Proteome Analysis of Lipid Rafts in Jurkat Cells Characterizes a Raft Subset That Is Involved in NF-KB Activation Xiaolin Tu,† Aaron Huang,† David Bae,† Ndaisha Slaughter,† Julian Whitelegge,‡ Timothy Crother,§ Perry E. Bickel,| and Andre Nel*,† Division of Clinical Immunology and Allergy, Department of Medicine, The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry and Biobehavioral Sciences, Division of Infectious Disease, Department of Medicine and Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, and Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110 Received September 24, 2003
Lipid rafts are detergent-insoluble membrane domains that play a key role in signal transduction by the T-cell antigen receptor. Proteome analysis revealed the presence of amidosulfobetaine-soluble signal transducing, integral membrane, cytoskeletal, heat shock, and GTP-binding proteins in rafts prepared from Jurkat cells. Several of these proteins were recruited to rafts by CD3/CD28 costimulation. Of particular interest is the inducible association of activated IκB kinase complexes with raft vesicles that could be captured with anti-flotillin-1 antibodies. Following amidosulfobetaine solubilization, flotillin-1 and IKKβ underwent reciprocal co-immunoprecipitation. Treatment of Jurkat cells with methyl-βcyclodextrin disrupted the assembly and activation of this raft complex and also interfered in CD3/ CD28-induced activation of a NF-κB response element in the IL-2 promoter. Keywords: lipid rafts • proteomics • T-cell antigen receptor • NF-κB signaling
Introduction Ligation of the T-cell antigen receptor (TCR) by peptide antigen-MHC complexes leads to T-cell activation.1-3 One of the earliest recognizable signaling events is activation of the protein tyrosine kinases (PTK), Lck, and the TCR ζ-chainassociated protein kinase ZAP-70.1-3 This early wave of protein tyrosine phosphorylation leads to the activation of downstream signaling pathways, including intracellular calcium flux and activation of the NF-κB and Ras-MAP kinase cascades.1-5 CD28 delivers important costimulatory signals that strengthen the TCR signal and are critical for IL-2 production, proliferation, survival, and prevention of anergy in naive T-cells.6-10 While the exact role of phosphatidylinositol-3 kinase (PI-3K), Tek kinases, Grb2, Jun kinases (JNK), and the NF-κB cascade in CD28 signaling still needs to be clarified,5-11 a major recent advance has been the demonstration that CD28 plays a role in the assembly of signaling molecules at the site of T-cell contact with the antigen presenting cell.12 In this contact zone or immunological synapse, TCR-associated signaling components, * To whom correspondence should be addressed. Tel: (310) 825-6620. Fax: (310) 206-8107. E-mail:
[email protected]. † Division of Clinical Immunology and Allergy, Department of Medicine, University of California. ‡ The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry and Biobehavioral Sciences, University of California. § Division of Infectious Disease, Department of Medicine and Department of Microbiology, Immunology and Molecular Genetics, University of California. | Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine. 10.1021/pr0340779 CCC: $27.50
2004 American Chemical Society
costimulatory receptors, and adhesion molecules are assembled into a large conglomerate of signaling molecules known as the supramolecular activation clusters (SMACs).13-16 CD28 assists in regulating this molecular assembly through its effect on the cortical cytoskeleton as well as recruitment of lipid rafts to the TCR synapse.2, 16-19 Lipid rafts are membrane microdomains that contain clusters of glycosphingolipids, cholesterol, and other lipids and are also known as glycolipid-enriched membrane domains (GEMs), or detergent-insoluble glycolipid-enriched domains (DIGs).20-25 Due to the tight packing between cholesterol and glycosphingolipids, lipid rafts interact with hydrophobic proteins as well as with molecules with glycophosphatidylinositol anchors or dual fatty acylations.20-24 This allows preassembly of TCRassociated signaling components such Lck (T-cell specific Src kinase), linker for activated T-cells (LAT), and small GTPbinding proteins such as Ras and Rac-1.22-25 Although lipid rafts are critical for TCR signaling, we still need to learn a great deal about their composition and function.12,23-25 Recent studies demonstrate the existence of heterogeneous rafts on the T-cell surface, suggesting that a variety of raft types could contribute to TCR signaling.26-31 CD28 could contribute to this process by regulating the trafficking and coalescence of different raft types at the TCR synapse.26-31 The power of proteomics has recently been used to identify new lipid raft components, with the potential to enhance our knowledge of the membrane and receptor-induced events that are required for optimal T-cell activation. We used novel approaches of raft preparation, solubilization, and immobilizaJournal of Proteome Research 2004, 3, 445-454
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research articles tion to identify raft-associated components by micro-liquid chromatography tandem mass spectrometry.26 This led to the identification of integral membrane, signaling and cytoskeletal proteins, a number of which were recruited to a flotillin-1associated raft subset. We focus on the functional significance of this raft subset in the activation of the IκB kinase (IKK) complex and transcriptional activation of the IL-2 promoter during CD3/CD28 costimulation.
