Association of GM3 with Zap-70 Induced by T Cell Activation in Plasma Membrane Microdomains

Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation.

Gangliosides are synthesized by virtually all the cells of peripheral blood (1). However, patterns of ganglioside cell expression depend on the species, cell type, and age of the individual. In human peripheral blood lymphocytes (PBL) 1 mono-sialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane (72% of total ganglioside content) (1), although not correlated with a particular lymphocyte subpopulation, i.e. both CD4 ϩ and CD8 ϩ cells express a similar amount of GM3 (2). Minor ganglioside constituents of human PBL include sialosyl paragloboside (about 14%) and sialosyl lactohexaosyl ceramide (about 7%) (1). In addition, disialoganglioside GD3 is also a minor component on a small subset of human peripheral blood T cells (3).
Previous immunofluorescence and immunogold electron microscopic studies revealed a clustered distribution of GM3 molecules on the cell surface of human PBL, clearly indicating the presence of glycosphingolipid (GM3)-enriched microdomains (GEM) (4,5). They are involved in modulating signal transduction by GSL-GSL interaction, binding with specific antibodies, or assembly with signal transducer molecules (6). The variety of proteins detected in these domains isolated from different cell types is extremely wide (7). The presence of tyrosine kinase receptors, mono (Ras, Rap)-and heterotrimeric G proteins, Src-like tyrosine kinases (LCK, LYN, FYN), protein kinase C isozymes, and glycosylphosphatidylinositol-anchored proteins (8,9) allows these portions of the plasma membrane to be considered as "glycosignaling domains" (6). We previously demonstrated that, in lymphocytes, not only GM3 and cholesterol but also CD4 and p56 lck are selectively recovered in GEM (4). The CD4-p56 lck complex represents one of the most important receptor systems in the T cell function, and CD4 is considered the TCR co-receptor in thymic selection, T cell activation, and cellular response (10,11). Most of the CD4 functions are due to CD4 interaction with p56 lck (12). Interestingly, in human T lymphocytes exogenous GM3 induces CD4 phosphorylation (13), dissociation from p56 lck , and internalization via endocytic pits and vesicles (14).
The role of GEM in lymphocyte activation became obvious after the demonstration that T cell activation by co-stimulation through CD28 led to the redistribution and clustering of membrane and intracellular kinase-rich microdomains at the site of TCR engagements (15). The hypothesis for the role of GEM in initiating TCR signaling has gained further support from the identification in them of two other crucial molecules, phosphatidylinositol 4,5-biphosphate (16) (a substrate of phospholipase C␥) and LAT (17,18) (one of the earliest and major tyrosine-phosphorylated proteins found following TCR triggering). LAT phosphorylated following Zap-70 activation. Zap-70, a Syk family kinase, is activated via both a self-and a Lck-dependent phosphorylation mechanism (19) and translocate from cytoplasmic compartment to the cell surface. It phosphorylates substrates, which in turn leads to the subsequent docking and activation of other Src homology 2 (SH2)-containing molecules involved in the amplification and diversification of TCR-initiated signaling. Zap-70 becomes GEM-associated (20,21) and binds phospholipase C␥, which, after phosphorylation, cleaves phosphatidylinositol 4,5-biphosphate.
Most of the studies aimed to the study of GEM during T cell activation were performed using cholera toxin as a marker of these microdomains (15, 20 -28), taking advantage of its capability to bind monosialoganglioside GM1. However, this molecule has been shown to be virtually undetectable (1) or present at very low levels (2 ϫ 10 Ϫ18 g/cell) in resting peripheral blood T lymphocytes (27). Thus, in this investigation we preliminarily analyzed the ganglioside pattern and composition of GEM and then focused on the distribution of the main ganglioside constituent of these domains and its interaction with the phosphorylation protein Zap-70 after T cell activation by CD3 and CD28 engagements.

MATERIALS AND METHODS
Cells-Human PBL were isolated from fresh heparinized blood by Lymphoprep (Nycomed AS Pharma Diagnostic Div., Oslo, Norway) density gradient centrifugation and washed three times in phosphate-buffered saline (PBS), pH 7.4. Human T lymphocytes were stimulated with anti-CD3 (10 g/ml, Ortho-Clinical Diagnostics, Raritan, NJ) and anti-CD28 (10 g/ml, PharMingen, La Jolla, CA) antibodies for 1 h at 37°C.
