HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ayllón, V. Articles by Rebollo, A. Search for Related Content PubMed PubMed Citation Articles by Ayllón, V. Articles by Rebollo, A.

Segregation of Bad from Lipid Rafts Is Implicated in the Induction of Apoptosis¹

Verónica Ayllón^*, Aarne Fleischer^*, Xavier Cayla, Alphonse García and Angelita Rebollo^2,*

^* Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain; Laboratoire de Physiologie de la Reproduction, Equipe Scientifique Associé Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, and Département d’Immunologie, Laboratoire de Signalisation Immunoparasitaire, Institut Pasteur, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl--cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptosis is a highly conserved and essential feature of development and homeostasis in higher organisms. Progress has been made in identifying extracellular, intracellular, and cell surface molecules that regulate apoptosis. Interestingly, positive and negative regulators have been identified at each level.

The Bcl-2 family proteins act as a decision point in the apoptotic pathway. The family is divided into two functional groups: antiapoptotic members and proapoptotic members, including the BH3-only member Bad, which promote apoptosis (1). Bad shares identity only in the BH3 domain and forms heterodimers with Bcl-2 and Bcl-x. Upon stimulation of cells with IL-3 and NGF, Bad becomes serine phosphorylated, resulting in association with 14-3-3 protein (2, 3). It has been shown that the association of 14-3-3 protein with Bad is dependent on serine 155 phosphorylation of Bad. In agreement, IL-2-stimulation of a murine T cell line induces serine 112 and 136 phosphorylation of Bad without association with 14-3-3 protein (4).

Localization of proteins to distinct subcellular compartments, including membranes, is a critical event in multiple cellular pathways such as apoptosis. Plasma membranes of many cell types contain microdomains, commonly referred to as lipid rafts, that are biochemically distinct from bulk plasma membranes (5). These domains are enriched in sphingolipids and cholesterol, and they can be isolated by density gradient ultracentrifugation. In T cells, a number of proteins involved in signal transduction copurify with lipid rafts isolated on sucrose gradient (6, 7). Lipid rafts were visualized in intact cells by confocal microscopy using fluorescently labeled cholera toxin (CTx)³ subunit B, which binds to the ganglioside GM1 (8). Disruption of raft integrity by a variety of methods inhibits early activation events, supporting a critical role for these domains in signaling.

Lipid rafts appear to play a central role in B cell activation. In mature B cells, signaling through the B cell Ag receptor is initiated from rafts and leads to activation. In immature B cells, B cell Ag receptor is excluded from rafts, and signaling leads to apoptosis (9, 10, 11). It has been shown that translocation and clustering of Fas into rafts triggers apoptosis in leukemic cells treated with the ether lipid ET-18-OCH(3) (12). In addition, Fas clustering in lipid rafts is a prerequisite for signaling and death (13, 14). CD24 induces apoptosis in human B cells via a raft-mediated signaling system (15). Finally, many receptors are inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. The sequestration of IL-2R -chain within lipid rafts restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2R - and -chains (16). In this manuscript we identify for the first time the attachment sites in the plasma membrane for the apoptotic molecule Bad. We also show that interaction of Bad with rafts is an active process regulated by IL-4. We propose that segregation of Bad from rafts is involved in the induction of apoptosis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, lymphokines, and reagents

TS1 is a murine T cell line that can be propagated independently in IL-2, IL-4, or IL-9. Cells were cultured in RPMI 1640 as previously described (17). Murine rIL-4 or supernatant of a HeLa subline transfected with PKCRIL-4.neo was used as a source of murine IL-4. FITC-labeled CTx B subunit, CTx-biotin, and methyl--cyclodextrin (M--CD) were obtained from Sigma-Aldrich (St. Louis, MO). Cy3- and Cy2-conjugated secondary Abs were purchased from Molecular Probes (Eugene, OR). Anti-mitochondria serum (mito 2813, pyruvate dehydrogenase) was a gift from Dr. A. Serrano (Centro Nacional de Biotecnologia, Madrid, Spain).

