Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells

BBRC Biochemical and Biophysical Research Communications 295 (2002) 540–546 www.academicpress.com

Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cellsq Lorri A. Morford,a Kathy Forrest,b Barbara Logan,a L. Kevin Overstreet,a Jens Goebel,b William H. Brooks,a and Thomas L. Roszmana,* a

Department of Microbiology and Immunology, University of Kentucky, Lexington, KY 40536-0298, USA b Department of Pediatrics, University of Kentucky, Lexington, KY 40536-0298, USA Received 3 June 2002

Abstract Calpain, a calcium-dependent cysteine protease, is known to associate with the T-cell plasma membrane and subsequently cleave a number of cytoskeletal-associated proteins. In this study, we report the novel observation that calpain II, but not calpain I, associates with membrane lipid rafts on human peripheral blood T-cells and Jurkat cells. Raft-associated calpain activity is enhanced with exogenous calcium and inhibited with calpeptin, a specific inhibitor of calpain activity. In addition, we demonstrate that calpain cleaves the cytoskeletal-associated protein, talin, during the first 30-min after cell stimulation. We propose that lipid raft associated-calpain II could function in early TCR signaling to facilitate immune synapse formation through cytoskeletal remodeling mechanisms. Hence, we demonstrate that the positioning of calpain II within T-cell lipid rafts strategically places it in close proximity to known calpain substrates that are cleaved during Ag-specific T-cell signaling and immune synapse formation. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Human; T lymphocyte; Jurkat; Cellular activation; Signal transduction; Calpain II; Lipid rafts

Lymphocyte plasma membrane contains specialized lipid microdomains that aid in various signaling events. These lipid microdomains (‘‘rafts’’) are enriched in sphingolipids, cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and doubly acylated proteins, e.g., src-family kinases [1–4]. Due to their low buoyant density and insolubility at low temperatures in nonionic detergents, such as Triton X-100 [5,6], these rafts can readily be isolated and studied to better understand their role in T-cell signaling events. Generally, lipid q Abbreviations: ‘‘rafts,’’ lipid microdomains; GPI, glycosylphosphatidylinositol; TCR, T-cell receptor; MHC, major histocompatability complex; APC, antigen presenting cell; LFA-1, leukocyte function associated molecule-1; c-SMAC, central supermolecular activation cluster; p-SMAC, peripheral supermolecular activation cluster; PKC, protein kinase C; PM, plasma membrane; ZAP-70, zeta-associated protein; TxR, Texas red; RT, room temperature; CTxB, cholera toxin B; OG, Oregon green; PNS, post nuclear supernatant; CYTO, cytosol; HRP, horseradish peroxidase. * Corresponding author. Fax: +1-859-257-8994. E-mail address: [email protected] (T.L. Roszman).

rafts appear to serve as signal transduction platforms in many cell types [7]. In T-cells, the coalescence of these rafts within the membrane is a prerequisite for appropriate signaling through a number of specialized receptors including the T-cell receptor (TCR)/CD3 complex [8–12] and leukocyte function associated molecule-1 (LFA-1) [13]. Accordingly, interactions between an Ag-specific T-cell and a peptide-primed antigen presenting cell (APC) induce raft aggregation within the T-cell membrane and ultimately lead to the formation of a specialized junction termed the immune synapse [14–19]. As proposed by Dustin and coworkers [14,17–19], several of the events required for immune synapse formation include: LFA-1 tethering, peptideMHC sampling by the TCR complex, lipid raft aggregation, and cytoskeletal rearrangements. These cytoskeletal rearrangements aid in the reorganization of proteins within the specialized contact region to form the peripheral and central supermolecular activation clusters (p- and c-SMAC) [16–19]. A mature immune synapse is defined by a specific ‘‘bulls-eye’’ pattern of