Isolation and Analysis of Glycosphingolipid-enriched Microdomain Fraction-GEM fraction from human PBL were isolated as described previously (29). Briefly, 2 ϫ 10 8 cells were suspended in 1 ml of lysis buffer containing 1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM NaVO 4 , and 75 units of aprotinin and allowed to stand for 20 min. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). The lysate was centrifuged for 5 min at 1300 ϫ g to remove nuclei and large cellular debris. The supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient centrifugation, i.e. the fraction was mixed with an equal volume of 85% sucrose (w/v) in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA). The resulting diluent was placed at the bottom of a linear sucrose gradient (5-30%) in the same buffer and centrifuged at 200,000 ϫ g for 16 -18 h at 4°C in a SW41 rotor (Beckman Instruments, Palo Alto, CA). After centrifugation, the gradient was fractionated, and 11 fractions were collected starting from the top of the tube. All steps were done at 0 -4°C. The amount of protein in each fraction was first quantified by Bio-Rad protein assay (Bio-Rad). The amount of cholesterol was evaluated as described previously (30). Free cholesterol was quantitated from TLC plates by densitometric scanning and comparison with the standard. The density of the bands used to quantitate cholesterol concentration fell within the linear range of compound concentration versus absorbance.
Finally, the GEM fraction was subjected to ganglioside extraction according to the method of Svennerholm and Fredman (31) with minor modifications. Briefly, the GEM fraction was extracted twice in chloroform:methanol:water (4:8:3, v:v:v) and subjected to Folch partition by the addition of water resulting in a final chloroform:methanol:water ratio of 1:2:1.4. The upper phase, containing polar glycosphingolipids, was purified of salts and low molecular weight contaminants using Supelclean LC-18 columns, 3 ml (Supelco Inc., Bellefonte, PA) according to the method of Williams and McCluer (32). The eluted glycosphingolipids were dried and separated by high performance thin-layer chromatography (HPTLC) using silica gel 60 HPTLC plates (Merck). Chromatography was performed in chloroform:methanol:0.25% aqueous KCl (5:4:1, v:v:v). Plates were then air-dried, and gangliosides were visualized with resorcinol (33).
Isolation and Gas Chromatographic Analysis of GM3 from GEM-GM3 was isolated by HPTLC from the GEM fraction ganglioside extract. After exposure to iodide vapors, the band comigrating with standard GM3 was scraped, eluted from silica with chloroform:methanol (2:1, v:v), and dried under nitrogen.
The identification of carbohydrates was carried out by methanolysis with 3 N methanolic HCl as reported (34). Trimethylsilylmethylglycosides were injected into a SPB-5 fused silica capillary column programmed at 5°C/min from 150 to 210°C, maintained isothermally for 5 min, and programmed at 5°C/min from 210 to 280°C. As standards, p-nitrophenyl ␤-D-galactopyranoside and p-nitrophenyl ␤-D-glucopyranoside (Sigma) were used.
The identification of sialic acid was performed as described (35) after methanolysis in 0.05 N methanolic-HCl. The thoroughly dried products were converted to trimethylsilyl ester derivatives with 50 l of trimethylsilylimidazole (Supelchem) at 70°C for 20 min. Aliquots of the reaction mixture were injected into a SPB-5 fused silica capillary column programmed at 3°C/min from 220 to 280°C. As standard, 2-O-(pnitrophenyl)-␣-D-N-acetylneuraminic acid (Sigma) was used.
The types and the percentages of fatty acids were determined as methyl esters after vigorous methanolysis with 0.5 N anhydrous HCl at 80°C for 18 h. After cooling, the solution was extracted four times with hexane. The hexane phases containing the fatty acids methyl esters were collected, dried, and injected (0.5 l in CH 2 Cl 2 ) into an SPB-2380 fused silica capillary column programmed at 5°C/min from 140 to 170°C.
Dry ganglioside was hydrolyzed using 5 N aqueous HCl:methanol:1:4, as described (36). The sphingosine bases were derivatized and analyzed as trimethylsilyl esters under the same conditions reported for the identification of carbohydrates.