Experimental Section Reagents. OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals (Raritan, NJ), and the 9.3 mAb (anti-CD28) was provided by Bristol-Meyer Squibb (Princeton, NJ). The primary stimulating Abs were cross-linked with mAb 187.1 (BristolMeyer Squibb). Polyclonal anti-IKKR, anti-IKKβ, as well as antivimentin mAb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-flotillin-1, anti-flotillin-2, and anti-IKKγ were obtained from Transduction Laboratories (Lexington, KY). Monoclonal SAPK/JNK antibody and phosphoSAPK/JNK antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit anti-actin was purchased from Sigma Aldrich (St. Louis, MO). Monoclonal anti-Lck and anti-LAT were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-HSP60 and anti-HSP90 were purchased from Stressgen (Victoria, BC, Canada). PMA and ionomycin were purchased from Sigma. GST-IκBR(1-100) was provided by Dr. Bryden Bennett (Signal Pharmaceuticals, San Diego, CA). HRPconjugated anti-mouse and anti-rabbit antibodies, as well as Protein A and G beads were purchased from Amersham Pharmacia (Little Chalfont, Buckinghamshire, England). ASB14 was purchased from CalBioChem (La Jolla, CA). Polyclonal anti-flotillin-1 antiserum was raised in rabbits.32,64 Cell Culture and Stimulation. The Jurkat T cell clone, BMS2, was grown in RPMI 1640 supplemented with 10% FCS and antibiotics. Cells were stimulated with 0.25 µg/mL of anti-CD3 (OKT3) or 0.25 µg/mL of OKT3 plus 2.5 µg/mL of anti-CD28 (9.3) mAb and secondarily cross-linked with 10 µg/mL of mAb 187.1 for various lengths of time as indicated. Sucrose Gradient Centrifugation and Preparation of Lipid Raft Pellets. Lipid rafts were prepared from Jurkat BMS2 cells by sucrose gradient flotation as previously described.9,33,34 Resting or stimulated Jurkat cells (2 × 108 cells/aliquot) were washed with PBS buffer. Cells were lysed for 30 min on ice in 2 mL of TNE/P buffer (25 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, 0.2 mM PMSF, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 µL/mL aprotinin, 1 mM Na3VO4, and 1 mM NaF) containing 1% Triton ×100 (TX100). The lysates were mixed with 2.5 mL of TNE/P buffer containing 80% (w/v) sucrose and transferred to SW41 ultracentrifuge tubes (Beckman). The mixture was overlaid with 6 mL of 35% sucrose in TNE/P buffer, followed by 3 mL of 5% sucrose in TNE/P, and ultracentrifuged at 100000g for 16 h at 4 °C. Eleven 1-mL fractions were sequentially collected from the top. To concentrate the lipid raft vesicles and eliminate the sucrose,the GM1-positive fractions (no. 3 in every case) were diluted with two volumes of TNE/1% TX100 in polycarbonate centrifuge tubes (13 × 51 mm).26 Samples were centrifuged at 100000g for 1 h at 4 °C in a Beckman TL-100 Ultracentrifuge. The pellets were washed 3× in TNE/1% TX-100 using ultracentrifugation at 100000g. Raft pellets were used for 2-D electrophoresis or were resuspended in TNE/1% TX100 for raft immune immobilization.26 446
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GM1 Ganglioside Staining. One microliter of each sucrose gradient fraction was dot-blotted onto Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk in blocking buffer (6% BSA, 10 mM Tris/HCl pH 7.2, 0.9% NaCl, 0.01% NaN3), membranes were washed in PBS/0.1% Tween 20 (PBST) and overlaid with HRP-conjugated cholera toxin B subunit (1:2,000 dilution) for 1 h. After washing in PBST, membranes were incubated with Supersignal Chemiluminescent substrate (Pierce, Rockford, IL) and subjected to autoradiography. Detergent Extraction of Lipid Raft Pellets with ASB-14. Lipid raft vesicles, concentrated by ultracentrifugation, were treated with 1% of ASB-14 in TNE/P buffer for 30 min on ice.26,35 After ultracentrifugation at 100000g for 1 h at 4 °C, the supernatant was collected as detergent-soluble material (DSM) and the pellet as detergent-insoluble material (DIM).26 Immune Isolation of Lipid Raft Vesicles. Lipid raft pellets were prepared from resting and stimulated Jurkat cells as described above.26 Raft pellets were resuspended in 200 µL of TNE/1% TX100 and incubated with 2 µg of polyclonal antiflotillin-1 or anti-IKKβ at 4 °C for 3 h during constant agitation.26 These suspensions were incubated with 20 µL of preblocked protein A-Sepharose beads overnight. Controls consisted of beads prior absorbed with nonimmune serum (NIS). Immobilized raft complexes were washed four times in TNE buffer containing 200 mM NaCl and prepared in 1x SDS sample buffer. Raft proteins and wash supernatants were electrophoresed by 10% SDS-PAGE and transferred to a PVDF membrane to identify raft components by immunoblotting. To determine whether flotillin-1 may directly interact with raft-associated LAT, Lck, and the IKKs, the raft pellets were extracted with 1% ASB-14 before anti-flotillin-1 or anti-IKKβ immune precipitation and immunoblotting for raft-associated components.26 Immunoprecipitation. These experiments used DSM, obtained by extracting lipid raft pellets with ASB-14 as described above. These extracts were incubated with 2 µg of the indicated antibodies, rocked for 1 h at 4 °C, and then incubated with 20 µL of the protein A-sepharose beads, prior blocked with 3% BSA.9,36 The immune complexes were washed 4× with PBS containing 200 mM NaCl and boiled in SDS sample buffer. SDS-PAGE and Western Blotting. Samples were examined by sodium sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described by Laemmli.37 Proteins were eletrophoretically transferred onto a PVDF membrane (0.45 µm pore size), which was blocked with 5% nonfat dry milk at room temperature for 1 h. Membranes were overlaid with the indicated concentrations of the primary Abs, followed by a 1:2000 dilution of the secondary HRP-conjugated Ab37,58 Blots were developed by enhanced chemiluminescense as described above. Two-Dimensional (2-D) Electrophoresis. 2-D electrophoresis was performed as previously described.39-42 Lipid raft pellets from 1.6 × 108 Jurkat cells were solubilized in 250 µL of isoelectric focusing (IEF) buffer (7 M urea, 2 M thiourea, and 1% ASB-14) at room temperature for 16 h. The solubilized extracts were replenished with 30 mM DTT and 0.5% ampholytes (pH 3-10) and incubated with commercial dry-strips (Immobiline, pH 3-10, Amersham Pharmacia) for 16 h. IEF was conducted at 0-500 V × 1 min, 500-4000 V × 90 min, and 8000 V for a total of 32 000 V h. The second dimension was performed in a 12% polyacrylamide gel at 80 V. After fixation in 10% methanol and 7% acetic acid, the gel was stained in Sypro Ruby and destained in 10% methanol and 7% acetic acid. Protein spots were visualized by scanning in an
