Translocation of tyrosine-phosphorylated TCR{zeta} chain to glycolipid-enriched membrane domains upon T cell activation

Atsushi Kosugi, Shin-ichiroh Saitoh1, Satoshi Noda2, Koubun Yasuda1, Fumie Hayashi, Masato Ogata1 and Toshiyuki Hamaoka1

School of Allied Health Sciences, Faculty of Medicine Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan
1 Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1141, Japan.

Correspondence to: : A. Kosugi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Recent studies point to glycolipid-enriched membrane (GEM) microdomains as the critical sites for TCR-mediated signal transduction. However, whether the TCR complex is localized in the GEM domain is not well-defined. In the present study, we analyzed localization of the TCR–CD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCR{zeta} chains was localized in the GEM fraction, most of the TCR complexes were excluded from the GEM before and after T cell activation, and the amount of TCR{zeta} in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCR{zeta} was strongly concentrated in the GEM fraction upon TCR engagement. A kinetic study revealed that tyrosine phosphorylation of TCR{zeta} occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCR{zeta} in the GEM. Thus, these results indicate that phosphorylated TCR{zeta} migrates into the GEM domains on T cell activation. We speculate that the GEM microdomains may function as a reservoir of activation signals from triggered TCR.

Keywords: glycolipid-enriched membrane, microdomain, phosphorylation, TCR, TCR{zeta} chain


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR is expressed on surface of T cells and functions as an apparatus for antigen recognition. The TCR has a multisubunit structure composed of the polymorphic {alpha}ß heterodimer and the invariant CD3 {gamma}, {delta}, {varepsilon}, and TCR{zeta} chains. Engagement of the TCR leads to a rapid rise in intracellular protein tyrosine phosphorylation, followed by a series of other biochemical events, eventually resulting in gene expression and effector function (1,2). The information as to what proteins are involved and what molecular interactions between these proteins exists in the TCR signaling pathway has been growing rapidly for several years. However, a mechanism responsible for the initial interaction between activated TCR and signal-transducing molecules is not clearly understood. In particular, it is unknown whether TCR signaling initiates in a specialized signaling compartment in the plasma membrane and how signal-transducing molecules are assembled to this compartment, if any.

We have been studying a role of thymic shared antigen (TSA)-1 in T cells and demonstrated that it regulates TCR-mediated signal transduction (3,4). TSA-1 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on lymphoid and non-lymphoid cells. We have further analyzed a mechanism of the regulation by TSA-1 on TCR signaling, and recently found a physical association between TSA-1 and the TCR complex (5). Since GPI-anchored proteins are known to be enriched in a glycolipid-enriched membrane (GEM) domain or a detergent-insoluble raft (68), we anticipated based on our findings that both TSA-1 and the TCR are co-localized and interact in the GEM domain. Moreover, because the GEM domain is known to represent a signaling compartment at the cell surface, it may have a role in TCR signaling.

Recently, Xavier et al. have reported the observation verifying our prospect (9). They demonstrated that TCR{zeta} chains are localized in detergent-resistant membrane rafts, and phosphorylated TCR{zeta} chains together with signal-transducing molecules including Lck, ZAP-70, Vav and phospholipase C-{gamma}1 are enriched in the rafts after T cell activation. In addition, Zhang et al. demonstrated that LAT, a critical linker molecule in TCR signaling, partitions into the GEM domain by its lipid modification (10). Thus, these studies suggest that the GEM domain and the proteins targeted to it are involved in TCR signaling. However, none of their data has documented localization of the TCR complex itself in the GEM domain before or after T cell activation.

In the present study, we analyzed the fate of the TCR complex upon TCR engagement in terms of its localization in the GEM domain. Our results demonstrate that, although the TCR complex is excluded from the GEM domain, phosphorylated TCR{zeta} is accumulated into the GEM domain after T cell activation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines and hybridomas
2B4 is a murine T cell hybridoma that is specific for pigeon cytochrome c plus I-Ek (11). LK35.2 is a B cell hybridoma and used as accessory cells (12). Cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO2.

mAb and reagents
The following mAb were used: 145-2C11 (13) and HMT3-1 (14), anti-CD3{varepsilon}; H146-968 (15), anti-TCR{zeta}; H28-710 (16), anti-TCR{alpha}; D7 (17), anti-Ly-6A/E; and MOL 171 (18), anti-human Lck. Normal hamster IgG was purchased from Cappel (Durham, NC). Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY).