Immunoprecipitation and Western blot

Cells (1 x 10⁷) were IL-4-stimulated or -deprived and lysed for 20 min at 4°C in lysis buffer (50 mM Tris-HCl (pH 8), 1% Nonidet P-40, 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10% glycerol, and protease inhibitor mixture). Lysates were immunoprecipitated with the corresponding Ab. Protein A-Sepharose was added for 1 h at 4°C, and after washing, immunoprecipitates were separated by SDS-PAGE. Alternatively, cells were lysed in Laemmli sample buffer, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 5% nonfat dry milk in TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) and incubated with primary Ab in TBS/0.5% nonfat dry milk. Membranes were washed with 0.05% Tween 20 in TBS and incubated with peroxidase-conjugated secondary Ab. After washing, proteins were developed using the ECL system.

Cell cycle analysis

A total of 2 x 10⁵ IL-4-stimulated cells treated with or without M--CD were washed, resuspended in PBS, permeabilized with 0.1% Nonidet P-40, and stained with 50 µg/ml propidium iodide. At different times, samples were analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). Apoptosis was measured as the percentage of cells in the sub-G₁ region of the fluorescence scale having a hypodiploid DNA content. Cell cycle was also analyzed by annexin staining. A total of 2 x 10⁵ cells were washed with ice-cold PBS diluted in ice-cold binding buffer and stained with annexin and propidium iodide. Samples were maintained on ice for 10 min in the dark and then analyzed by flow cytometry.

Subcellular fractionation

Subcellular fractionation was performed as previously described (18, 19). Briefly, IL-4-stimulated or -deprived cells were washed in PBS and then resuspended for 2 min in extraction buffer STE (10 mM HEPES (pH 7.4), 1 mM EDTA, 0.25 mM sucrose, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 µg/ml pepstatin). The extract was inspected under the microscope, and >95% of the cells were lysed. The homogenates were applied to a linear gradient sucrose (0.73–1.9 M) and ultracentrifuged at 20,000 x g overnight. The banded organelles were recovered by syringe, diluted with an equal volume of 10 mM HEPES buffer, and sedimented at the speed appropriated for the respective organelles. The purity of the organelles was determined by Western blot using Abs against specific markers: anti-cytochrome c for mitochondria, anti-Lck and CTx-biotin for rafts, anti-calnexin for endoplasmic reticulum, and anti-caspase 3 for cytosol. For preparation of cytosol, the homogenate was precentrifuged at 750 x g for 10 min to remove nuclei and unbroken cells, followed by centrifugation at 100,000 x g for 1 h to clear off the membranes.

CTx-FITC labeling

IL-4-stimulated or -deprived cells were fixed with 1% paraformaldehyde for 5 min on ice, permeabilized, and then incubated with CTx-FITC (20 min, 6 µg/ml) and anti-Bad Ab for 1 h in PBS-BSA. Cy3-labeled secondary Ab was added and incubated for 1 h. Finally, and after several washing steps, cells were incubated with methanol at -20°C for 10 min, mounted with Vectashield medium, and analyzed by confocal microscopy. The program used for quantification of samples was Leica TSC NT version 1.5.451 (Leica, Lasertechnik, Heidelberg, Germany).

Cholesterol depletion

IL-4-stimulated serum-deprived cells were treated for 30 min at 37°C with 10 mM M--CD, washed, and then incubated with CTx-FITC and anti-Bad or anti-Lck Abs as described above. Secondary Ab was added and incubated for 1 h. Finally, cells were incubated with methanol at -20°C for 10 min and mounted as described above.

Triton X-100 flotation

IL-4-stimulated or -deprived cells were lysed in TXNE buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.2% Triton X-100) containing protease inhibitor mixture. Detergent-insoluble membranes were isolated by ultracentrifugation (17,000 x g, 4 h, 4°C) in a 30–35% gradient of Optiprep as previously described (Sigma-Aldrich, St. Louis, MO) (20).

