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Tumor Biology |
Department of Pathology, Faculty of Medicine, Centre médical universitaire, 1211 Geneva 4, Switzerland [I. S., C. P., K. K., N. K., B. B., D. C. H.], and Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, D-53121 Bonn, Germany [G. v. E-D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The coisolation of CD20 with tyrosine and serine/threonine kinases on incubation of cells with anti-CD20 antibodies suggested an implication of the molecule in transmembrane signaling pathways (6 , 7) . However, no direct modular interaction of the cytoplasmic portions of CD20 with signaling components has been defined, and the involvement of CD20 in transmembrane signaling probably results from the association of the molecule with raft signaling platforms. Such a redistribution of CD20 to rafts is induced by several bivalent antibodies recognizing an extracellular epitope on CD20 (4) . The efficient raft redistribution of CD20 by bivalent monoclonal antibodies without cross-linking with secondary antibodies suggests that dimerization with appropriate anti-CD20 antibodies stabilizes interactions of CD20 with raft membrane components and connects CD20 to the transmembrane signaling machinery.
Rafts are liquid-ordered domains of the plasma membrane composed of high melting temperature lipid aggregates, wherein interactions between receptors and signaling proteins are facilitated. Such structures function as signaling platforms in plasma membranes, and the raft platform was shown to coordinate functions as diverse as cellular adhesion (8) , transmembrane signaling (9) , and virus budding (10) . Sphingolipids and GPI-linked surface proteins are enriched in liquid-ordered domains of the plasma membrane outer leaflet (11) , and the acylation of a variety of signaling proteins is necessary for targeting those proteins to the raft inner leaflet (12) . Transmembrane proteins such as CD4 (13) , LAT (14) , and Cbp/PAG (15 , 16) are also acylated and display the propensity to associate with rafts, but a protein sequence common to all transmembrane proteins associated with rafts has not been identified. In lymphocytes, rafts participate in the functional organization of the immune synapse of T cells (17) and allow the recognition and endocytosis of soluble antigen by B-cell receptors (18) . Acylated protein tyrosine kinases associated with lymphocyte rafts are in a higher state of activation (19) and function in signaling pathways that effect a variety of cellular responses in different cell types (20, 21, 22) .
Given the pivotal role of rafts in transmembrane signaling, we set out to analyze the consequences of CD20 accumulation in rafts induced by the therapeutic monoclonal antibody RTX in the Burkitt lymphoma-derived Raji B-cell line. Rafts containing CD20 (isolated from cells incubated with RTX) were compared with rafts free of CD20 (isolated from control cells) for: (a) protein kinase activities; (b) overall lipid contents and phosphoprotein patterns; (c) behavior toward raft perturbants such as OTG or MBCD; and (d) susceptibility of the raft-residing, GPI-linked CD55 to phospholipases C and D. Our results show that RTX induced redistribution of CD20 to rafts without modifying the overall contents in lipids, surface, and signaling proteins but increased the resistance of sphingolipids and Cbp/PAG toward OTG, modulated the catalytic activity of the raft-associated Lyn kinase, and increased the sensitivity of the GPI-linked, CD55 C’-defense protein to phospholipases C and D.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RTX (a humanized IgG1 anti-CD20 antibody) was a gift from Roche (Basel, Switzerland). Fab fragments of RTX were generated using the Immunopure Fab kit (Pierce). Human IgG1
was from Sigma Chemical Co. (St. Louis, MO). Polyclonal anti-Lyn (NH2 terminal), anti-Syk, and anti-Csk antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse anti-CD20 mAb (L-26) was from DAKO. The anti-CD55 mAb IA-10 was from PharMingen, and the anti-Cbp/PAG mAb MEM-255 was provided by Dr. V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic). The antiganglioside antibody 2B5 that reacts with alkali-resistant sphingolipids present in mast cell rafts (23)
was provided by Dr. I. Pecht (Weizmann Institute, Rehovot, Israel). The GM1 ganglioside was detected with the cholera toxin B subunit as described previously (24)
. Triton X-100 was from Merck (Geneva, Switzerland); OTG and MBCD were from Sigma-Aldrich (Buchs, Switzerland). Phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis) was purchased from Oxford GlycoSystems (Oxford, United Kingdom). Human phosphatidylinositol-specific phospholipase D was supplied by Dr. P. Bütikofer, Institute of Biochemistry and Molecular Biology, University of Berne (Berne, Switzerland).
