Translocation of tyrosine-phosphorylated TCR
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
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Abstract
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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 TCRCD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCR
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
in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCR
was strongly concentrated in the GEM fraction upon TCR engagement. A kinetic study revealed that tyrosine phosphorylation of TCR
occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCR
in the GEM. Thus, these results indicate that phosphorylated TCR
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
chain
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Introduction
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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
ß heterodimer and the invariant CD3
,
,
, and TCR
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
chains are localized in detergent-resistant membrane rafts, and phosphorylated TCR
chains together with signal-transducing molecules including Lck, ZAP-70, Vav and phospholipase C-
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
is accumulated into the GEM domain after T cell activation.
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Methods
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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
; H146-968 (15), anti-TCR
; H28-710 (16), anti-TCR
; 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 ASepharose (Pharmacia, Piscataway, NJ) and immunoprecipitated with anti-TCR
mAb (H146968) prebound to Protein ASepharose. 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
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
, 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.
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Results
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Localization of TCR
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. 1A
). We also demonstrated that almost all of the GPI-anchored Ly6A/E (Fig. 1B
) and >30% of Lck (Fig. 1C
) 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
ß heterodimers, CD3
chains and TCR
homodimers in the density gradient fraction was examined by immunoblotting analysis using specific antibodies. Interestingly, although a significant amount of TCR
homodimers was in the GEM fraction, almost all of the TCR
ß and CD3
was in the Triton X-100-soluble fractions. In three independent experiments, ~10% of TCR
homodimers was associated with the GEM fraction, whereas the amounts of TCR
ß and CD3
in the GEM were <1% (data not shown). Thus, whereas most TCR complexes are excluded from the GEM fraction, 10% of TCR
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).

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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.
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Localization of phosphorylated TCR
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. 2
, no accumulation of TCR
ß and CD3
was observed and the amount of TCR
detected with anti-TCR
antibody was not changed in the GEM fraction following activation. However, when we analyzed tyrosine phosphorylation of TCR
chains following activation, we observed that the tyrosine-phosphorylated form of TCR
was strongly concentrated in the GEM fraction (Fig. 3
). Although a small amount of phosphorylated TCR
was detectable in the Triton X-100-soluble membrane fraction, most of the phosphorylated TCR
was localized in the GEM fraction after activation (Fig. 3A
, top, right). About 80% of total phosphorylated TCR
was associated with the GEM fraction in this experiment. In contrast, the amount of TCR
in the GEM fraction detected with anti-TCR
antibody remained constant before and after activation (Fig. 3A
, bottom). A tyrosine-phosphorylated 16 kDa TCR
, 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
was not consistent. Figure 3
(B) shows the average of three experiments where phosphorylated 23 kDa TCR
in the GEM and the Triton X-100-soluble fractions after activation was measured. The phosphotyrosine content of the TCR
in each sample is represented by the ratio of the phosphotyrosine signal divided by the TCR
signal. On average, the GEM-associated CD3
had a 16-fold greater phosphotyrosine content compared with that of the Triton X-100-soluble TCR
after T cell activation. Since TCR
phosphorylation is considered to occur within the TCR complex after TCR cross-linking (1,2), these data may suggest that phosphorylated TCR
is dissociated from the TCR complex and migrates into the GEM domains on T cell activation. In the GEM domains, phosphorylated TCR
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).
Distribution of phosphorylated TCR
in COS-7 cells
Although the data presented above may suggest the physical dissociation of phosphorylated TCR
from the TCR complex upon T cell activation, it is possible that a small fraction of TCR
ß and CD3
is localized in the GEM domains, and phosphorylated TCR
in the GEM is associated with TCR
ß and CD3
to form the TCR complex. To address this possibility, we next examined whether phosphorylated TCR
can be localized in the GEM in the absence of other TCR chains. COS-7 cells were transfected with TCR
cDNA together with Lck and Syk cDNAs, stimulated with pervanadate, and subjected to fractionation and analysis. It is known that tyrosine phosphorylation of TCR
chains can be induced by cooperative enzymatic activities of Lck and ZAP-70 /Syk kinases (1,2). As shown in Fig. 4
, a considerable amount of phosphorylated TCR
was detectable in the GEM fraction when transfected COS-7 cells were stimulated. The ratio of phosphorylated TCR
in the GEM and Triton X-100-soluble fractions is not so impressive as that observed in Fig. 3
(A). However, whereas phosphorylated TCR
in the Triton X-100-soluble fractions mostly migrated between 16 and 23 kDa, the size of phosphorylated TCR
in the GEM was predominantly 23 kDa. This result seems to be very intriguing since phosphorylated 23 kDa TCR
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
can be effectively localized in the GEM domains in the absence of other TCR chains.
Kinetic changes of phosphorylated TCR
appearance in the GEM fraction upon T cell activation
In order to demonstrate the movement of phosphorylated TCR
into the GEM domains, we investigated kinetics of TCR
phosphorylation in each membrane fraction of 2B4 T cell hybridomas after TCR engagement. As shown in Fig. 5
, phosphorylated TCR
first appeared in the Triton X-100-soluble fractions 5 min after TCR cross-linking. At this time, no phosphorylated TCR
was observed in the GEM fraction. However, a strong phosphotyrosine signal of TCR
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
is transferred into the GEM domains during T cell activation.
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Discussion
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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
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
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-
1, LAT and TCR
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
ß and CD3
in the GEM were basically undetectable by densitometric scanning (Figs 1 and 2
). Only 10% of TCR
was associated with the GEM domain and no redistribution of TCR
reactive with anti-TCR
antibody was observed after activation (Fig. 2
). The amount of TCR
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
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
is localized to the GEM domain (Fig. 3
). A kinetic study revealed that tyrosine phosphorylation of TCR
occurs initially in the Triton X-100-soluble fractions followed by the accumulation of phosphorylated TCR
in the GEM fraction (Fig. 5
). These findings indicate that phosphorylated TCR
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
is thus far unknown. However, it is possible that phosphorylated TCR
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
in an experiment in which membrane localization of various TCR
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
ß, CD3
and phosphorylated TCR
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
ß and CD3
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
ß and CD3
is localized in the GEM in contrast to the high content of phosphorylated TCR
following TCR engagement. We believe that these data support the physical dissociation of phosphorylated TCR
from the TCR complex.
Since TCR
in the GEM of transfected COS-7 cells can be efficiently phosphorylated without other TCR chains (Fig. 4
), it is possible that TCR
present within the GEM domain before stimulation is the major TCR
subset that becomes phosphorylated following activation. However, there is a report suggesting that TCR
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
phosphorylation was clearly observed in this Lck mutant cells after activation. Since Lck is known to be indispensable for TCR
phosphorylation (1,2), TCR
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
phosphorylation could be occur outside the GEM in this mutant. Based on this study in addition to our kinetic study (Fig. 5
), we think that TCR
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 peptideMHC complexes (33). Viola et al. demonstrated that T cells `count' the number of TCR engaged by the peptideMHC 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 TCRpeptideMHC 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.
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Acknowledgments
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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.
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Abbreviations
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GEM | glycolipid-enriched membrane |
GPI | glycosylphosphatidylinositol |
MBS | MES-buffered saline |
TSA | thymic shared antigen |
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Notes
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The first two authors contributed equally to this work
Transmitting editor: A. Singer
Received 8 January 1999,
accepted 10 May 1999.
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