Isolation of a GEM fraction in equilibrium density gradients
GEM fractions were prepared as described by Rodgers and Rose with minor modifications (19). Briefly, 1x108 cells were washed with PBS containing 1 mM sodium orthovanadate and 5 mM EDTA, and lysed with 1 ml MES-buffered saline (MBS; 25 mM MES, pH 6.5/150 mM NaCl) containing 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 1 mM sodium orthovanadate and 5 mM EDTA. The lysate was homogenized with 20 strokes of a Dounce homogenizer, gently mixed with an equal volume of 80% sucrose (w/v) in MBS and placed in the bottom of a 14x95 mm clear centrifuge tube (344060; Beckman, Palo Alto, CA). The sample was then overlaid with 6.5 ml of 30% sucrose and 3.5 ml of 5% sucrose in MBS, and centrifuged at 200,000 g in a Beckman SW40Ti rotor at 4°C for 16 h. Following centrifugation, 12 fractions of 1 ml (excluding the pellet) were collected from the top of the gradient.

Immunoprecipitation and immunoblotting analysis
A half of each gradient fraction was diluted with 10% digitonin in MBS at a final concentration of digitonin to 1%, precleared with Protein A–Sepharose (Pharmacia, Piscataway, NJ) and immunoprecipitated with anti-TCR{zeta} mAb (H146–968) prebound to Protein A–Sepharose. Immunoprecipitates were washed with washing buffer (0.1% digitonin in MBS) and eluted. To examine the presence of cell surface and intracellular proteins in the density gradient fractions, 16 µl of each fraction was solubilized in 5xsample buffer and electrophoresed. Immunoblotting analysis and detection of tyrosine phosphorylation of TCR{zeta} were performed as previously described (5).

Cell stimulation
2B4 cells (1x108) were stimulated with 2C11 (10 µg/ml) in the presence of LK cells (5x107) for 45 min at 37°C. In some experiments, 2B4 cells were stimulated for various time periods. Stimulated cells were harvested and the lysates were subjected to equilibrium gradient centrifugation.

Transfection and stimulation of COS-7 cells
A total of 1x107 COS-7 cells were washed with HEPES-buffered saline and resuspended in 1 ml ice-cold HEPES-buffered saline. A total of 25 µg of plasmid DNA (10 µg of TCR{zeta}, 10 µg of Lck and 5 µg of Syk) was added to the cell suspension in a cuvette (Gene Pulser Cuvette; BioRad, Richmond, CA) and the electric pulse (250 V, 960 µF) was applied by a Gene Pulser (BioRad). After 2 days culture in DMEM supplemented with 10% FCS, transfected COS-7 cells were stimulated with pervanadate at a concentration of 30 µM for 5 min. Unstimulated and stimulated cells were harvested, and the lysates were subjected to equilibrium gradient centrifugation.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Localization of TCR{zeta} chains without other TCR subunits in a GEM fraction in 2B4 T cell hybridomas
To isolate a GEM fraction in the plasma membrane of T cells, the Triton X-100 lysate of 2B4 T cell hybridomas was subjected to equilibrium density gradient centrifugation in a sucrose density gradient. It has been shown that the GEM fraction localizes at the interface between the top (5%) and middle (30%) sucrose layers (19), corresponding to fractions 4 and 5 in our density gradient fraction. The Triton X-100-soluble material remained at the bottom of the gradient in fractions 11 and 12. The detergent-resistant membranes are known to be enriched for glycolipids (68). We confirmed the enrichment of the glycosphingolipids in fractions 4 and 5 by reactivity with cholera toxin (20), which recognizes ganglioside GM1 (Fig. 1AGo). We also demonstrated that almost all of the GPI-anchored Ly6A/E (Fig. 1BGo) and >30% of Lck (Fig. 1CGo) was recovered from the GEM fraction, which is consistent with the previous reports by others (21). To investigate whether the TCR is present in the same membrane compartment as GPI-anchored proteins and Lck, localization of TCR{alpha}ß heterodimers, CD3{varepsilon} chains and TCR{zeta} homodimers in the density gradient fraction was examined by immunoblotting analysis using specific antibodies. Interestingly, although a significant amount of TCR{zeta} homodimers was in the GEM fraction, almost all of the TCR{alpha}ß and CD3{varepsilon} was in the Triton X-100-soluble fractions. In three independent experiments, ~10% of TCR{zeta} homodimers was associated with the GEM fraction, whereas the amounts of TCR{alpha}ß and CD3{varepsilon} in the GEM were <1% (data not shown). Thus, whereas most TCR complexes are excluded from the GEM fraction, 10% of TCR{zeta} disassembled from the TCR complex is localized in the glycolipid-enriched membrane domains. An identical profile was obtained for the distribution of each TCR chain from freshly isolated thymocytes and splenic T cells (data not shown).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1. Isolation of GEM domains from 2B4 cells. 2B4 cells (1x108) were lysed with MBS containing 1% Triton X-100 and the lysates were subjected to equilibrium gradient centrifugation as described in Methods. Twelve 1 ml fractions were collected from the top, and 16 µl of each fraction was electrophoresed under non-reducing conditions and immunoblotted with horseradish peroxidase-conjugated cholera toxin or mAb against each of the proteins as indicated. Fraction 1 represents the top of the gradient. Fractions 4 and 5 correspond to the 5/30% sucrose interface. Molecular sizes (kDa) are shown on the left.