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Identification of Lipid Raft-Associated Proteins
Alpha Innotech Imager (San Leandro, CA). Protein spots were sliced from the gel, dehydrated in acetonitrile for 30 min, and centrifuged in a Speedvac (Savant, Farmingdale, NY). After rehydration in 100 mM ammonium bicarbonate for 30 min, gel slices were dehydrated a second time and swelled overnight in an equal volume of 12.5 µg/µL of modified trypsin (Promega, Madison, MI) at 37 °C.43 The digested peptides were extracted with 5% formic acid and 50% acetonitrile and dried in a Speedvac. Mass Spectrometry. Protein identification was performed by micro-liquid chromatography tandem mass spectrometry (µLC/ MS/MS).40-42 Dried samples were dissolved in 5 µL of 70% acetic acid and loaded onto the µLC system using a 5 µL loop. A splitter (Accurate, LC Packings, Dionex Corp) was used to provide a 2 µL/min flow rate for µLC provided by a Surveyor HPLC (ThermoFinnigan, San Jose) running at 200 µL/min. The system was equilibrated in 95% buffer A and 5% buffer B (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) for 20 min prior to sample injection and then eluted with a linear gradient to 100% B over 70 min. A 180 µm × 15 cm reversed-phase column was used (5 µm 300A PLRP/ S, LC Packings, Dionex Corp). Column eluent was delivered directly to the electrospray source via a 50 µM coated emitter (New Objective) which required around 3 kV for stable spray performance. Data dependent acquisition was achieved using an ion-trap mass spectrometer (LCQ-DECA, Ion Trap Agilent), operated according to the manufacturer’s instructions. Single charged ions were excluded from MS/MS experiments. Data sets were screened against protein sequence databases (NCBI) using appropriate software (Mascot, Matrix Sciences; Sequest, ThermoFinnigan and Sonar, Proteometrics). Sequence tags were inspected manually and would only rarely be accepted where the X-correlation score was less than 3.0 (Sequest). Depending on which instrument was used for data acquisition, a single high quality sequence tag was sufficient to identify a protein if a BLAST search revealed the peptide to have a unique sequence. Luciferase Assays. A total of 107 Jurkat cells were transiently transfected with 10 µg of CD28RE/AP-1 luciferase construct together with 10 µg of the Renilla reporter vector (Promega, Madison, MI). The cells were incubated for 10 min on ice in a 0.4 cm cuvette in a total volume of 250 µL of RPMI 1640 containing 20% FCS. Cells were pulsed at 240 V and 950 µF using a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, CA).36,37 After resting for 24 h, an aliquot of the transfected cells was treated with 10 mM MβCD for 15 min at 37 °C, following which MβCD-treated and untreated cells were stimulated with anti-CD3 or anti-CD3 + antiCD28 mAb as described above. After 1 h at 37 °C, the cells were lysed in luciferase buffer (Promega, Madison, MI) and dual-luciferase activities measured by using 20 µg of lysate protein in a Lmax Luminometer (Molecular Devices Corp. Sunnyvale, CA). Renilla luciferase values were used to correct for the efficiency of transfection and the firefly luciferase values adjusted accordingly. Immune Complex Kinase Assays. Kinase assays were performed as previously described.37,44 Briefly, lipid rafts were treated with 1% ASB-14 in TNE/P buffer. DSM was incubated with 2 µg of anti-IKKβ for 1 h at 4 °C. Protein A beads (20 µL) were added to the mixture, rocked overnight at 4 °C, following which the immune complexes were washed 3× with kinase buffer. Kinase reactions were initiated by the addition of 10 µCi [γ-32P]ATP and 3 µg GST-IκBR and carried for 30 min at 30
Figure 1. Preparation and detergent extraction of lipid raft pellets. Stimulated and resting Jurkat cells (1 × 108 cells/aliquot) were extracted in a TNE/P buffer containing 1% TX-100 and used for sucrose gradient centrifugation as previously described.26,33,34 Eleven 1 mL fractions were collected, and the presence of GM1 was determined by spotting on a PVDF membrane which was overlaid with HRP-conjugated CtxB. (A): Immunoblotting of selected sucrose fractions shows the presence of Lck, LAT, and PKCθ. 50 µL of each fraction was used for immunoblotting as described in the Experimental Section. Notice that Lck migrates as a 56 and 60 kD species in activated cells. (B): 700 µL of fraction 3 was mixed with two volumes TNE/P buffer plus 1% TX-100 and centrifuged at 100000g for 1 h. The pellets were washed 3× in the same buffer, resuspended in 200 µL of TNE/P buffer, aliquoted, and replenished with 1% Triton X-100, 1% Triton X-114, 60 mM octyl glucoside (OctG), 1% CHAPS, or 1% ASB-14 for 30 min. These extracted materials were re-ultracentrifuged at 100000g, and the detergent-soluble material (DSM or supernatant) and detergent-insoluble material (DIM or detergent resistant pellets) was used for immunoblotting with anti-LAT, anti-Lck, and antiflotillin-1 as described in the Experimental Section. The above data were reproduced three times. IB ) immunoblot.