Purification and Mass Spectrometric Analysis of GM3 from GEM-GM3 was isolated from the GEM fraction ganglioside extract by HPLC on amino silica columns, as already reported (37). The use of an on-line variable wavelength diode array detector allows the identification and quantitative determination of GM3 on the basis of its UV spectrum and accurately acquired analytical data. The GM3 peak was collected and analyzed by mass spectrometry (MS) using an Applied Biosystems Sciex API III tandem mass spectrometer (Applera Italia, Monza, Italy), equipped with articulated ion spray source. Mass calibration and resolution were checked with a polypropylene glycol solution. The MS experiments were run with a resolution better than 0.8 atomic mass unit; for the MS/MSMS runs, resolution for both quadrupoles was set at 1 atomic mass unit. All of the instrumental operating parameters were standard except the orifice voltage, which was operated at 50 V. MS/ MSMS gas collision fragmentation was run at a collision energy of 50 eV with argon at a thickness of 280 ϫ 10 12 molecules/cm 2 . Sample was dissolved in an aqueous solution of 50% ethanol containing 0.1% formic acid and 2 mM ammonium acetate. The introduction was operated by a Harvard infusion pump at a flow rate of 2 l/min. MS and MSMS spectra were collected at a rate of 10 ms/atomic mass unit with a step size of 0.1 atomic mass unit for MS and 1 atomic mass unit for MSMS experiments. Acquired data were processed by MacSpec software; the Hypermass option was used to handle data concerning multiply charged ions.
TLC Immunostaining Analysis-Immunostaining was performed using as antigen about 2 g of the ganglioside extract from either untreated or anti-CD3 and anti-CD28-treated lymphocytes. Gangliosides were separated by TLC using HPTLC aluminum-backed silica gel 60 plates (Merck). Plates were soaked in a 0.2% solution of polyisobutylmethacrylate in hexane for 90 s, air-dried, and incubated in the blocking solution (3% bovine serum albumin in 20 mM Tris, 0.5 M NaCl, pH 7.5) for 1 h at room temperature. The blocking solution was removed and replaced by washing buffer (TBS). The plates were then incubated for 1 h at room temperature with anti-GD3 mAb (R24, Matreya Inc., Pleasant Gap, PA) in 1% bovine serum albumin-TBS. The antibody was removed, and plates were washed three times for 10 min with TBS. Horseradish peroxidase-conjugated sheep anti-mouse IgG (Sigma) in 1% bovine serum albumin-TBS was added and incubated for 1 h at room temperature. The color reaction was obtained by adding 200 mg of sodium nitroprusside (Sigma) and 80 mg of o-dianisidine (Sigma) dissolved in 100 ml of H 2 0 containing 35 l of 30% H 2 O 2 . The reaction was stopped by washing in distilled water. As a control for nonspecific reactivity, parallel blots were performed as described above using an anti-mouse IgG3 (Sigma).
Immunofluorescence Analysis-Human PBL were incubated with GMR6 anti-GM3 mAb (38) (a gift from Dr. T. Tai, Tokyo Metropolitan Institute of Medical Science, Tokyo) for 1 h at 4°C followed by three washes in PBS and the addition (30 min at 4°C) of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Sigma). Cells were then fixed in 4% formaldehyde in PBS and analyzed by immunofluorescence microscopy. In parallel experiments, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature before incubation with anti-GM3 mAb.
Analysis of GM3-Zap-70 Colocalization on the Cell Surface by Scanning Confocal Microscopy-Human PBL were fixed with 4% formaldehyde in PBS for 30 min at 4°C and labeled with anti-Zap-70 mAb (Upstate Biotechnology, Lake Placid, NY) for 1 h at 4°C followed by the addition (30 min at 4°C) of Texas red-conjugated anti-mouse IgG (Calbiochem, La Jolla, CA). After three washes in PBS, cells were incubated with GMR6 anti-GM3 mAb (38) for 1 h at 4°C followed by three washes in PBS and the addition (30 min at 4°C) of FITC-conjugated goat anti-mouse IgM (Sigma). In parallel experiments, cells were stained with anti-GM3 mAb before fixing the cells. Cells were finally washed three times in PBS and resuspended in glycerol/Tris-HCl, pH 9.2. The images were acquired through a confocal laser scanning microscope (Sarastro 2000, Molecular Dynamics) equipped with a Nikon Optiphot microscope and an argon ion laser. Simultaneously, the green (FITC) and the red (Texas red, which reduces greatly overlapping) fluorophores were excited at 488 and 518 nm. Acquisition of single FITC-stained samples in dual fluorescence scanning configuration did not show overlapping. Images were collected at 512 ϫ 512 pixels.