Isolation of mitochondria and S-100 fraction

Mitochondria were isolated using a modification of the method described by Yang et al. (21). Briefly, 20 x 10⁶ cells were IL-4-stimulated or -deprived, harvested, and washed with ice-cold PBS. Cell pellet was suspended in 5 vol ice-cold buffer A (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 250 mM sucrose) supplemented with protease inhibitors. Cells were disrupted in a Dounce homogenizer (Kontes, Vineland, NJ), the nuclei were centrifuged (1,000 x g, 10 min, 4°C), and the supernatant was further centrifuged (10,000 x g, 15 min, 4°C). The resulting mitochondrial pellet was resuspended in buffer A and stored at -80°C. The supernatant was centrifuged (100,000 x g, 1 h, 4°C), and the resulting S-100 fraction was stored at -80°C.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bad associates with lipid rafts in IL-4-stimulated cells

We have previously reported that Bcl-2 is expressed in IL-2-stimulated cells and Bcl-x in IL-4-cultured cells (22). When IL-4-maintained cells are deprived of lymphokine, they undergo apoptosis (Fig. 1). As early as 4 h after IL-4 deprivation, 9% of the cells were apoptotic, reaching 40% at 24 h, whereas control IL-4-stimulated cells showed no significant level of apoptosis.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Effect of IL-4 deprivation on apoptosis. Cells were cultured in the presence of IL-4, deprived for the indicated times, and harvested. To analyze apoptosis, cells were diluted in ice-cold binding buffer, stained with annexin and propidium iodide, and analyzed by flow cytometry. The SD is shown where n = 4.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of IL-4 on association of Bad with 14-3-3 protein. Cytoplasmic extracts from 10 x 106 IL-4-stimulated or -deprived cells were immunoprecipitated with anti-Bad or anti-Raf Abs and blotted with anti-14-3-3, anti-Raf, and anti-Bad. Total extracts (lane T) were used as a positive control of 14-3-3 and RAFT expression. Similar results were obtained in three independent experiments.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Subcellular localization of Bad in IL-4-stimulated or -deprived cells. A, Anti-Bad, anti-Lck (rafts), CTx-biotin (GM1 ganglioside, rafts), anti-caspase 3 (cytosol), anti-calnexin (endoplasmic reticulum), and anti-cytochrome c (mitochondria) immunoblot analysis of subcellular fractions from IL-4-stimulated or -deprived cells. The fractions (nos. 1–4) were prepared by sucrose gradient ultracentrifugation and tested for purity using Abs against mitochondria, rafts, endoplasmic reticulum, and cytosol. The nuclear fraction is not shown in the blot (fraction 5). The protein loaded per well in each gradient fraction corresponds to that of 5 x 106 cells. Total extracts contained 30 µg protein. Similar results were obtained in three independent experiments. B, IL-4-stimulated or -deprived cells were Triton X-100-extracted and fractionated in Optiprep flotation gradient. Fractions were collected from the top to the bottom of the gradient and were analyzed by Western blot. Only the first, insoluble proteins (I) and the last fraction, soluble proteins (S) are shown. Similar results were obtained in two independent experiments.