°C. Products were resolved by SDS-PAGE, and the phosphorylated substrate was detected by autoradiography.
Results Presence of Previously Characterized as well as novel Membrane Proteins in Lipid Rafts Prepared from Jurkat T-cells As Shown by Protein Mass Spectrometry. Membrane domains that are highly enriched in lipid raft components can be purified on the basis of TX100 insolubility and buoyancy in a sucrose gradient.21-25 Rafts collect at the 5-30% sucrose interface (fractions 3 and 4), as demonstrated by immunoblotting for the ganglioside GM1 (not shown), as well as the presence of the TCR-associated signaling molecules, Lck and LAT (Figure 1A). While these proteins show constitutive raft association, a key downstream signaling component, PKCθ, was recruited upon CD3/CD28 costimulation, as well as to a lesser degree during anti-CD3 treatment (Figure 1A). While it is possible to use the GM1-positive fractions to identify raft components by immunoblotting, the sucrose in the medium interferes in 2-D electrophoresis and raft immune immobilization.26 This problem was overcome by diluting the raft vesicles in a sucrose-free buffer and performing high-speed centrifugaJournal of Proteome Research • Vol. 3, No. 3, 2004 447
research articles tion (100000g) as previously described.26 This dilution and centrifugation leads to the collection of the raft vesicles at the bottom of the centrifuge tube.26 Quantitatively, these raft vesicles maintain a full complement of raft-associated proteins (e.g., GM1, LAT and Lck), while nonraft components are washed away.26 These raft pellets were extensively washed in a sucrose-free buffer for further biochemical analysis.26 To identify novel raft components by protein mass spectrometry, it was necessary to solubilize the TX100-insoluble raft components. We experimented with a range of different detergents, including octylglucoside, Triton X-114, CHAPS, and ASB-14. The best results were obtained with ASB-14, a zwitterionic amidosulfobetaine detergent with linear 14C alkyl chains (Figure 1B35). ASB-14 solubilized >75% Lck and almost all the LAT molecules (Figure 1B) which is in agreement with its ability to solubilize hydrophobic membrane proteins, including difficult-to-purify bacterial lipoproteins and lymphocyte membrane proteins.26,35 ASB-14 was therefore included in the solubilization buffer for all 2-D electrophoresis procedures. 2-D electrophoresis resolved approximately 60 polypeptides in lipid rafts prepared from unstimulated Jurkat cells (Figure 2A). These spots were sliced from the gel, digested with modified trypsin, and used for micro-liquid chromatography tandem mass spectrometry with data-dependent acquisition. To date, we have positively identified 40 of these polypeptides (Table 1). These include proteins belonging to the following major families or classes: (i) TCR-associated signaling molecules (TCR-R, Lck, calmodulin); (ii) cytoskeletal proteins or proteins involved in locomotion and membrane function (lamin B1, vimentin, tubulin, actin, tropomyosin, myosin regulatory light chain); (iii) heatshock proteins (HSP60, HSP90); (iv) GTP-binding regulatory proteins (Gs(R), Gs(R)-1, Gs(R)-2, Gi(R)-2, Gq, Gβ2, Gβ4); (v) intrinsic membrane proteins (flotillin-2, stomatin-like protein); and (vi) transport/channel proteins (Kv3.1 potassium channel subunit, H+ transporting ATPase). While anti-CD3/CD28 treated Jurkat cells showed a similar array of raft proteins, proteome analysis revealed the recruitment or increased abundance of HSP60, HSP90, vimentin, Rho GDI, and calmodulin (Figure 2B). Taken together, these results indicate that lipid rafts contain a range of proteins with diverse biological activity, including components that contribute to TCR signal transduction such as the receptor itself, Lck, calmodulin, and actin. Confirmation of the Constitutive and/or Inducible Association of Heat Shock Proteins, Cytoskeletal Proteins, and Flotillins with Lipid Rafts by Immunoblotting. Flotillins are integral membrane proteins that have been shown to be present in caveolae and lipid rafts.26,45-48 Immunoblotting of sucrose gradient fractions demonstrated the presence of flotillin-1 and flotillin-2 in GM1-positive rafts obtained from resting Jurkat cells (Figure 3A). These GM1-positive fractions include the TCR-associated adapter protein LAT (Figure 3A). No flotillins were present in the detergent-soluble membrane fractions (e.g., fraction # 10), confirming their constitutive raft association (Figure 3A).26 Although cellular stimulation with anti-CD3 or anti-CD3/CD28 mAb did not affect the raft association of the flotillins (Figure 3A), these stimuli did shift some flotillin-2 (as well as LAT) to fractions 3 and 4 (Figure 3A). Although the explanation for this change in migration is not clear, it could signify the formation of higher density rafts due to increased recruitment of signaling complexes to the membrane (i.e., formation of the SMACs). This notion is in 448