Exposure of Cells to RTX and Preparation of DRMs.
Cells (50 x 106) were incubated with increasing concentrations of RTX or control IgG1 in 10 ml of RPMI complete medium for 15 min at 37°C. After centrifugation, cell pellets were lysed for 1 h at 4°C in 0.5 ml of TKM [50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, and 1 mM EGTA] containing 1% TX-100, protease inhibitors (10 µM leupeptin, 25 µM aprotinin, and 2 mM Pefabloc), and 1 mM Na vanadate as a phosphatase inhibitor. Cell extracts were placed at the bottom of Beckman SW41 tubes, made 40% sucrose w/v in a 1.5-ml volume, and overlaid with 6 ml of 35% (w/v) sucrose and 3.5 ml of 5% (w/v) sucrose. Ultracentrifugation was carried out for 16–20 h at 38,000 rpm (250,000 x g) at 4°C. Eleven 1-ml fractions were collected from the top of the gradient. The RTX IgG1 coisolating with the raft fraction was detected by Western blotting using an antihuman IgG antibody. The amount of RTX in a given raft aliquot was estimated by comparing the signals generated by known amounts of purified RTX and calculated for the raft preparation obtained from 50 x 106 cells and total amount of RTX applied.
Exposure of DRMs to OTG or MBCD.
Low-density gradient fractions 3–5 (DRMs) were pooled, diluted in TKM, and ultracentrifuged at 38,000 rpm for 2 h at 4°C in a SW 41 Beckman rotor. To validate the purification of rafts, the gradient fractions were controlled for the presence of GM1 and Cbp/PAG in raft fractions and the presence of CD45 in soluble fractions. The TX-100-resistant membrane pellet was resuspended in TKM, and aliquots of isolated rafts were incubated with increasing concentrations of OTG or MBCD. All incubations were carried out at 37°C for 30 min; the membranes were diluted in TKM and sedimented by ultracentrifugation as above. The proteins recovered in the high-speed pellet were considered to be part of DRMs resistant to the disruptive effects of OTG or MBCD.
The proteins in the sedimentable membrane pellet were separated on a 10% minigel and transferred to nitrocellulose (Hybond-C; Amersham Pharmacia Biotech) with a semidry blotting apparatus (Bio-Rad). After 2 h of blocking at room temperature in TTBS [Tris-HCl 10 mM (pH 7.4), 100 mM NaCl, and 0.05% Tween 20] containing 5% low-fat, dry milk powder (TTBS-5% MP), the filters were incubated with antibodies in TTBS-5% MP overnight at 4°C. Thoroughly washed filters were incubated with alkaline-phosphatase-conjugated second antibodies for 1 h at room temperature. Chemiluminescence development was carried out with the Immun-Star Pack reagents (Bio-Rad), and the filters were exposed to X-OMAT Kodak films. Quantitation of the protein bands was achieved by laser scanning and ImageQuant analysis.
Analysis of DRM Lipids.
DRMs from RTX-exposed (10 µg/ml: +) or control (-) cells were extracted in chloroform:methanol:pyridine:water (60:30:1:6 by volume) for 48 h at 50°C and analyzed by thin-layer chromatography as described earlier (25)
. DRMs recovered from sucrose density gradients were deposited on nitrocellulose filters, and the GM1 ganglioside was detected with cholera toxin B subunit (peroxidase-labeled; Sigma) and other, raft-associated gangliosides, with the 2B5 mAb (23)
. The dot blots were developed as described previously (24)
. Intensity of the spots was measured by laser scanning and analyzed with ImageQuant.