The subcellular localization of Bad was also analyzed in mitochondrial and cytosolic fractions of IL-4-stimulated or -deprived cells. Bad was detected in the mitochondrial fraction of IL-4-stimulated cells (Fig. 4A). The amount of Bad associated with mitochondria increased upon IL-4 deprivation. Traces of Bad were detected in the cytosolic fraction of IL-4-stimulated or -deprived cells. The antiapoptotic molecule Bcl-x_L was weakly detected in the mitochondrial fraction of IL-4-stimulated cells, increasing after IL-4 deprivation. As an internal control of protein fractionation, the blot was probed with anti-caspase 3 (cytosolic marker), anti-mitochondria Mito 2813 (pyruvate dehydrogenase, mitochondrial marker), and anti-calnexin to show the lack of endoplasmic reticulum contamination in mitochondrial preparation. Total extracts (lane T) were used as a positive control of calnexin expression. Finally, we explored the association of Bad with some Bcl-2 family members. We performed coimmunoprecipitation experiments of cytoplasmic proteins under IL-4 stimulation or deprivation conditions using specific Abs. Bad was detected by Western blot in anti-Bcl-x_L immunoprecipitates of IL-4-stimulated cells, decreasing throughout the starvation period analyzed (Fig. 4B). Probing the membrane with anti-Bcl-x_L Ab showed similar levels in all analyzed conditions.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Association of Bad with mitochondria and Bcl-xL. A, Mitochondrial and cytosolic proteins were isolated from IL-4-stimulated or -deprived cells, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Bad, anti-Bcl-xL, anti-caspase 3 (cytosolic marker), anti-mitochondria Mito 2813 (pyruvate dehydrogenase, mitochondria marker), and anti-calnexin (endoplasmic reticulum marker). Protein bands were detected using the ECL system. Similar results were obtained in three independent experiments. B, Cytoplasmic lysates from IL-4-stimulated or -deprived cells were immunoprecipitated with anti-Bcl-xL, transferred to nitrocellulose, and blotted with anti-Bad and anti-Bcl-xL Abs. Protein bands were detected as described in A. Similar results were obtained in three independent experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Raft localization of Bad in IL-4-stimulated cells. A, IL-4-stimulated or -deprived cells were stained with CTx-FITC and either anti-Lck or anti-Bad Abs as indicated, followed by Cy3-labeled secondary Ab, and were analyzed by confocal microscopy. Similar results were obtained in three independent experiments. Single confocal sections show fluorescence in green (FITC) and red (Cy3). B, The profile of green and red fluorescence colocalization in a surface of the cell membrane was analyzed using quantification software (Leica). The peaks of fluorescence colocalization are labeled with stars. C, Freshly isolated thymocytes from mice were stained as described in A, followed by Cy3-labeled secondary Ab, and analyzed by confocal microscopy. Similar results were obtained in two independent experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Mitochondrial localization of Bad. IL-4-stimulated or -deprived cells were stained with anti-Bad and anti-mitochondria Abs, followed by FITC- and Cy3-labeled secondary Abs, and analyzed as described above. Similar results were obtained in three independent experiments. B, The profile of green and red fluorescence colocalization in a surface of the cell membrane was analyzed using quantification software (Leica). The peaks of fluorescence colocalization are labeled with stars.

