounts of total protein of each fraction (1 2 3 4 5 6 7 8 9 10 11 12 ) were immunoblotted with anti-cholera toxin B Ab (CTX), anti-Cbp Ab, and anti-PY mAb (4G10). Lower panels, The same fraction as in A was immunoprecipitated with anti-HA mAb. C, Association of Cpb and ERM in lipid raft and its dissociation upon TCR stimulation. After subcellular fraction of the lysates from stimulated (+) or unstimulated (-) Jurkat as in B, detergent-insoluble fraction (fraction 4–6, insoluble) and soluble fraction (fraction 10–12, soluble) were pooled. Total lysates (upper two panels) or immunoprecipitants with anti-moesin Ab (lower panel) were blotted with anti-Cbp Ab or anti-moesin Ab.

 
Because the C terminus of EBP assembles with ERM (16, 17, 18, 19) and moesin is the dominant ERM in T cells, the formation of Cbp-EBP-moesin complex in lipid raft was examined. Although the majority of moesin was located in detergent-soluble fraction, a minor fraction was detected in raft fractions, and the moesin-Cbp complex was observed only in detergent-insoluble fractions (Fig. 2GoC). This association disappeared after TCR stimulation. Thus, Cbp is connected to ERM though EBP binding, and the Cbp-EBP-ERM complex was disrupted upon TCR stimulation.

Constitutive formation of the Cbp-EBP-ERM complex may affect the mobility of lipid raft on the cell surface membrane. To test this hypothesis, fluorescence-labeled GM1 was incorporated into the lipid raft on the T cell membrane (10), and a part of the membrane was subjected to photobleaching (11). The fluorescence recovered within 2 min after bleaching in Jurkat cells (Fig. 3GoA). We quantitated the recovery kinetics. Whereas Jurkat cells expressing parental and 294A-Cbp showed similar kinetics of recovery, the recovery of WT-Cbp was significantly delayed (Fig. 3GoB), suggesting that Cbp reduces the mobility of surface GM1, presumably lipid raft.



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  		FIGURE 3. Cbp-EBP assembly reduces mobility of lipid raft and inhibits IS formation. A, Photobleaching of GM1-BODIPY-labeled raft on Jurkat cell membrane. The boxed area on the GM1-BODIPY-labeled Jurkat membrane (10 ) was photobleached (30 bleaches), and the recovery of fluorescence was measured (11 ). Bars, 5 µm. B, Quantitation of fluorescence recovery from photobleached Jurkat transfectants of WT and 429A-Cbp. The ratio of the fluorescence intensity at the center of the bleached area to that of the opposite side was calculated. All fluorescence was recovered after 2 min. C, sAg-induced IS formation in Jurkat cells. Jurkat cells were cocultured with Raji cells in the presence or absence of SEE on a coverslip coated with poly-L-lysine, fixed, and stained with anti-talin Ab (green) and anti-CD19 Ab (red) for Raji cells. D, IS formation by Jurkat cells expressing WT or 429A-Cbp. Transfectants were treated similarly to the method in C. E, Quantitation of sAg-induced IS formation in Jurkat cells expressing WT and 429A-Cbp. The number of cells exhibiting IS in the interface of Jurkat and Raji cells was counted, and the percentage of IS-forming cells among all remaining Jurkat cells was calculated. One hundred samples per each transfectant were analyzed.

 
The formation of IS appears to be initiated by dynamic reorganization of membrane proteins and aggregation of lipid rafts. Together with our observation that lipid raft containing phosphorylated Cbp anchors to the cytoskeleton through binding to EBP-ERM, we postulated that Cbp may regulate IS formation. To analyze Ag-induced IS formation using Jurkat cells, we developed a system of sAg-induced IS formation. When Jurkat cells were mixed with Raji cells as APC in the presence of SEE, Jurkat cells formed IS (Fig. 3GoC). However, there was no IS formation in the absence of either Raji cells or SEE, indicating specific IS formation in this system. WT-Cbp-expressing Jurkat cells failed to form IS, whereas 429A-Cbp showed IS formation (Fig. 3GoD). Quantitation of the percentages of IS-forming cells supported the same conclusion, namely, that Cbp inhibits but 429A mutant restores IS formation (Fig. 3GoE). This is consistent with the concept that the Cbp-EBP-ERM assembly inhibits dynamic arrangement for IS formation.