 
Localization of phosphorylated TCR{zeta} chains in the GEM fraction upon T cell activation
It has been suggested recently that detergent-resistant membrane microdomains play important roles in the TCR-signaling pathway (9,10). Although most TCR complexes do not reside in the GEM fraction at the resting stage, they could move into the GEM after activation. To address this possibility, 2B4 cells were stimulated with 2C11 and subjected to fractionation and analysis. As shown in Fig. 2Go, no accumulation of TCR{alpha}ß and CD3{varepsilon} was observed and the amount of TCR{zeta} detected with anti-TCR{zeta} antibody was not changed in the GEM fraction following activation. However, when we analyzed tyrosine phosphorylation of TCR{zeta} chains following activation, we observed that the tyrosine-phosphorylated form of TCR{zeta} was strongly concentrated in the GEM fraction (Fig. 3Go). Although a small amount of phosphorylated TCR{zeta} was detectable in the Triton X-100-soluble membrane fraction, most of the phosphorylated TCR{zeta} was localized in the GEM fraction after activation (Fig. 3AGo, top, right). About 80% of total phosphorylated TCR{zeta} was associated with the GEM fraction in this experiment. In contrast, the amount of TCR{zeta} in the GEM fraction detected with anti-TCR{zeta} antibody remained constant before and after activation (Fig. 3AGo, bottom). A tyrosine-phosphorylated 16 kDa TCR{zeta}, which was reported previously as the one associated with cytoskeleton (22), was detected in the GEM fraction before activation. However, the appearance of this tyrosine-phosphorylated 16 kDa TCR{zeta} was not consistent. Figure 3Go(B) shows the average of three experiments where phosphorylated 23 kDa TCR{zeta} in the GEM and the Triton X-100-soluble fractions after activation was measured. The phosphotyrosine content of the TCR{zeta} in each sample is represented by the ratio of the phosphotyrosine signal divided by the TCR{zeta} signal. On average, the GEM-associated CD3{zeta} had a 16-fold greater phosphotyrosine content compared with that of the Triton X-100-soluble TCR{zeta} after T cell activation. Since TCR{zeta} phosphorylation is considered to occur within the TCR complex after TCR cross-linking (1,2), these data may suggest that phosphorylated TCR{zeta} is dissociated from the TCR complex and migrates into the GEM domains on T cell activation. In the GEM domains, phosphorylated TCR{zeta} co-localizes with many signal-transducing molecules involved in TCR signaling as reported recently (9,10). We have observed the relative abundance of the active-converted 60 kDa form of Lck in the GEM fraction following activation (data not shown).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2. Distribution of each TCR subunit in the GEM or the Triton X-100-soluble fractions is not changed after TCR cross-linking. 2B4 cells (1x108) were stimulated either with normal hamster IgG (5 µg/ml) or with 2C11 (10 µg/ml) in the presence of LK cells (5x107) for 45 min at 37°C. Normal hamster IgG-stimulated or 2C11-stimulated 2B4 cells were lysed and the lysates were subjected to equilibrium gradient centrifugation. Each 10 µl from fractions 4 and 5 (the GEM fractions; GEM) or that from fractions 11 and 12 (the Triton X-100-soluble fractions; TXS) was combined, electrophoresed, and tested for their protein levels of TCR{alpha}, CD3{varepsilon} and TCR{zeta} by immunoblotting analysis.