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keeping with our demonstration that the flotillins cluster at the site of CD3/CD28 ligation.26 Heatshock proteins are molecular chaperones that play an important role in signaling processes, including the intracellular assembly and transport of protein kinase complexes.49,50 In contrast to the constitutive raft association of the flotillins, HSP60 and HSP90 where recruited to detergent-resistant membrane domains during CD3/CD28 costimulation (Figure 3A). For cytoskeletal proteins the picture was mixed (Figure 3B). While actin and vimentin were associated with GM1positive fractions in resting cells, there was increased vimentin staining in fraction 3 during CD3/CD28 costimulation (Figure 3B). Vimentin also appeared to increase in fractions 8-10 during CD3/CD28 costimulation (Figure 3B). The latter fractions contain detergent-soluble membrane components. Talin was recruited to fraction 3 as well as to fractions 8-10 during CD3/CD28 costimulation (Figure 3B). This is compatible with digital imaging studies which show that talin is recruited to the periphery of the SMACs.14 The recruitment of components of the IκB kinase (IKK) complex will be discussed later (Figure 4B). Use of Flotillin-1 to Immune-Immobilize Raft Domains That Contain IKK-Components. Although much needs to be discovered about the role of flotillins, these proteins have been shown to co-localize with Fyn-kinase and stimulatory GPIlinked proteins and have been found in neurons and T cells.47 This suggests that the flotillins may be important structural proteins that play a role in signal transduction.26,47 To further explore that notion, we asked whether the flotillin-1 containing rafts include other signaling molecules. The purification of raft vesicles that are decorated with flotillin-1 is a theoretical possibility, based on the demonstration that anti-caveolin-1 antibodies, immobilized on a solid support, can capture caveolae.51 Polyclonal anti-flotillin-1 antibodies, raised in rabbits,32 were immobilized on protein A beads and incubated with a suspension of sucrose-free rafts. This incubation of flotillin1-conjugated beads with the raft suspension led to the capture of flotillin-1 containing rafts (Figure 4A), whereas the incubation of the flotillin-1-conjugated beads with nonimmune serum (NIS) did not capture any raft components (Figure 4A). Further immunoblotting showed that the flotillin-1 containing rafts, prepared from CD3/CD28 costimulated cells, include LAT, Lck, vimentin and a component of the IKK complex, IKKβ (Figure 4A). Except for LAT, which was present in lesser abundance in resting cells, the above components were absent from the flotillin-1 rafts prepared from resting or anti-CD3 treated cells (Figure 4A, lanes 1 and 2). This finding is in agreement with the demonstration that CD28 costimulation is required to recruit IKKR, IKKβ, and IKKγ to GM1-positive sucrose fractions (Figure 4B). The presence of flotillin-1 and IKKβ in the same raft subset was confirmed by reciprocal immune precipitation using immobilized anti-IKKβ to capture flotillin-1 containing rafts from CD3/CD28 costimulated cells (Figure 4C). In contrast to IKKβ, the HSPs (HSP60 and HSP90), failed to coprecipitate with flotillin-1, but could be detected in raft vesicles that remained in the supernatant (Figure 4A). This suggests that these proteins are associated with a flotillin-1 independent raft subset (Figure 4A). Please notice that anti-CD3/CD28 costimulation leads to increased HSP60 and -90 association with this subset. The above results do not imply direct IKKβ interaction with flotillin-1, vimentin, LAT, or Lck, but merely indicate that these proteins reside in the same raft subset. To determine if direct
Identification of Lipid Raft-Associated Proteins
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Figure 2. 2-D electrophoresis and protein mass spectrometry to identify novel raft proteins. Lipid raft pellets were prepared from 1.6 × 108 resting and stimulated Jurkat cells as described in Figure 1. After solubilization in IEF buffer, the extracted material was used to conduct 2-D electrophoresis. Protein spots were identified by Sypro Ruby staining. These spots were sliced from the gel and used to obtain protein sequence data by µLC/MS/MS. Polypeptides with a high probability score in one of the three protein sequence databases are numbered and their identities disclosed in Table 1: (A) lipid rafts obtained from unstimulated cells; (B) lipid rafts obtained from TCR/CD28 stimulated cells.
protein-protein binding may be involved, ultracentrifuged raft pellets were extracted with ASB-14 and the DSM used for immune precipitation and Western blotting. ASB-14 successfully extracted most flotillin-1 (Figure 1B) as well as the majority of the IKKs (Figure 5A). Using the DSM to perform flotillin-1 immune precipitation, we showed coprecipitation of IKKβ in
rafts prepared from CD3/CD28 costimulated but not control cells (Figure 5B). No immune precipitation was obtained with NIS (Figure 5B). Similar results were obtained using reciprocal immune precipitation, namely flotillin-1 coprecipitation with IKKβ (Figure 5C). A considerable amount of flotillin-1 remained in the supernatant, indicating that flotillin-1 is more abundant Journal of Proteome Research • Vol. 3, No. 3, 2004 449
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Table 1. Lipid Raft Proteome Analysis, Jurkat BMS2 Cells 2D no.