In Vitro Kinase Assay of DRMs.
To measure kinase activities, aliquots of the pooled DRMs (fractions 3–5) were diluted 
20-fold with TKM buffer and centrifuged at 250,000 x g for 2 h. The sedimented membranes were suspended directly in kinase buffer [20 mM MOPS (pH 7.0), 5 mM MgCl2, 5 mM MnCl2, and 0.1 mM Na3VO4]; the kinase assay was set up in a final volume of 30 µl and initiated by the addition of 10 µCi of [
-32P] ATP (Amersham; 5000 Ci/mmol) in 5 µl of kinase buffer. To measure the protein tyrosine kinase activity, 1 µg of acid denatured enolase was included in the assay. After incubation for 15 min at 30°C, the reaction was stopped by the addition of 6x sample buffer and boiling in the presence of DTT. Phosphorylated proteins separated in 10% minigels were fixed in 10% trichloroacetic acid for 15 min and washed in 10% methanol: 10% acetic acid solution for 6–12 h with several changes before drying and autoradiography. Quantitation of the phosphorylated bands was carried out with PhosphorImager and ImageQuant.
Alternatively, the kinase assay was stopped by dilution in PBS, the rafts were collected by ultracentrifugation, and the two-dimensional analysis of phosphorylated raft proteins was carried out as in Ref. 26 .
Exposure of DRMs and Whole Cells to PIPLC or PIPLD.
DRMs were resuspended in PBS for PIPLC incubation and in 50 mM MES (pH 6.5) buffer containing 0.05 mM CaCl2, TX-100 0.02% for incubation with PIPLD. Incubation with the phospholipases was carried at 37°C for 30 min (PIPLC) or 1 h (PIPLD). Raft membranes were then diluted in PBS or MES buffer, sedimented by ultracentrifugation, and subjected to Western blotting analysis with anti-CD55 antibody.
Raji cells were incubated with 10 µg/ml RTX or no antibody, washed in PBS, and incubated with increasing concentrations of PIPLC (30 min in PBS at 37°C) or PIPLD (60 min in MES buffer at 37°C). After one PBS wash, 3 x 105 cells were stained with FITC-conjugated anti-CD55 antibody (DAKO) for 30 min at 4°C, fixed in PBS containing 0.5% formaldehyde, and analyzed on a FACScan instrument (Becton Dickinson). Negative controls consisted of cells stained with the same concentration of an isotype-matched, FITC-labeled irrelevant antibody. Mean channel fluorescence of CD55 was determined for each concentration of RTX after subtracting the value obtained with the isotype-matched antibody from the specific anti-CD55 antibody value.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The amounts Lyn and Cbp/PAG remained constant both in rafts and soluble fractions from cells preincubated with RTX (Fig. 1A)
. At the same concentration of antigen-binding sites, the monovalent Fab fragment of RTX was only 10–20% as effective as the bivalent antibody in redistributing CD20, and the IgG1 control was without effect on CD20 distribution (data not shown). The RTX antibody remained bound to CD20 in rafts, and 6–7% of the input antibody coisolated with the detergent-resistant raft fractions obtained from 50 x 106 Raji cells (data not shown).
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without changes in the contents of Lyn and Cbp/PAG (Fig. 1A)
. The tyrosine phosphorylation of the exogenous substrate enolase was likewise decreased (Fig. 1, B and C)
, indicating a parallel decrease of the tyrosine kinase activity (Fig. 1C)
. On the contrary, the normal IgG1 control used at the same concentration as the anti-CD20 RTX antibody resulted in a 35% increase of Lyn autophosphorylation at 2.5 µg/ml, an increase which progressively returned to baseline values with 10 and 25 µg/ml (Fig. 1C)
. This increased protein kinase activity after incubation of rafts with the IgG1 control is probably attributable to interaction of the IgG1 with Fc receptors.