Depletion of cellular cholesterol impairs the ability of glycosyl phosphatidylinositol-anchored proteins to associate with lipid rafts. To examine whether there is a similar requirement of cholesterol for the association of Bad with rafts, IL-4-stimulated cells were treated for 30 min with or without 10 mM M--CD in serum-free medium to deplete cellular cholesterol. Cells were then incubated with CTx-FITC and labeled with anti-Bad or anti-Lck Abs. Serum depletion alone weakly disrupted the association of Lck or Bad with lipid rafts (Fig. 7A). However, M--CD treatment caused a severe disruption of raft formation and association of Lck and Bad with rafts in IL-4-stimulated cells (Fig. 7A). This result indicates that disruption of raft formation by cholesterol depletion induces segregation of Bad and Lck from rafts in IL-4-stimulated cells. The profile of green and red fluorescence colocalization in control or M--CD-treated cells is shown in Fig. 7B. Background staining with secondary Abs, anti-rabbit Cy3, or anti-mouse Cy3 is shown in Fig. 7C.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. M--CD treatment abolishes association of Bad with rafts. A, IL-4-stimulated cells were serum-starved for 30 min and then treated with or without 10 mM M--CD for 30 min at 37°C before incubation with CTx-FITC and anti-Bad or anti-Lck Abs, followed by Cy3-labeled secondary Ab. Then cells were analyzed by confocal microscopy. Similar results were obtained in two independent experiments. Single confocal sections show green (FITC) and red (Cy3) fluorescence. B, The profile of green and red fluorescence colocalization in a surface of the cell membrane was analyzed using quantification software (Leica). The peaks of fluorescence colocalization are labeled with stars. C, Background staining with anti-rabbit Cy3 or anti-mouse Cy3.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. M--CD treatment induces apoptosis. IL-4-stimulated cells were serum-starved for 30 min, then treated with or without 10 mM M--CD for 30 min at 37°C, washed, and transferred to complete medium supplemented with IL-4. At different times, apoptosis was measured. The sub-G1 region of the fluorescence scale was used to determine the percentage of cells present in the initial step of apoptosis. Similar results were obtained in two independent experiments. , control cells; , M--CD-treated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our data show that 14-3-3 protein does not control the proapoptotic role of Bad. On the basis of this result, we analyzed thesubcellular distribution of Bad in IL-4-stimulated or -deprived cells, because localization of proteins is an important event in apoptosis. Our results show that different plasma membrane fractions can be separated using a subcellular fractionationsucrose ultracentrifugation gradient, because raft markers were successfully resolved from non-raft markers. We also isolated rafts and mitochondria by Triton X-100 flotation gradient and differential centrifugation, respectively. There are precedents for reversible raft association, as has been shown following the movement of single-fluorescence lipid molecules (23). In addition, after activation by ligand binding the epidermal growth factor migrates out of rafts into bulk plasma membrane (24). The association of proteins with lipid rafts can be modulated, because some proteins may be excluded from rafts by association with other proteins (25). Association of Bad with rafts may be involved in steps leading to Bad inactivation, because rafts do not constitute the final site of activation. IL-4 deprivation induces the segregation of Bad from rafts. This result suggests a two-step apoptotic process: segregation of Bad from rafts, which triggers apoptosis, and disorganization of lipid rafts during the apoptotic process. This is strongly suggested by results showing that disruption of cholesterol-rich rafts prevents Bad association and induces apoptosis in IL-4-stimulated M--CD-treated cells. Addition of FCS to IL-4-supplemented medium restores the lipid components of the plasma membrane, preventing the progression of apoptosis.

Localization of proteins to distinct subcellular fractions is an essential step in multiple signaling pathways, including apoptosis. According to this, it has been shown that some signaling molecules are sequestered in rafts. Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T lymphocytes (26). Our results strongly suggest that in the absence of association of Bad to 14-3-3 protein, Bad is sequestered in rafts, avoiding a proapoptotic role and association with partners. IL-4 deprivation-induced segregation of Bad from rafts correlates with translocation to mitochondria and induction of apoptosis. Restriction of intermolecular interactions by sequestration in lipid rafts has been also described for the -chain of the IL-2R, avoiding its association with the - and -chains of the IL-2R (16). We show for the first time the sequestration of Bad into lipid rafts as an IL-4-dependent mechanism that controls the availability of proapoptotic Bad.

	Footnotes

 
¹ This work was supported in part by grants from the Association pour la Recherche sur le Cancer (ARC 4437) and Institut Pasteur (PTR-60). The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council and Pharmacia Corp.

² Address correspondence and reprint requests to Dr. Angelita Rebollo, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, UAM 28049 Madrid, Spain. E-mail address: arebollo{at}cnb.uam.es

³ Abbreviations used in this paper: CTx, cholera toxin; M--CD, methyl--cyclodextrin.