1a 2 3a b c d e 4 5a b 6a 7a b ca d 8 9 10 11a b c 12 13a b 14 15 16a b 17 18 19 20 21 22 23 24 25 26a 27 28a
protein
access no.
MW (kD)
probability scoreb
calcd MW (kD)
HSP90 lamin B1 human TCRR (R/δ constant region) human TCRR (R/δ constant region) human TCRR (R/δ constant region) human TCRR (R/δ constant region) human TCRR (R/δ constant region) Kv3.1 potassium channel subunit albumin precursor albumin precursor HSP60 vimentin vimentin vimentin vimentin Dehydrolipoamide dehydrogenase Lck H+-transporting ATPase tubulin, β5 tubulin, R tubulin, β5 ATP synthase β chain dihydrolipoamide succinyltransferase dihydrolipoamide succinyltransferase flotillin-2 eukaryotic transaction inhibition factor 4A actin γ actin stomatin-like protein GTP-binding regulatory protein, Gs(R)-2 guanine nucleotide-binding protein, G(s)R guanine nucleotide-binding protein, Gq guanine nucleotide-binding protein, G(s)R-1 guanine nucleotide binding protein, G(i)R-2 guanine nucleotide binding protein, Gβ2 guanine nucleotide binding protein, Gβ4 Tropomyosin Rho GDP dissociation inhibitor (GDI) beta myosin regulatory light chain calmodulin
P10413 NP_005564 M94081 M94081 M94081 M94081 M94081 AAD34618 AF130117 CAA41735 CAA37654 P20152 AAA61282 XP_042950 AAA61281 NP_000099 AAA18225 B44138 AAD33992 XP_028720 BAB27292 P06576 Q9N0F1 NP_001924 NP_004466 NP_001407 ATBOG P18603 NP_038470 RGBOGA P24799 XP_040972 NP-536351 NP_002061 P11017 NP_038559 AAH29186 NP_001166 AAH32748 1003191A
86 66.5 62 62 54.5 46.2 38.2 57.9 57.9 57.9 57.0 54.8 54.8 54.8 52.3 50.8 50.1 49.2 48.9 48.9 48.1 48.5 48.0 48.0 43.6 44.7 42.6 42.1 38.2 40.0 41.0 38.2 38.2 33.8 33.5 32.5 34.8 26.1 19.2 14.0
3.0541 (Sequest) 5.1 × 10-5 0.16 0.35 0.31 0.14 0.13 0.32 1.6 × 10-2 1.9 × 10-17 88 (Mascot) 5.7 × 10-16 3.2 × 10-13 3.2 × 10-22 248 (Mascot) 4.4 × 10-2 88 (Mascot) 224 (Mascot) 3.5 × 10-31 3.0 × 10-15 5.3 × 10-25 440 (Mascot) 1.5 × 10-2 68 (Mascot) 2.3 × 10-44 7.3 × 10-3 3.2 × 10-31 181 (Mascot) 3.898 (Sequest) 5.3 × 10-8 46 (Mascot) 3.4459 (Sequest) 1.8 × 10-3 2.2 × 10-2 7.1 × 10-11 4.4 × 10-4 3.0 × 10-3 7.3 × 10-5 5.7 × 10-16 5.1 × 10-14
90 66.6 2.3 2.3 2.2 2.2 2.3 65.9 56.7 69.3 57.9 53.7 53.7 53.7 41.5 54.8 56.0 56.5 50.2 51.2 50.0 56.5 48.9 48.9 47.4 46.1 42.0 41.8 38.5 41.0 44.5 42.0 44.7 41.0 36.5 37.0 33.1 23.0 18.9 16.7
a New or increased expression during the co-stimulation with anti-CD3 and CD28. b The probability scores were obtained using Sonar (Service.proteometrics.com/prowl) unless otherwise stated. Sonar scores < 1 are regarded as significant matches, with confidence increasing as the score gets smaller. Sequest scores > 3.0 are regarded highly significant. Mascot scores > 60 are regarded as confident identifications.