 



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 3. Phosphorylated TCR{zeta} chains are highly enriched in the GEM fraction after TCR cross-linking. (A) 2B4 cells (1x108) were stimulated with 2C11, lysed and the lysates were subjected to equilibrium gradient centrifugation as described. Half of each gradient fraction was immunoprecipitated with anti-TCR{zeta} mAb, H146-968. Eighty percent of each immunoprecipitate was electrophoresed under reducing conditions and blotted with an anti-phosphotyrosine mAb, 4G10 (upper panels), whereas 20% was electrophoresed under non-reducing conditions and blotted with an anti-TCR{zeta} mAb (lower panels). Total cell lysates (8x106 cell equivalent for anti-phosphotyrosine blot and 2x106 cell equivalent for anti-TCR{zeta} blot) before and after TCR cross-linking are shown in left. A closed arrowhead indicates a phosphorylated 23 kDa TCR{zeta}, whereas an open arrowhead indicates a phosphorylated 16 kDa TCR{zeta}. The bands migrating >30 kDa in total cell lysates and in lane 11 and 12 of the density gradient fractions (upper panels) are likely to be the light chains of hamster IgG used for stimulation. An arrow indicates TCR{zeta} dimers. Molecular sizes (kDa) are shown on the left. (B) The graph shows the average of three experiments where the phosphotyrosine content of 23 kDa TCR{zeta} in the GEM fractions (4 plus 5) and in the Triton X-100-soluble fractions (11 plus 12) after TCR cross-linking was measured. Each of the experiments was done as described in (A). The phosphotyrosine content of the TCR{zeta} in each sample is represented by the ratio of the phosphotyrosine signal divided by the TCR{zeta} signal.

 
Distribution of phosphorylated TCR{zeta} in COS-7 cells
Although the data presented above may suggest the physical dissociation of phosphorylated TCR{zeta} from the TCR complex upon T cell activation, it is possible that a small fraction of TCR{alpha}ß and CD3{varepsilon} is localized in the GEM domains, and phosphorylated TCR{zeta} in the GEM is associated with TCR{alpha}ß and CD3{varepsilon} to form the TCR complex. To address this possibility, we next examined whether phosphorylated TCR{zeta} can be localized in the GEM in the absence of other TCR chains. COS-7 cells were transfected with TCR{zeta} cDNA together with Lck and Syk cDNAs, stimulated with pervanadate, and subjected to fractionation and analysis. It is known that tyrosine phosphorylation of TCR{zeta} chains can be induced by cooperative enzymatic activities of Lck and ZAP-70 /Syk kinases (1,2). As shown in Fig. 4Go, a considerable amount of phosphorylated TCR{zeta} was detectable in the GEM fraction when transfected COS-7 cells were stimulated. The ratio of phosphorylated TCR{zeta} in the GEM and Triton X-100-soluble fractions is not so impressive as that observed in Fig. 3Go(A). However, whereas phosphorylated TCR{zeta} in the Triton X-100-soluble fractions mostly migrated between 16 and 23 kDa, the size of phosphorylated TCR{zeta} in the GEM was predominantly 23 kDa. This result seems to be very intriguing since phosphorylated 23 kDa TCR{zeta} represents the one that is phosphorylated at all six tyrosine residues and is induced following full T cell activation (23). Thus, the result demonstrated that completely phosphorylated TCR{zeta} can be effectively localized in the GEM domains in the absence of other TCR chains.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. Distribution of phosphorylated TCR{zeta} chains in transfected COS-7 cells. COS-7 cells were transfected with DNA encoding TCR{zeta}, Lck and Syk as described in Methods. At 48 h after transfection, the cells were stimulated with pervanadate for 5 min (right panels) or left untreated (left panels). The cells were lysed, the lysates were subjected to equilibrium gradient centrifugation and each gradient fraction was immunoprecipitated with anti-TCR{zeta} mAb as described. Eighty percent of each immunoprecipitate was blotted with an anti-phosphotyrosine mAb, 4G10 (upper panels), whereas 20% was blotted with an anti-TCR{zeta} mAb (lower panels). The amount of unphosphorylated TCR{zeta} in the GEM fraction before stimulation is 4.7%, whereas that after stimulation is 2.5%. A closed arrowhead indicates a phosphorylated 23 kDa TCR{zeta}, whereas open arrowheads indicate incompletely phosphorylated TCR{zeta} species. Molecular sizes (kDa) are shown on the left.