Accumulation of CD20 in Rafts Does Not Alter the Lipid and Phosphoprotein Compositions.
In an attempt to understand how the accumulation of CD20 in rafts functionally modulates the raft signaling platform, we compared by two-dimensional electrophoresis the rafts containing CD20 (obtained from Raji cells incubated with 10 µg/ml RTX) with rafts from control Raji cells for their overall patterns of lipids and 32P in vitro labeled phosphoproteins.
Equal amounts of phosphoproteins from CD20-containing and control rafts gave superimposable two-dimensional patterns, and the ratios of phosphoLyn to phosphoPAG were the same (Fig. 2)
, suggesting that accumulation of CD20 did not perturb kinase/substrate relationships in rafts but rather influenced the catalytic activity of Lyn. Moreover, the in vitro labeled phosphoproteins were not detectable as metabolically labeled proteins (data not shown) and therefore constitute a very select subset of raft proteins. The lack of detectable phosphorylation of metabolically labeled proteins strongly suggests that promiscuous phosphorylation did not occur in the isolated rafts, neither in absence, nor in presence of CD20. It is therefore reasonable to assume that the organization of kinases and substrates in isolated rafts had not been randomized by the isolation procedure and thus resembles the in vivo organization of signaling proteins or signalosome (28)
.
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. The lipid analysis of CD20-free and CD20-containing Raji rafts showed that both types of rafts were very similar in cholesterol-free fatty acid, phosphatidylethanolamine, neutral glycosphingolipids, phosphatidylcholine, and sphingomyelin contents. Sphingomyelin and alkali-resistant neutral glycosphingolipids were the major sphingolipids detected, a finding consistent with the notion that sphingolipids (in association with cholesterol) are the major components of liquid-ordered membranes (29)
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Indeed, incubation of isolated rafts with 20 mM OTG reduced GM1 and the sphingolipids reactive with the 2B5 mAb to 11 and 21%, respectively, in CD20-free rafts, whereas in CD20-containing rafts, about two to three times that amount of either lipid (27 versus 11% for GM1 and 60 versus 21% for 2B5-reactive sphingolipids) were recovered after OTG exposure (Fig. 4A)
, suggesting that CD20 accumulation increases the stability of raft sphingolipids toward OTG.
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. The Cbp/PAG protein was efficiently solubilized (
90%) in CD20- rafts at all concentrations of OTG tested, but in CD20+ rafts, the full solubilization of Cbp/PAG was only achieved at 50 mM. At 2.5 mM OTG, 55% of Cbp/PAG from CD20+ rafts remained sedimentable instead of 11% in CD20- rafts. The protective effect of CD20 was also measurable at 10 mM OTG (12% sedimentable in CD20- rafts versus 29% in CD20+ rafts). The GPI-anchored CD55 surface protein was progressively solubilized with increasing concentrations of OTG in CD20- and + rafts. The more efficient OTG extraction of CD55 from CD20+ rafts probably reflects the increased instability of CD55 in rafts containing CD20 (see below). The Lyn, Syk, and Csk kinases were detectable in similar amounts in CD20+ and CD20- rafts. For all three kinases, OTG extracted 25% of the proteins at the highest concentration of perturbant (data not shown). Sonication of rafts in the presence of OTG did not dissociate the kinases from sedimentable membranes (data not shown), strongly suggesting that Lyn, Syk, and Csk were not artifactually trapped in membrane vesicles.
The redistributed CD20 was almost as resistant to OTG as the intracellular Lyn, Syk, and Csk kinases, with 56% remaining sedimentable at 50 mM OTG (Fig. 4B)
, and this resistance of redistributed CD20 to OTG suggests a stable interaction with raft components.
Both CD20-enriched and CD20-free Rafts Are Sensitive to Extraction with MBCD.