The immunoprecipitates were split into two aliquots. A portion was then subjected to ganglioside extraction as reported above. Another portion of the immunoprecipitates was electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) after 10% SDS-PAGE and then probed with polyclonal anti-Zap-70 (Santa Cruz Biotechnology, Santa Cruz, CA) or, alternatively, with GMR6 anti-GM3 mAb. The bound antibodies were then visualized with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgM (Sigma), respectively. Immunoreactivity was assessed by chemiluminescence reaction using the ECL Western blocking detection system (Amersham Biosciences).

Ganglioside Pattern and Composition of GEM in Human
PBL-We investigated the ganglioside composition of GEM fraction of lymphocyte plasma membrane. Gangliosides were extracted in chloroform:methanol:water and separated in HPTLC as reported above. Three main resorcinol-positive bands were shown: (a) comigrating with GM3, (b) between GM3 and GM1, and (c) between GM1 and GD1a. The GM3 content, determined as lipid-bound sialic acid, was 17.5 Ϯ 1.4 g/mg of protein in the GEM fraction as compared with 0.864 Ϯ 0.1 g/mg of protein in total lymphocytes (Table I). The two other main bands, migrating between GM1 and GD1a (4.375 Ϯ 0.3 versus 0.168 Ϯ 0.02 g/mg of protein) and between GM3 and GM1 (1.2 Ϯ 0.1 versus 0.084 Ϯ 0.01 g/mg of protein) also revealed a significant enrichment in the GEM fraction as compared with the total cell lysate.
The identity of the GM3 comigrating band was verified by gas liquid chromatographic (GLC) analysis as reported previously (2). In the hydrophilic head galactose, glucose (Fig. 1a), and sialic acid in a molar ratio of 1:1:1 were found. Lymphocytic GM3 showed the same retention time peaks (␣ and ␤ anomers of methylketosides of sialic acid) as those of standard N-acetylneuraminic acid (Fig. 1b); in the hydrophobic part, the main fatty acids were C16:0, C18:0, C18:1, and C15:0 (Fig. 1c). Long-chain bases were analyzed by GLC as O-trimethysilyl derivatives. GM3 exclusively gave n-C18-sphingenine, which accounted for more than 95% of the long-chain bases.
The MS analysis of GM3 from GEM fraction ganglioside extract confirmed the identity of the molecule (Fig. 2a). The linkage of sialic acid and sugars was analyzed both by GM3 fragmentation after mild alkaline hydrolysis at pH 7 (Fig. 2b) and by GM3 fragmentation under acidic conditions (Fig. 2c).
Expression and Distribution of GM3 after T Cell Activation by Co-stimulation through CD28 -We investigated the ganglioside composition of human PBL in the absence and presence of anti-CD3 and CD28 stimulation. Gangliosides were extracted in chloroform:methanol:water and separated in HPTLC as reported above. Again, three main resorcinol-positive bands were shown: (a) comigrating with GM3, (b) between GM3 and GM1, and (c) between GM1 and GD1a (Fig. 3a). In both cell types, the GM3 double band is due to the heterogeneity of fatty acid composition as described (40). No significant differences in the amount of single gangliosides were observed (Fig. 3a).
The identity of the band migrating between GM1 and GD1a was verified by TLC immunostaining using the R24 mAb. The analysis revealed that in both untreated and in anti-CD3-and anti-CD28 treated cells the band was immunostained by the anti-GD3 mAb (Fig. 3b).