in these rafts than IKKβ (Figure 5C). HSP60, HSP90, actin, vimentin, and Lck did not coprecipitate with IKKβ and was recovered in the supernatant (Figure 5C). These data suggest that CD28 costimulation leads to the recruitment of LAT, IKKβ, and vimentin to flotillin-1 containing rafts. After extraction with ASB-14, there is evidence for proteinmediated interactions between flotillin-1 and IKKβ. We do not exclude the possibility that low affinity protein interactions may be disrupted in the process. Although HSPs were recruited to GM1-positive sucrose fractions, we found no evidence that they associated with the flotillin-1 containing rafts. It is interesting, however, that HSP60 and HSP90 coprecipitate with IKKβ in detergent-soluble cellular extracts (not shown). This is compatible with the notion that heatshock proteins play a role in the assembly of the 900 kD cytosolic IKK heterocomplex.44,50 Activation of the Raft-Associated IKK Complex by CD28 Costimulation and Functional Disruption by Methyl-β-Cyclodextrin (MβCD) Treatment. We asked whether the ASB-14 extractable IKK complexes are functionally linked to TCR signaling. To answer this question, in vitro kinase assays were performed in which anti-IKKR and anti-IKKβ immune complexes, prepared from the raft DSM, were reconstituted with a substrate (GST-IκBR) and 32P-ATP. This showed anti-CD3 stimulation leads to weak activation of the raft-associated IKK 450
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complex, and CD28 costimulation further increases this activity (Figure 6A). To determine whether the activated IKK complexes are functionally relevant, MβCD was used to deplete membrane cholesterol. This leads to raft disruption and subsequent interference in the recruitment of IKKβ to the hypodense sucrose fractions (Figure 6B). Raft disruption also interferes in subsequent IκBR degradation (not shown), which is necessary to release Rel proteins to the nucleus. To determine whether MβCD disrupts a NF-κB-dependent nuclear event, we looked at a downstream event at the level of the IL-2 promoter.7,11 The CD28RE/AP-1 is a combinatorial response element in this promoter that is dependent on JNK and IKK activity for its transcriptional activation.7,11 Utilizing an exogenously transfected CD28RE/AP-1 luciferase promoterreporter construct, we demonstrated that MβCD treatment decreases reporter gene activity by 45% in anti-CD3/CD28 treated Jurkat cells (Figure 6C). This likely represents interference in the NF-κB pathway since there is little change in JNK activity after MβCD treatment (Figure 6D). This phosphopeptide blot shows that anti-CD3 and anti-CD3/CD28 treatments induce the phosphorylation of the 54 kD JNK isoform, which is not affected by MβCD treatment (Figure 6D). CD3/CD28 costimulation induces weak phosphorylation of the 46 kD JNK isoform, which is affected by MβCD treatment (Figure 6D).
Identification of Lipid Raft-Associated Proteins
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Figure 3. Immunoblotting reveals the presence of flotillins, heat shock proteins, and cytoskeletal proteins in lipid rafts. 50 µL from each of the indicated sucrose gradient fractions was separated by SDS-PAGE and used to immunoblot for the presence of flotillins, HSPs, IKKs, and cytoskeletal proteins as described in the Experimental Section: (A): immunoblotting for flotillin-1, flotillin-2, LAT, HSP90, and HSP60; (B) immunoblotting for talin, vimentin, and actin. These results were confirmed in an independent experiment.
Discussion 2-D electrophoresis and mass spectrometry led to the identification of novel raft-associated signaling, cytoskeletal, heat shock, and GTP-binding proteins. Western blotting confirmed that a number of these proteins are recruited to GM1positive sucrose fractions during CD3/CD28 costimulation. Of particular interest is the raft localization of flotillins, cytoskeletal proteins and components of the NF-κB kinase complex. Capture of raft vesicles with immobilized anti-flotillin-1 or antiIKKβ antibodies demonstrated the colocalization of the flotillins and IKKs in a raft subset that contains other TCR-associated signaling molecules, including Lck and LAT. Flotillin-1 and IKKβ coprecipitated from the ASB-14-soluble raft material while the other raft components remained in the supernatant. The raftassociated IKK complexes were activated by CD3/CD28 costimulation. Treatment of Jurkat cells with methyl-β-cyclodextrin disrupted IKK assembly in the rafts, and interfered in the activation of the CD28 response element in the IL-2 promoter. Although limited proteome analysis of TCR signaling components has been accomplished, this is a difficult undertaking due to the tendency of hydrophobic membrane components to precipitate during 2-D electrophoresis.52-54 Consequently, this type of analysis has often resorted to 1-D protein fractionation.52,53 We have partially overcome this problem by including ASB-14 in the solubilization buffer (Figure 2). This zwitterionic detergent leads to efficient solubilization and resolution of difficult-to-purify integral membrane and bacterial lipoproteins.35,42 Mass spectrometry analysis of detergent-resistant Jurkat membrane domains has been reported by Von Haller et al. and Bini et al. 52,54 These analyses confirmed the presence of cytoskeletal, GTP-binding, heat shock, and TCR-associated signaling molecules (Table 1).52,54 Von Haller et al. reported the
Figure 4. Use of flotillin-1 to immune-immobilize raft domains that contain TCR-associated signaling components, including the IKKs. (A) Lipid raft pellets were prepared from 5 × 107 resting or stimulated Jurkat cells as described in Figure 2. After resuspension in 200 µL of TNE/1% TX-100, lipid raft vesicles were incubated with 2 µg of polyclonal anti-flotillin-1 for 3 h before the addition of 20 µL of protein A-Sepharose beads for 16 h. Immobilized raft vesicles were washed four times and boiled in 1x SDS sample buffer. The accompanying raft proteins were used for immunoblotting to detect the presence of flotillin-1, IKKβ, LAT, Lck, HSP90, HSP60, and vimentin. These results were confirmed in an independent experiment. The panel on the right-hand side shows lipid raft vesicles that failed to bind to anti-flotillin-1, therefore remaining in the supernatant. These supernatants were subjected to HSP60 and HSP90 immunoblotting; lanes 1-3 represent the same conditions as panels 1-3 in the main figure. (B) Immunoblot from sucrose gradient fractions probed with antiIKKR, -β, and -γ. CL ) crude lysate. (C) Reverse IP of lipid raft vesicles from CD3/CD28-stimulated cells using rabbit anti-IKKβ and immunoblotting for the presence of flotillin-1 in stimulated cells.