 
Kinetic changes of phosphorylated TCR{zeta} appearance in the GEM fraction upon T cell activation
In order to demonstrate the movement of phosphorylated TCR{zeta} into the GEM domains, we investigated kinetics of TCR{zeta} phosphorylation in each membrane fraction of 2B4 T cell hybridomas after TCR engagement. As shown in Fig. 5Go, phosphorylated TCR{zeta} first appeared in the Triton X-100-soluble fractions 5 min after TCR cross-linking. At this time, no phosphorylated TCR{zeta} was observed in the GEM fraction. However, a strong phosphotyrosine signal of TCR{zeta} was detected in the GEM fraction 20 min after TCR cross-linking, whereas that in the Triton X-100-soluble fractions was not increased. Thus, this result supports the idea that activated form of TCR{zeta} is transferred into the GEM domains during T cell activation.



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 5. Kinetics of phosphorylated TCR{zeta} appearance in the GEM fraction. 2B4 cells (1x108) were stimulated with 2C11 at the indicated time, lysed and the lysates were subjected to equilibrium gradient centrifugation. Each gradient fraction was immunoprecipitated with anti-TCR{zeta} mAb, electrophoresed under reducing conditions and blotted with an anti-phosphotyrosine mAb. As a negative control, 2B4 cells were stimulated with normal hamster IgG for 20 min (top). Phosphorylated TCR{zeta} chains are indicated by arrowheads. Molecular sizes (kDa) are shown on the left.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The plasma membrane of many cell types is known to contain specific microdomains enriched in glycosphingolipids, sphingomyelin and cholesterol but poor in phospholipids (6). These GEM domains are relatively resistant to solubilization with non-ionic detergent such as Triton X-100 and have been given many different names (7). With regard to proteins in the GEM domains, GPI-anchored proteins and signal-transducing proteins, such as GTP-binding proteins, small G proteins, and non-receptor-type tyrosine kinases, have been shown to localize to these membrane domains (68). Moreover, some transmembrane proteins such as epidermal growth factor receptors and CD44 are also enriched in the GEM (24,25).

Although it has been proposed that the GEM domains could represent a specialized signaling compartment at the cell surface (68), little is known about a role of these microdomains in hematopoietic cells. In a rat mast cell line, a fraction of Fc{varepsilon}RI, the high-affinity IgE receptor, is associated with detergent-resistant membrane domains when the receptors are cross-linked with IgE plus antigen (26,27). Tyrosine phosphorylation is detected only for the receptors that associate with these domains, suggesting that specialized membrane domains are involved in Fc{varepsilon}RI-mediated signal transduction. Recently, several groups have documented the importance of the GEM domains in TCR signaling (9,10,28). Upon TCR engagement, the activated form of Lck and tyrosine-phosphorylated Vav, phospholipase C-{gamma}1, LAT and TCR{zeta} are shown to be enriched in the GEM domains. Moreover, disruption of the GEM domain structure with agents that deplete membrane cholesterol content impairs early TCR signaling events (9). The mechanism responsible for localization of these signal-transducing molecules to the GEM domain is largely unknown. However, palmitoylation seems to be essential for LAT and Lck targeting to membrane microdomains (10,21). When an Lck-negative Jurkat cell line is reconstituted by Lck that have a mutation of N-terminal palmitoylation sites, the cell line shows a defect in late TCR signaling events (29). This study also points to the GEM domain as the critical site for TCR signaling. However, none of these studies address whether the TCR complex itself is localized to the GEM domain before or after TCR engagement. If the TCR complex is not present in the GEM domain even after TCR engagement, it becomes very important to elucidate how signals from triggered TCR are transmitted and accumulated to the membrane microdomain where many signaling molecules co-localize and interact each other.