The cyclic carbohydrate MBCD extracts cholesterol from membranes and perturbs the raft membranes differently than OTG (31)
. In contrast to OTG, CD20+ and CD20- rafts exposed to MBCD (10 mM) retained 57 and 56% of GM1 and all of the 2B5-reactive sphingolipids (Fig. 4A)
; thus, the presence or absence of CD20 did not influence the extractability of sphingolipids toward MBCD. Approximately 50% of the cholesterol in raft membranes was removed by 30 mM MBCD (data not shown). Exposure of either CD20-enriched or CD20-free rafts to MBCD (Fig. 4C)
released Cbp/PAG from sedimentable rafts with increasing concentrations of the drug (21% Cbp/PAG remaining sedimentable at 50 mM), whereas the amounts of sedimentable Lyn, Syk, and Csk were minimally affected after MBCD incubation at any concentration (data not shown). CD55 remained partially sedimentable (58–65%) at the highest MBCD concentration (50 mM) from either kind of raft. At 50 mM MBCD, 57% of the raft-redistributed CD20 remained sedimentable to the same extent as with 50 mM OTG (56%). MBCD at 30 and 50 mM released 80% of Cbp/PAG (21% remaining sedimentable), with minimal protective effect of CD20 in rafts.
The Presence of Cbp/PAG Is Important for Mediating the Effect of RTX on Raft-associated Lyn Kinase Activity.
To evaluate the contribution of Cbp/PAG to the effect of RTX, SKW6.4 lymphoblastoid cells expressing very low amounts of Cbp/PAG (Fig. 5, A and B)
were compared with Raji cells for their response to RTX (Fig. 5, C and D)
. The CD20 protein was redistributed to rafts in SKW6.4 cells (Fig. 5C)
with an efficiency comparable with that measured in Raji cells (Fig. 1A)
, and redistribution of CD20 to rafts was accompanied by a proportional decrease of CD20 in the soluble fractions (data not shown). Rafts isolated from SKW6.4 cells were enriched in GM1 and CD55 and free of CD45, similarly to Raji cells, but contained almost no Cbp/PAG. However, after incubation with RTX, the SKW6.4 lymphoblastoid cell line failed to respond with decreased protein tyrosine kinase activity (Fig. 5D)
, although the Lyn kinase was detectable by Western blotting as in Raji cells (Fig. 5A)
and was capable of autophosphorylation (Fig. 5B)
. As expected, minimal phosphorylation of Cbp/PAG was achieved in the in vitro kinase assay of SKW6.4 rafts, as compared with Raji rafts (Fig. 5B)
. To confirm the role of Cbp/PAG in the signaling response to RTX, the tumoral cell line DOHH2 (a follicular lymphoma cell line containing similar amounts of Cbp/PAG as Raji cells) was compared with EBV13 (a lymphoblastoid cell line containing reduced amounts of Cbp/PAG, as SKW6.4). Only in DOHH2 did RTX exposure significantly decrease the raft-associated kinase activity, whereas in EBV13 cells, no change in raft-associated kinase activity could be measured (data not shown). In both DOHH2 and EBV13, exposure to RTX resulted in the redistribution of CD20 to rafts, as in all CD20-positive cells (data not shown).
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). Similarly, the presence of CD20 in rafts correlated with an increase in the sensitivity of CD55 to both the bacterial PIPLC (B. thuringiensis) and the human PIPLD (Fig. 6)
. In rafts free of CD20 (isolated from control cells), CD55 remained insensitive to either PIPLC (1:1000; Fig. 6A
) or PIPLD (1:5000; Fig. 6B
), whereas in rafts containing CD20 (isolated from cells exposed to 10 µg/ml RTX), incubation with the same concentrations of PIPLC (in PBS) or PIPLD (in 0.02% TX-100) efficiently released CD55 from sedimentable rafts. Either kind of raft exposed to 10-fold higher concentrations of PIPLC or PIPLD showed complete cleavage of CD55 (data not shown). As a control, the Lyn kinase was similar in all samples, irrespective of CD20 content or phospholipase exposure.