The analysis of the ganglioside pattern of sucrose gradient fractions from lymphocytes revealed that T cell stimulation TABLE I Ganglioside composition of GEM Quantitative analysis of ganglioside scraped bands was performed by measuring the lipid-bound sialic acid. Cholesterol was quantified according to the method described by Huber et al. (30). Band x, migrating between GM1 and GD1a, was immunostained by anti-GD3 mAb. It does not exclude the presence of sialosyl paragloboside, as described (1,40). Band y, migrating between GM3 and GM1, was characterized as sialosyl lactohexaosyl ceramide, as reported (1,40 1. GLC analysis of GM3 from lymphocytic GEM fraction. GM3 was isolated from a GEM fraction of about 2 ϫ 10 8 human PBL. a, GM3 carbohydrates are identified as ␥, ␣, and ␤ forms of trimethylsilylmethyl-D-galactoside and ␥, ␣, and ␤ forms of trimethylsilylmethyl-D-glucoside. They showed the same retention time peaks as those of standards. b, ␣ and ␤ anomers of methylketosides of sialic acid from isolated GM3 showed the same retention time peaks as those of standard N-acetylneuraminic acid. c, fatty acids from isolated GM3 were analyzed as methyl esters. with anti-CD3 and anti-CD28 did not cause translocation of gangliosides (GM3, sialylparagloboside, and GD3) within or from the GEM fraction (Fig. 4).
The results by immunofluorescence microscopy analysis re-vealed that most of the cells showed an uneven signal distribution of ganglioside molecules over the cell surface, indicating the presence of plasma membrane microdomains with GM3 enrichment. Fig. 5 shows the GM3 distribution in untreated (Fig. 5a) and in anti-CD3-and anti-CD28 treated cells (Fig. 5b). The analysis of permeabilized cells revealed an intracellular GM3 localization, resembling specific cytoplasmic compartment(s), such as the Golgi complex (Fig. 5, c and d, respectively).

Analysis of the Association between GM3 and Zap-70 after T Cell Activation by Co-stimulation through CD28 -
To study the possible GM3-Zap-70 interaction after T cell activation, we analyzed their distribution on the plasma membrane by scanning confocal microscopy (Fig. 6). The results revealed that most of the cells showed an uneven signal distribution of ganglioside mole- cules over the cell surface either constitutively (Fig. 6, lane A2) or after treatment with anti-CD3 (10 g/ml) and anti-CD28 (10 g/ml) for 1 h at 37°C (Fig. 6, lane B2). As expected, in untreated cells Zap-70 staining was mostly diffuse in the cytoplasm (Fig. 6,  lane A1) whereas, after T cell activation, it appeared uneven and punctate over the plasma membrane, indicating that the protein translocates mostly in correspondence to specific membrane domains (Fig. 6, lane B1). This change of localization pattern is generally associated with protein phosphorylation (21). To determine the possible association between Zap-70 and GM3, we superimposed the double immunostaining of anti-Zap-70 and anti-GM3 in the absence or presence of CD3 and CD28 engagements. In the absence of T cell stimulation, GM3 and Zap-70 showed weak colocalization (Fig. 6, lane A3). This finding suggests that GM3 and Zap-70 are not physically associated in quiescent human PBL. After T cell activation by co-stimulation through CD28, the merged image of anti-Zap-70 and anti-GM3 staining revealed yellow areas, resulting from the overlap of green and red fluorescence, which correspond to nearly complete colocalization areas (Fig. 6, lane B3). Therefore, T cell activation preferentially promotes translocation of Zap-70 in selective membrane domains in which Zap-70 may interact with GM3.

Co-immunoprecipitation of GM3 and Zap-70 after T Cell Activation by Co-stimulation through CD28 -To verify whether
Zap-70 binds directly to GM3, cell-free lysates from anti-CD3and CD28-treated and untreated cells were immunoprecipitated with the anti-Zap-70 mAb, followed by protein G-acrylic beads. Acidic glycosphingolipids were then extracted from the Zap-70 immunoprecipitates, and HPTLC analysis showed that a GM3 comigrating band was selectively detectable in the extracts from activated cells (Fig. 7, lane b). The absence of other gangliosides in the immunoprecipitates indicates that the interaction of gangliosides with Zap-70 is specific for GM3. On the contrary, no resorcinol-positive bands were detectable in the immunoprecipitates from untreated cells (Fig. 7, lane d). In control samples the immunoprecipitation with a mouse IgG with irrelevant specificity, under the same condition, did not result in detectable levels of the ganglioside (Fig. 7, lanes c and e).