The data presented here demonstrated that the TCR complex is excluded from the GEM domain before or after TCR cross-linking. The TCR{alpha}ß and CD3{varepsilon} in the GEM were basically undetectable by densitometric scanning (Figs 1 and 2GoGo). Only 10% of TCR{zeta} was associated with the GEM domain and no redistribution of TCR{zeta} reactive with anti-TCR{zeta} antibody was observed after activation (Fig. 2Go). The amount of TCR{zeta} associated with the GEM seems to be much smaller than that observed by Xavier et al. (9). However, because only 3% of total cellular proteins is present in the GEM domain (data not shown), they could have overestimated the amount of TCR{zeta} in the GEM by comparing equal amounts of protein from the Triton X-100-soluble and the GEM fractions. Upon TCR engagement, most of the phosphorylated TCR{zeta} is localized to the GEM domain (Fig. 3Go). A kinetic study revealed that tyrosine phosphorylation of TCR{zeta} occurs initially in the Triton X-100-soluble fractions followed by the accumulation of phosphorylated TCR{zeta} in the GEM fraction (Fig. 5Go). These findings indicate that phosphorylated TCR{zeta} could be dissociated from the TCR complex and redistributed to the GEM domain on T cell activation. The mechanism underlying the translocation of phosphorylated TCR{zeta} is thus far unknown. However, it is possible that phosphorylated TCR{zeta} may bring TCR-associated ZAP-70 to the GEM domain where this kinase phosphorylates LAT in cooperation with the GEM-associated Lck. Studies are in progress to elucidate the mechanism for the translocation of phosphorylated TCR{zeta} in an experiment in which membrane localization of various TCR{zeta} mutants is analyzed upon activation using COS-7 cells.

With regard to the TCR complex in the GEM domain, Montixi et al. recently published a paper showing that the TCR complex including TCR{alpha}ß, CD3{varepsilon} and phosphorylated TCR{zeta} is recruited to low-density detergent-insoluble membrane domains upon TCR stimulation (30). The discrepancy between their data and our lack of detection of TCR{alpha}ß and CD3{varepsilon} is likely due to different methods for separation of the glycolipid-enriched fraction or to different cells used for analysis. They used mouse thymocytes as a model, whereas T cell hybridomas were analyzed in this study. However, they also observed that only a small fraction of TCR{alpha}ß and CD3{varepsilon} is localized in the GEM in contrast to the high content of phosphorylated TCR{zeta} following TCR engagement. We believe that these data support the physical dissociation of phosphorylated TCR{zeta} from the TCR complex.

Since TCR{zeta} in the GEM of transfected COS-7 cells can be efficiently phosphorylated without other TCR chains (Fig. 4Go), it is possible that TCR{zeta} present within the GEM domain before stimulation is the major TCR{zeta} subset that becomes phosphorylated following activation. However, there is a report suggesting that TCR{zeta} can be phosphorylated outside the GEM after activation (29). It has been shown by several investigators that Lck is localized in the GEM domain by virtue of palmitoylation at N-terminal cysteine residues (31,32). In the aforementioned Lck-negative Jurkat cell line that was reconstituted with Lck with mutation of N-terminal palmitoyllation sites, Lck mutant protein was not localized in the GEM domain, resulting in a defect in the late activation event of TCR signaling. Nonetheless, TCR{zeta} phosphorylation was clearly observed in this Lck mutant cells after activation. Since Lck is known to be indispensable for TCR{zeta} phosphorylation (1,2), TCR{zeta} already present within the GEM before stimulation could not be phosphorylated in this Lck mutant cells because of the absence of Lck in the GEM. Thus, TCR{zeta} phosphorylation could be occur outside the GEM in this mutant. Based on this study in addition to our kinetic study (Fig. 5Go), we think that TCR{zeta} is phosphorylated outside the GEM and translocated to the GEM during activation.