presence of flotillins, while Bini et al. demonstrated the presence of a stomatin-like protein.52,54 Protein mass spectrometry also identified the presence of flotillin and stomatin in lipid rafts obtained from erythrocytes.55 It is possible that through their SPFH (stomatin, prohibitin, flotillin and HflK/C) domains, flotillins, and stomatin participate in protein-protein interactions in the membrane.56 The presence of lamin B1, Lck, ATP synthase β-subunit, and numerous small G-proteins in Jurkat rafts is also consistent with previous observations (Table Journal of Proteome Research • Vol. 3, No. 3, 2004 451
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Figure 5. Use of ASB-14 extracted rash supernatants to show physical association of flotillin-1 with IKKs. (A) Immunoblot to show that most IKKs are ASB-14 extractable. Lipid rafts from 5 × 107 were extracted in 1% ASB-14 as described in Figure 1B. The DSM and DIM were subjected to immunoblotting with antiIKKR, -β, and -γ. (B) Immunoblot to show the coprecipitation of IKKβ with flotillin-1. A similar amount of raft vesicles were extracted with 1% ASB-14, following which the DSM was used to immune precipitate flotillin-1. These immune complexes were probed for the presence of IKKβ. No IKKβ coprecipitation was obtained with NIS. These results were confirmed in an independent experiment. (C) Reverse immune precipitation performed with anti-IKKβ showing that HSP60, HSP90, actin, vimentin, Lck, and PKCθ do not coprecipitate, while flotillin-1 does. CL ) crude lysate.
1).52-55 While the role of regulatory GTP-binding proteins in lipid rafts is unclear (Table 1), it has been demonstrated that CD28 can activate small G-proteins, including Cdc42 and Rac1.5,57 These components play a role in cytoskeletal assembly. Despite resolving ∼60 raft polypeptides, it is interesting that some raft components that were identified by immunoblotting did not show up in our 2-D analysis. These includes raft components such as LAT, IKKs, ZAP-70, and PKCθ (Figures 1 and 3). Noteworthy, coverage of these signaling components were also absent from previous proteome analysis of Jurkat cell 452
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rafts.52,54 While it is possible that some of these components could be present in sub-threshold amounts, others could be poorly solubilized or may have less stable membrane association in the presence of nonionic detergents.58 Integral membrane protein coverage by 2D-gel electrophoresis is still a contentious issue. Several recent reports describe improved characterization of these proteins through use of chaotropes such as thiourea, detergents such as ASB-14 and other additives.59 The conservative conclusion is that while our 2D raft analysis has produced useful data, integral membrane proteins are under-represented. Alternative first dimensions and/or nongel based approaches may be required to achieve full coverage, and we note the success achieved by Wu and coworkers, who used proteinase K at high pH to yield soluble peptides from known integral proteins of the mammalian Golgi.61 We used our proteome analysis to clarify the functional significance of a limited number of raft-associated proteins. Of particular interest is the recruitment of the IKK complex to lipid rafts during CD3/CD28 costimulation (Figure 4B). Classically, the IKK complex has been depicted as a 900 kD cytosolic heterocomplex that can be activated by diverse stimuli such as cytokines (e.g., TNFR), chemokines (e.g., MIP-1R), and adhesion molecules (e.g., VCAM).44,50 In addition, it has been shown that the IKK complex is recruited to the surface membrane during engagement of the type 1 TNFR receptor (TNF-R1).62,63 This is compatible with our demonstration that IKKβ is recruited to a flotillin-1 decorated raft subset during CD3/CD28 costimulation (Figure 4A), as well as the recent observations that NF-κB activation by the TCR requires IKKγ recruitment to the immunological synapse.64 These data raise the interesting question whether the membrane and cytosolic IKK complexes are functionally related. In this regard, it is known that TNF-R1 ligation induces shuttling of IKK complexes between the surface membrane and the cytosolic compartment.62,63 A suggestion that the same is true in Tlymphocytes, is the observation that raft disruption by MβCD interferes in NF-κB activation and transcriptional activation of the CD28RE (Figure 6C). This response element is dependent on the release of Rel proteins from the cytosol to the nucleus as well as the activation of the JNK pathway, which was minimally affected by MβCD (Figure 6D).7,11 The presence of heatshock proteins in lipid rafts is compatible with the role of these chaperones in signal transduction (Figure 3A), including IKK activation. HSP90 is an integral component of the cytosolic IKK complex and plays a key role in TNFR-induced IKK activation.50 The presence of HSP90 in these heterocomplexes is a prerequisite for trafficking between the cytoplasm and the membrane during TNF-R1 stimulation.50 However, since HSP90 and HSP60 do not appear to be associated with the flotillin-1 and IKKβ-associated raft subset, it would appear as if these chaperones are not critical for IKK activation in the membrane. Immune purification of lipid rafts has also been accomplished by using immobilized anti-CD3 or anti-Thy1 antibodies.31 These studies resulted in the characterization of a raft subset that contains preassembled TCR/CD3, CD4, Lck, and ZAP-70.31 We demonstrate a flotillin-1-associated raft subset that includes IKKβ, LAT, and Lck, as well as the cytoskeletal proteins vimentin and actin (Figures 3 and 4). We propose that this raft subset is one of many different raft types that play a role in the formation of the TCR synapse. Considering that rafts are small structures (