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. Higher concentrations of PIPLC abolished the differences between RTX+ and RTX- cells, similarly to the responses of isolated rafts. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptor, a CD20 homologous tetraspan protein, also redistributes to rafts on cross-linking of the Fc receptor (35
, 36)
, and it is possible that a common transmembrane sequence motif or a common conformation of the two proteins facilitates their redistribution to rafts (37)
. The association of the multispan CD20 membrane proteins with rafts is induced by anti-CD20 antibody binding to the extracellular portion of the protein in the plasma membrane (4) . Here we show that the therapeutic anti-CD20 antibody RTX also redistributes CD20 to rafts with an efficacy comparable with that of murine anti-CD20 mAbs. In Raji cell rafts, RTX-cross-linked CD20 results in structural changes that cause: (a) increased resistance of sphingolipids and Cbp/PAG to the disruptive effects of OTG; (b) decreased activities of the raft-associated protein kinases and of the Lyn protein tyrosine kinase; and (c) increased sensitivity of CD55 to suboptimal concentrations of PIPLC and PIPLD.
Accumulation of CD20 in rafts neither modifies the amounts nor the nature of the sphingolipids that constitute those rafts. CD20 cross-linked by the IgG1 RTX monoclonal antibody probably assumes a configuration that favors its interaction with sphingolipids and diminishes the dissociating effects of OTG on isolated rafts. It can be envisioned that cross-linking of CD20 favors the formation of a lipid shell at one or several of CD20’s transmembrane domains and thus promotes the association of CD20 with lipid rafts (38) .
The removal of cholesterol efficiently releases Cbp/PAG, CD55, and GM1, but the membrane disruption consecutive to cholesterol removal is not mitigated by the presence of CD20. Cholesterol removal thus perturbs rafts through a different mechanism than OTG and does not directly dissociate sphingolipid aggregates or sphingolipid-protein complexes. The functional effect on rafts resulting from inclusion of cross-linked CD20 affects essentially the Lyn protein kinase and the GPI-anchored, C’ defense protein CD55.
The Lyn kinase is the main protein tyrosine kinase in Raji rafts; it cross-phosphorylates other Lyn kinases and phosphorylates Cbp/PAG. The accumulation of CD20 apparently does not prevent Lyn cross-phosphorylation, nor phosphorylation of Cbp/PAG, because the levels of phosphorylation of Lyn and Cbp/PAG decrease symmetrically after CD20 accumulation. The catalytic activity of Lyn is decreased after CD20 accumulation, and this catalytic modulation appears to depend on the presence of Cbp/PAG, as this down-modulation is not measurable in CD20+ cells that express suboptimal amounts of Cbp/PAG. It can be speculated that Cbp/PAG provides a specific environment around the Lyn kinase that relays the perturbation in the membrane caused by the accumulation of CD20. Src kinases are known to "breathe" and respond to very slight changes in their membrane environment (39) . Our interpretation of the present data would be that Lyn still contacts its substrates effectively when CD20 is in rafts but breathes less well when the transmembrane Cbp/PAG substrate is caught in between aggregates of sphingolipids and cross-linked CD20. The Lyn kinase docks at the raft inner leaflet and is minimally perturbed by OTG and MBCD that mostly affect molecules in the outer leaflet (sphingolipids, CD55, CD20, and Cbp/PAG). Cbp/PAG is a transmembrane, raft-residing protein that extends mostly intracellularly to make contacts with Src kinases and Csk (16) . In this respect, the sensitivity of Cbp/PAG to OTG underlies its capacity to interact with outer leaflet sphingolipids via the transmembrane portion. The present study further suggests that Cbp/PAG may communicate changes occurring in the extracellular aspect of rafts to kinases associated with the raft inner leaflet.