Received for publication November 28, 2001. Accepted for publication January 29, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Downward, J.. 1999. How BAD phosphorylation is good for survival. Nat. Cell. Biol. 1:33.[Medline]
2. Hsu, H. Y., L. Kaipai, L. Zhu, A. J. Hsueh. 1997. Interference of Bad-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11. Mol. Endocrinol. 11:1858.[Abstract/Free Full Text]
3. Yang, E., J. Zha, E. J. Jockel, L. H. Boise, C. B. Thompson, S. J. Korsmeyer. 1995. Bad, a heterodimeric partner for Bcl-x_L and Bcl-2, displaces Bax and promotes cell death. Cell 80:285.[Medline]
4. Ayllón, V., X. Cayla, A. Garcia, F. Roncal, R. Fernandez, J. P. Albar, C. Martinez, A. Rebollo. 2001. Bcl-2 targets protein phosphatase 1 to Bad. J. Immunol. 166:7345.[Abstract/Free Full Text]
5. Brown, D. A., E. London. 1998. Functions of lipid rafts in biological membranes. Annu. Rev. Cell. Dev. Biol. 14:111.[Medline]
6. Langlet, C., A. M. Bernard, P. Drevot, H. Y. He. 2000. Membrane rafts and signaling by the multichain immune recognition receptors. Curr. Opin. Immunol. 12:250.[Medline]
7. Anderson, H. A., E. M. Hiltbold, P. A. Roche. 2000. Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation. Nat. Immunol. 2:156.
8. Harder, T., P. Scheiffele, P. Verkade, K. Simons. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141:929.[Abstract/Free Full Text]
9. Cheng, P. C., M. L. Dykstra, R. N. Mitchel, S. K. Pierce. 1999. A role for lipid rafts in BCR signaling and antigen targeting. J. Exp. Med. 190:1549.[Abstract/Free Full Text]
10. Aman, M. J., K. S. Ravichandran. 2000. A requirement for lipid rafts in B cell receptor induced Ca⁺⁺ flux. Curr. Biol. 10:393.[Medline]
11. Petrie, R. J., P. P. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165:1220.[Abstract/Free Full Text]
12. Gajate, C., F. Mollinedo. 2001. The antitumor ether lipid ET-18-OCH(3) induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells. Blood 98:3860.[Abstract/Free Full Text]
13. Grassme, H., A. Jekle, A. Richle, H. Schwarz, J. Berger, K. Sandhoff, R. Kolesnick, E. Gulbins. 2001. CD95 signaling via ceramide rich membrane rafts. J. Biol. Chem. 276:20589.[Abstract/Free Full Text]
14. Grassme, H., V. Jendrossek, J. Block, A. Reihle, E. Gulbins. 2002. Ceramide-rich membrane rafts mediate CD40 clustering. J. Immunol. 168:298.[Abstract/Free Full Text]
15. Suzuki, T., N. Kiyokawa, T. Taguchi, T. Sekino, Y. Katagiri, J. Fujimoto. 2001. CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system. J. Immunol. 166:5567.[Abstract/Free Full Text]
16. Marmor, M., M. Julius. 2001. Role of lipid rafts in regulating IL-2 receptor signaling. Blood 98:1489.[Abstract/Free Full Text]
17. Pitton, C., A. Rebollo, J. Van Snick, J. Theze, A. Garcia. 1993. High affinity and intermediate affinity forms of the IL-2R expressed in an IL-9-dependent murine T cell line deliver proliferative signals via differences in their transduction pathways. Cytokine 5:362.[Medline]
18. Hacki, J., L. Egger, L. Monney, S. Coms, T. Rosse, I. Fellay, C. Borner. 2000. Apoptotic crosstalk between the endoplasmic reticulum and mitochondria. Oncogene 19:2286.[Medline]
19. Millan, J., M. A. Alonso. 1998. MAL, a novel integral membrane protein of human T lymphocytes, associates with glycosylphosphatidylinositol-anchored proteins and Src-like tyrosine kinases. Eur. J. Immunol. 28:3675.[Medline]
20. Mañes, S., E. Mira, C. Gomez, R. Lacalle, P. Keller, P. Labrador, C. Martinez-A. 1999. Membrane rafts mediate front-rear polarity migrating cells. EMBO J. 18:6211.[Medline]
21. Yang, J., X. Liu, K. Bhalla, C. N. Kim, A. M. Ibarro, J. Cai, T. Peng, D. P. Jones, X. Wang. 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria. Science 275:1129.[Abstract/Free Full Text]
22. Gomez, J., C. Martinez-A, A. Gonzalez, A. Rebollo. 1998. Bcl-2 is differentially regulated by IL-2 and IL-4: role of the transcription factor NFAT. Oncogene 17:1235.[Medline]
23. Schutz, G. J., G. Kada, V. P. Pastushenko, H. Schindler. 2000. Properties of lipid microdomains visualized by single molecule microscopy. EMBO J. 19:892.[Medline]
24. Mineo, C., G. N. Gill, R. W. Anderson. 1999. Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem. 274:30636.[Abstract/Free Full Text]
25. Field, K. A., D. Holowka, B. Baird. 1995. FcRI-mediated recruitment of p53/p56^lyn to detergent resistant membrane domains accompanies cellular signaling. Proc. Natl. Acad. Sci. USA 92:9201.[Abstract/Free Full Text]
26. Kabouridis, P. S., J. Janzen, A. L. Magee, S. C. Ley. 2000. Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T cells. Eur. J. Immunol. 30:954.[Medline]