Finally, we analyzed IL-2 production of these Jurkat transfectants as functional consequence. IL-2 production by WT-Cbp transfected Jurkat cells was inhibited, but Cbp-429A significantly restored the response (Fig. 4Go).



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  		FIGURE 4. Association of Cbp with EBP inhibits T cell activation. Jurkat cells (105) transfected with a vector (mock), WT, and 429A-Cbp was stimulated with immobilized anti-TCR mAb (C305) (left, at 1/300 dilution {blacksquare} and at 1/1000 dilution {square}) or PMA plus ionophore for 24 h. IL-2 concentration in the supernatant of triplicate cultures was determined by ELISA. The amounts of IL-2 produced during TCR stimulation were normalized by those with PMA-ionophore stimulation (PMA-ionophore stimulation of WT was used as 1.0 standard). The responses after PMA-ionophore stimulation were 2798 ± 98, 3610 ± 150, and 2793 ± 98 (nanograms per milliliter) for mock, WT, and 429A, respectively. Statistic analysis indicates that all differences among mock-WT, mock-429A, and WT-429A were significant (p < 0.01).

 
The association of EBP with several surface receptors such as {beta}-adrenergic receptor and CFTR is mediated through PDZ-1, whereas that with ERM is through the C-terminal domain (16). EBP was also identified as NHE-RF, a regulatory cofactor for the Na+-H+ exchanger (NH3) (20) that associates through PDZ-2. Ligand binding to {beta}-adrenergic receptor induces the association of the receptor with EBP, and the complex regulates Na+-H+ exchange (15). Upon CFTR activation, CFTR assembles with EBP and then associates with ERM (14, 18).

In contrast to these systems, our results demonstrated that the Cbp-EBP association appears to function in a reverse manner in T cells. Cbp is constitutively associated with EBP and then ERM, and the association is disrupted by TCR stimulation. Therefore, the association of Cbp-, EBP-, and ERM-containing cortical cytoskeleton in T cells has the special function of inhibiting T cell activation by anchoring lipid rafts to the cytoskeleton. Because Cbp is localized exclusively in lipid rafts regardless of its phosphorylation status, the activation-induced dissociation of EBP from Cbp may result in the liberation of lipid rafts from being anchored to the ERM-cytoskeleton and their subsequent susceptibility to IS formation. T cell activation induces dephosphorylation of Cbp including Y314, the Csk-binding site, dissociates Csk, and results in activation of src family kinases (4). T cell activation is induced by two mechanisms regarding Cbp and lipid raft; one by uncoupling of the assembly of Cbp with EBP-ERM, which allows lipid rafts to aggregate and form IS, and the other by dissociation of Csk as previously suggested (4, 5).

IS formation appears to require active reorganization of the cytoskeleton. TCR stimulation induces phosphorylation of LAT and SLP-76, which then associate with crucial molecules for cytoskeleton reorganization such as N-WASP and Arp2/3 (21) and may induce aggregation of lipid rafts. In contrast, Cbp recruits negative regulatory molecules such as Csk and EBP. The cooperative regulation by LAT and Cbp for lipid raft and assembly with cytoskeleton will have to be clarified to understand the dynamic regulation of T cell activation.


   Acknowledgments
 
We thank S. Taki for discussion, M. Sakuma and R. Shiina for technical help, and H. Yamaguchi and Y. Kurihara for secretarial assistance.


   Footnotes
 
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. Back

2 K.I. and M.S. contributed equally to this work. Back

3 Current address: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143. Back

4 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: saito{at}med.m.chiba-u.ac.jp Back

5 Abbreviations used in this paper: IS, immune synapse; ERM, ezrin-radixin-moesin; EBP, ERM-binding phosphoprotein of 50 kDa; sAg, superantigen; SEE, Staphylococcus enterotoxin E; WT, wild-type; GFP, GST fusion protein; CFTR, cystic fibrosis transmembrane conductance regulator; NHE-RF, Na+-H+ exchanger-regulatory factor. Back

Received for publication October 16, 2001. Accepted for publication November 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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