It is now becoming clear that T cell activation can be induced by serial triggering of many TCR by a few peptide–MHC complexes (33). Viola et al. demonstrated that T cells `count' the number of TCR engaged by the peptide–MHC complex and become activated when that number exceeds a certain threshold (34). However, the mechanisms that account for the `counting' ability in T cells is not yet defined. Although it has been shown that the TCR complex is internalized and eventually degraded after antigenic stimulation (35), it is totally unknown how and where T cells store the activation signal induced by the single TCR–peptide–MHC interaction. A fascinating hypothesis is that the GEM microdomains may function as a reservoir of activation signals. Investigating a role of these membrane microdomains may provide a clue toward resolving the mechanism for the initiation of TCR signaling.


    Acknowledgments
 
The authors are grateful to Drs Ralph T. Kubo and Yasuhiro Koga for providing mAb, and to Dr Yasuhiro Minami for helpful discussions. This work was supported by grants-in-aid for Science Research from the Ministry of Education, Science and Culture (Japan), and by a Research Grant of the Ryoichi Naito Foundation for Medical Research.


    Abbreviations
 
GEMglycolipid-enriched membrane
GPIglycosylphosphatidylinositol
MBSMES-buffered saline
TSAthymic shared antigen

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: A. Singer

Received 8 January 1999, accepted 10 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Weiss, A. and Littman, D. R. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[ISI][Medline]
  2. Wange, R. L. and Samelson, L. E. 1996. Complex complexes: signaling at the TCR. Immunity 5:197.[ISI][Medline]
  3. Saitoh, S.-I., Kosugi, A., Noda, S., Yamamoto, N., Ogata, M., Minami, Y., Miyake, K. and Hamaoka, T. 1995. Modulation of T cell receptor-mediated signaling pathway by thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2). J. Immunol. 155:5574.[Abstract]
  4. Noda, S., Kosugi, A., Saitoh, S.-I., Narumiya, S. and Hamaoka, T. 1996. Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2. J. Exp. Med. 183:2355.[Abstract]
  5. Kosugi, A., Saitoh, S.-I., Noda, S., Miyake, K., Yamashita, Y., Kimoto, M., Ogata, M. and Hamaoka, T. 1998. Physical and functional association between thymic shared antigen-1/stem cell antigen-2 and the T cell receptor complex. J. Biol. Chem. 273:12301.[Abstract/Free Full Text]
  6. Simons, K. and Ikonen, E. 1997. Functional rafts in cell membranes. Nature 387:569.[ISI][Medline]
  7. Brown, D. A. and London, E. 1997. Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240:1.[ISI][Medline]
  8. Anderson, R. G. W. 1993. Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr. Opin. Cell Biol. 5:647.[Medline]
  9. Xavier, R., Brennan, T., Li, Q., McCormack, C. and Seed, B. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723.[ISI][Medline]
  10. Zhang, W., Trible, R. P. and Samelson, L. E. 1998. LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity 9:239.[ISI][Medline]
  11. Samelson, L. E., Germain, R. N. and Schwartz, R. H. 1983. Monoclonal antibodies against the antigen receptor on a cloned T-cell hybrid. Proc. Natl Acad. Sci. USA 80:6972.[Abstract]
  12. Kappler, J., White, J., Wegmann, D., Mustain, E. and Marrack, P. 1982. Antigen presentation by Ia+ B cell hybridomas to H-2-restricted T cell hybridomas. Proc. Natl Acad. Sci. USA 79:3604.[Abstract]
  13. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374.[Abstract]
  14. Born, W., Miles, C., White, J., O'Brien, R., Freed, J. H., Marrack, P., Kappler, J. P. and Kubo, R. T. 1987. Peptide sequences of T cell receptor {delta} and {gamma} chains are identical to predicted X and {gamma} proteins. Nature 330:572.[ISI][Medline]