The complement defense protein CD55 remains in similar amounts in rafts after CD20 redistribution, but its sensitivity to suboptimal concentrations of both GPI-specific phospholipases C and D is increased, as well as its extractability to OTG. This may be caused by the interaction of CD20 with sphingolipids that leaves the GPI-anchored CD55 unprotected toward phospholipases, because it was shown for the GPI-anchored CD14 in ganglioside-deficient cells (40) . Increased sensitivity of CD55 to GPI-specific phospholipases is documented both in isolated rafts and in whole cells after RTX treatment. The increased susceptibility of CD55 on the intact cell surface is moderate compared with that measured in rafts and reflects that cell surface CD55 is both in and out of rafts; only a portion of the CD55 proteins is sequestered in rafts and susceptible to cleavage by PIPLC. The phospholipase-induced decrease of CD55 in rafts is expected to alter the complement defense of cells that bind RTX in vivo.
The efficiency of RTX in depleting lymphoma B cells in vivo is thought to depend both on the binding of its IgG1 Fc portion to complement or to Fc receptors (41) on phagocytes and on its capacity to modulate the cellular responses of the CD20+ B lymphocyte (42) . We show that RTX modifies the functional organization of raft components by inducing CD20 to associate with the stable sphingolipid clusters that form the backbone of rafts and cause modulation of Lyn. The modulation of Lyn also appears to depend on the presence of PAG and suggests that the catalytic activity of Lyn is controlled in part by Cbp/PAG.
In this study, we suggest that RTX-induced cross-linking of the tetraspan CD20 membrane protein causes a conformational change in the protein that promotes its stable association with lipid rafts. Consequently, the gangliosides and the Cbp/PAG protein in such lipid rafts become increasingly resistant to the disruptive effects of OTG. Because OTG selectively dissociates sphingolipid aggregates, it is likely that CD20 somehow strengthens the cohesion of sphingolipid aggregates and thus alters the structure of rafts. The functional consequences of such structural changes appear to influence interactions between the Cbp/PAG transmembrane signaling protein and the intracellular Lyn kinase. Such impact on rafts should be taken into account to explain the long-term effects of RTX on lymphoma cell survival in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Swiss National Science Foundation Grant 31-57696.99, Swiss Cancer League, Aargauische Krebsliga Grant SKL 426-2-1997, OncoSwiss Grant OCS 1117-02-2001, and Fondation Henri Dubois Ferrière Dinu Lipatti. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Faculty of Medicine, Centre médical universitaire, 1, rue Michel-Servet, 1211 Genève 4, Switzerland. Phone: 41-22-7025893; Fax: 41-22-7025746; E-mail: Daniel.Hoessli{at}medecine.unige.ch
3 The abbreviations used are: GPI, glycosylphosphatidyl inositol; Cbp/PAG, Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain; MBCD, methyl-ß-cyclodextrin; MES, 4-morpholinepropanesulfonic acid; PIPLC, phosphatidylinositol-specific phospholipase C; PIPLD, phosphatidylinositol-specific phospholipase D; TTBS, tween-containing tris-buffered saline; TKM, Tris, KCl and Mg-containing buffer; DRM, detergent-resistant membranes; mAb, monoclonal antibody; OTG, n-octyl-ß-D-glucopyranoside; RTX, rituximab; MOPS, 3-morpholinopropane sulfonic acid.
Received 6/20/02. Accepted 11/14/02.
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, CD20 (dotted line) in TX-insoluble DRM fractions 3–5; 
, CD20 (dotted line) in TX-soluble fractions 10–11, as shown in A. RTX-induced phosphorylation of Lyn (
), Cbp/PAG (
), and enolase (
), as shown in B, and IgG1-induced phosphorylation of Lyn (
) in DRMs. The phosphorylation and CD20 redistribution data were obtained in five independent experiments that yielded comparable results.
) rafts induced by increasing concentrations of RTX. The results shown in A–D are representative of three independent experiments.