In parallel experiments we performed Western blot analysis of the anti-Zap-70 immunoprecipitates with anti-GM3 mAb. Our results revealed the presence of a 70-kDa band in the immunoprecipitates from activated cells (Fig. 7, lane f). To confirm the positive band as Zap-70, the anti-GM3 antibody was stripped from the nitrocellulose, and the membrane was then reprobed with rabbit polyclonal anti-Zap-70 antibody. The results showed the positive band as Zap-70 (not shown). On the contrary, no bands were detectable in the immunoprecipitates with IgG with irrelevant specificity (Fig. 7, lane g) or from untreated cells (Fig. 7, lane h). DISCUSSION In this investigation we provide evidence that GM3 is the main constituent of GEM in human PBL and can be considered a marker of these specialized portions of cell plasma membrane. This finding is in agreement with previous reports on both total cell extract (1,2,41) and low density Triton-insoluble fraction (4). Here, we further characterized the GM3 comigrating band as GM3 by MS and GLC. Galactose, glucose, and sialic acid in a molar ratio of 1:1:1 were revealed in the hydrophilic head, and in the hydrophobic part, a heterogeneous fatty acid composition was revealed. Immunofluorescence and scanning confocal microscopic observations showed the presence of GM3 clusters. As very little isolated immunostaining was present, our results strongly suggest the existence on the cell surface of glycosphingolipidenriched microdomains with GM3 molecule concentration. These observations are consistent with previously reported thermodynamic results (42) and with our immunoelectron microscopic and immunofluorescence data showing that GM3, CD4, and p56 lck were selectively recovered in the same microdomains in human PBL. The presence of GM3, Src family proteins, and CD4 indicated that GEM may be involved in signal transduction and cell activation in lymphocytes. This hypothesis gained further support from the demonstration that disruption of GEM inhibits CD3-induced phosphorylation and association with the cytoskeleton (43). In addition, a large number of studies indicate the role of GEM in initiating of TCR signaling; they demonstrate the recruitment of TCR to GEM upon receptor stimulation and the redistribution and clustering of plasma membrane and intracellular kinase-rich microdomains at the site of TCR engagement (15, 20 -28).
Our study points out the role of GM3 as the main ganglioside constituent of GEM in human lymphocytes. Its concentration is about 10,000-fold higher than that reported for GM1 in the same cells (27). However, our scanning confocal observations of permeabilized cells also revealed an intracellular staining of GM3 molecules. It is tempting to speculate that newly synthesized GM3, in both resting and activated cells, can be associated with the Golgi apparatus.
Taking advantage of the possibility of staining the main ganglioside component of GEM, and following our previous observation that in human lymphocytes GM3, CD4, and p56 lck are selectively recovered (4) and interact (44) in the same microdomains of the plasma membrane, we now provide evidence that GM3 tightly binds to the phosphorylation transducer protein Zap-70 after CD3 and CD28 engagements. In the present study, GM3-Zap-70 association was demonstrated by both Western blot and TLC analyses, showing that GM3 was co-immunoprecipitated by anti-Zap-70 mAb. These findings demonstrate that CD3 and CD28 engagements induce not only Zap-70 translocation to the cell surface but also its high affinity binding with the GEM component GM3. This SDS-resistant interaction is similar to that described between Trk A receptor and GM1 (45). Specific high affinity SDS-resistant gangliosideprotein interactions reported in different cell types (44 -48) appear in fact to be involved in transducing stimulatory and/or inhibitory signals (6), although the precise mechanism involved remains to be elucidated. To this end, it has been hypothesized that the interaction of ganglioside with some transducers (e.g. c-Srk, Rho) may be due to the presence of aliphatic chain (fatty acyl or farnesyl group) linked to the transducer, although other transducers present in glycosignaling domains (e.g. FAK, Ras) may require a different mechanisms (6).
In conclusion, this study shows that CD3 and CD28 engagements induce a high affinity association between Zap-70 and the microdomain-specific component, ganglioside GM3. Interestingly, T cell activation does not modify GM3 distribution, revealing that co-stimulation through CD28 does not promote a redistribution of GEM as has been suggested (15,21,23,24,27,28) but induces a preferential translocation of Zap-70 to discrete microdomains of the plasma membrane in which it may interact with GM3. These findings strongly suggest a role for GM3 as a structural component of the membrane multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.