This article has been cited by other articles:

				S. Kierszniowska, B. Seiwert, and W. X. Schulze Definition of Arabidopsis Sterol-rich Membrane Microdomains by Differential Treatment with Methyl-{beta}-cyclodextrin and Quantitative Proteomics Mol. Cell. Proteomics, April 1, 2009; 8(4): 612 - 623. [Abstract] [Full Text] [PDF]

				D. Sikder and T. Kodadek The neurohormone orexin stimulates hypoxia-inducible factor-1 activity Genes & Dev., November 15, 2007; 21(22): 2995 - 3005. [Abstract] [Full Text] [PDF]

				M. Hekman, S. Albert, A. Galmiche, U. E. E. Rennefahrt, J. Fueller, A. Fischer, D. Puehringer, S. Wiese, and U. R. Rapp Reversible Membrane Interaction of BAD Requires two C-terminal Lipid Binding Domains in Conjunction with 14-3-3 Protein Binding J. Biol. Chem., June 23, 2006; 281(25): 17321 - 17336. [Abstract] [Full Text] [PDF]

				Y. Sun, S. Orrenius, S. Pervaiz, and B. Fadeel Plasma membrane sequestration of apoptotic protease-activating factor-1 in human B-lymphoma cells: a novel mechanism of chemoresistance Blood, May 15, 2005; 105(10): 4070 - 4077. [Abstract] [Full Text] [PDF]

				A. Fleischer, A. Ghadiri, F. Dessauge, M. Duhamel, X. Cayla, A. Garcia, and A. Rebollo Bad-Dependent Rafts Alteration Is a Consequence of an Early Intracellular Signal Triggered by Interleukin-4 Deprivation Mol. Cancer Res., December 1, 2004; 2(12): 674 - 684. [Abstract] [Full Text] [PDF]

				Y.-Y. Fan, L. H. Ly, R. Barhoumi, D. N. McMurray, and R. S. Chapkin Dietary Docosahexaenoic Acid Suppresses T Cell Protein Kinase C{theta} Lipid Raft Recruitment and IL-2 Production J. Immunol., November 15, 2004; 173(10): 6151 - 6160. [Abstract] [Full Text] [PDF]

				T. Garofalo, R. Misasi, V. Mattei, A. M. Giammarioli, W. Malorni, G. M. Pontieri, A. Pavan, and M. Sorice Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas. EVIDENCE FOR CASPASE-8-GANGLIOSIDE INTERACTION IN T CELLS J. Biol. Chem., February 28, 2003; 278(10): 8309 - 8315. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ayllón, V. Articles by Rebollo, A. Search for Related Content PubMed PubMed Citation Articles by Ayllón, V. Articles by Rebollo, A.