  15. Punt, J. A., Kubo, R. T., Saito, T., Finkel, T. H., Kathiresan, S., Blank, K. J. and Hashimoto, Y. 1991. Surface expression of a T cell receptor ß (TCR-ß) chain in the absence of TCR-{alpha}, -{delta}, and -{gamma} proteins. J. Exp. Med. 174:775.[Abstract]
  16. Becker, M. L. B., Near, R., Mudgett-Hunter, M., Margolies, M. N., Kubo, R. T., Kaye, J. and Hedrick, S. M. 1989. Expression of a hybrid immunoglobulin–T cell receptor protein in transgenic mice. Cell 58:911.[ISI][Medline]
  17. Ortega, G., Korty, P. E., Shevach, E. M. and Malek, T. R. 1986. Role of Ly-6 in lymphocyte activation I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity. J. Immunol. 137:3240.[Abstract/Free Full Text]
  18. Moroi, Y., Koga, Y., Nakamura, K., Ohtsu, M., Kimura, G. and Nomoto, K. 1991. Accumulation of p60lck in HTLV-I-transformed T cell lines detected by an anti-Lck monoclonal antibody, MOL 171. Jpn. J. Cancer Res. 82:909.[ISI][Medline]
  19. Rodgers, W. and Rose, J. K. 1996. Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains. J. Cell Biol. 135:1515.[Abstract]
  20. Schnitzer, J. E., Mclntosh, D. P., Dvorak, A. N., Liu, J. and Oh, P. 1995. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science 269:1435.[ISI][Medline]
  21. Rodgers, W., Crise, B. and Rose, J. K. 1994. Signals determining protein tyrosine kinase and glycosylphosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol. Cell. Biol. 14:5384.[Abstract]
  22. Caplan, S., Zeliger, S., Wang, L. and Baniyash, M. 1995. Cell-surface-expressed T-cell antigen-receptor {zeta} chain is associated with the cytoskeleton. Proc. Natl. Acad. Sci. USA 92:4768.[Abstract]
  23. Kersh, E. N., Shaw, A. S. and Allen, P. M. 1998. Fidelity of T cell activation through multiple T cell receptor {zeta} phosphorylation. Science 281:572.[Abstract/Free Full Text]
  24. Mineo, C., James, G. L., Smart, E. J. and Anderson, R. G. W. 1996. Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J. Biol. Chem. 271:11930.[Abstract/Free Full Text]
  25. Ilangumaran, S., Briol, A. and Hoessli, D. C. 1998. CD44 selectively associates with active Src family protein tyrosine kinases Lck and Fyn in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes. Blood 91:3901.[Abstract/Free Full Text]
  26. Field, K. A., Holowka, D. and Baird, B. 1995. Fc{varepsilon}RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc. Natl Acad. Sci. USA 92:9201.[Abstract]
  27. Field, K. A., Holowka, D. and Baird, B. 1997. Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains. J. Biol. Chem. 272:4276.[Abstract/Free Full Text]
  28. Brdicka, T., Cerny, J. and Horejsi, V. 1998. T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem. Biophys. Res. Commun. 248:356.[ISI][Medline]
  29. Kabouridis, P. S., Magee, A. I. and Ley, S. C. 1997. S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes. EMBO J. 16:4983.[Abstract/Free Full Text]
  30. Montixi, C., Langlet, C., Bernard, A.-N., Thimonier, J., Dubois, C., Wurbel, M.-A., Chauvin, J.-P., Pierres, M. and He, H.-T. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.[Abstract/Free Full Text]
  31. Rodgers, W., Crise, B. and Rose, J. K. 1994. Signals determining protein tyrosine kinase and glycosylphosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol. Cell Biol. 14:5384.[Abstract]
  32. Kwong, J. and Lublin, M. 1995. Amino-terminal palmitate or polybasic domain can provide required second signal to myristate for membrane binding to p56lck. Biochem. Biophys. Res. Commun. 207:868.[ISI][Medline]
  33. Valitutti, S. and Lanzavecchia, A. 1997. Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition. Immunol. Today 18:299.[ISI][Medline]
  34. Viola, A. and Lanzavecchia, A. 1996. T cell activation determined by T cell receptor number and tunable thresholds. Science 273:104.[Abstract]
  35. Valitutti, S., Müller, S., Salio, M. and Lanzavecchia, A. 1997. Degradation of T cell receptor (TCR)–CD3-{zeta} complexes after antigenic stimulation. J. Exp. Med. 185:1859.[Abstract/Free Full Text]