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receptor organization, where the c-SMAC contains TCR/CD3, p56lck , p59fyn , and protein kinase C (PKC) h and excludes such molecules as LFA-1 and talin to the p-SMAC [16,17,19]. Formation of the immune synapse may be important for efficient TCR-mediated signaling [8–12], polarized cytokine secretion by T-cells and/or TCR downregulation [20–22]. The mechanisms by which cytoskeletal reorganization is induced to permit movement of various plasma membrane (PM) proteins and assembly of SMACs and the immune synapse remain conjectural. Calpain is a calcium-dependent cysteine protease that is ubiquitously expressed in mammalian cells [23]. Two major isoforms have been identified which differ according to the concentrations of Ca2þ required for their activation. Calpain I (l-calpain) requires micromolar levels of Ca2þ for activation whereas calpain II (m-calpain) requires millimolar levels [23,24]. Both isoforms of calpain are heterodimers, consisting of a unique 80-kDa catalytic subunit coupled to a 30-kDa regulatory subunit common to both [23,25,26]. Calpain is located largely, but not exclusively in the cytosol. In addition, some calpain is present in the PM and is associated with the cortical actin cytoskeleton subsequent to appropriate T-cell activation where cleavage of a variety of substrates occurs [27–29]. Calpain appears to be important in T-cell function based on studies demonstrating that calpain-specific inhibitors block T-cell proliferation and early T-cell shape changes required for activation [28]. For example, calpain-specific inhibitors block actin polymerization normally associated with early T-cell activation [28]. Thus, releasing the constraints of the cytoskeleton by calpain would facilitate T-cell shape changes thereby establishing a more stable contact between appropriately adjacent receptors and their ligands. This conjecture is supported by the observation that calpain is activated as a consequence of TCR/CD3 complex ligation and integrin binding to various substrates [28–30]. In addition, calpain is capable of cleaving a variety of cytoskeletal (e.g., talin [31], a-actinin [28], paxillin [32], actin-binding protein [33], ezrin [34]) and immune synapse-associated proteins (e.g., talin [31], PKC [35], zeta-associated protein (ZAP-70) [36]). These observations led us to hypothesize that calpain is located in lipid rafts and may have a role as an endogenous mediator of raft clustering and hence in immune synapse formation. This report demonstrates for the first time the presence of calpain II in T-cell lipid rafts using both biochemical and confocal microscopy techniques. Calpain is found in rafts after either CD59 crosslinking or CD3 crosslinking. In addition, we show that calpain cleaves talin after anti-CD3 mAb stimulation of T-cells. The association of calpain II with rafts in T-cells and its intrinsic ability to cleave cytoskeletal proteins suggest that this protease may play an important role in the initiation of T-cell signaling.

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Materials and methods Isolation of human T-cells. Peripheral blood lymphocytes were isolated from heprinized (Elkins–Sinn, Cherry Hill, NJ) venous blood from healthy adult human donors by Ficoll–Hypaque gradient centrifugation at 4 °C [37]. T-cells were isolated by sheep erythrocyte rosetting as previously described [37]. Cell culture. The human T-cell leukemia line, Jurkat, was obtained from American Type Culture Collection (ATCC; Rockville, MD) and was cultured in RPMI supplemented with penicillin/streptomycin (Gibco, Grand Island, NY), 10 mM HEPES (Gibco), and 10% FBS (Summit Biotechnology, Fort Collins, CO). The cells were passaged twice weekly. Detection of raft formation in Jurkat cells by confocal microscopy. The Jurkat cells were washed in serum-free media and plated onto TESPA-coated coverslips (3-aminopropyltriethoxy silane; Sigma, St. Louis, MO) [10] in the cold. Raft aggregation was induced by incubating these cells with anti-CD59 mAb (PharMingen, San Diego, CA) for 20 min on ice followed by the addition of horse anti-mouse IgGTexas red (2°Ab-TxR; Vector Laboratories, Burlingame, CA) for 10 min on ice, and 20 min at 37 °C in a 5% CO2 environment. Similarly, TCR/CD3 aggregation was achieved using 10 lg=ml mouse antihuman CD3 mAb (OKT3 clone, ATCC) followed by 5 lg/ml of 2°AbTxR. Cells were either directly fixed in 3% paraformaldehyde (Sigma) for at least 30 min (control) or extracted with 1% Triton X-100 (Sigma) for 5 min on ice prior to fixation. Cells were permeablized with 0.1% Nonidet P-40 (Sigma) prepared in PBS/HEPES for 5 min at room temperature (RT) followed by the addition of PBS/HEPES containing 4% goat normal sera (Sigma), 2% BSA (Sigma) for 15 min at RT. The cells were stained with primary Abs: mouse anti-p59fyn mAb (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-calpain II polyclonal antiserum (Chemicon, Temecula, CA), mouse anti-ZAP-70 mAb (Transduction Laboratories, Lexington, KY), mouse anti-CD3 mAb (OKT3), mouse anti-CD45 Ab (DAKO, Carpinteria, CA) for 30 min at RT. Cholera toxin B subunit (CTxB) staining was performed with CTxB-FITC immunoconjugate (10 lg/ml; Sigma) for 30 min at RT. After extensive washing with PBS/HEPES, the slides were incubated, when appropriate, with goat anti-rabbit IgG-OG secondary Ab (Molecular Probes, Eugene, OR) for 15 min at RT, washed and mounted with ProLong Antifade reagents (Molecular Probes). The cells were examined by confocal microscopy on the Leica TCS confocal microscope with 100 objectives, using laser excitation at 488 and 568 nm (University of Kentucky Medical Center Electron Microscopy & Imaging Facility, Lexington, KY). Sequential scanning was utilized to eliminate bleed through between fluorescent channels. Cell lysis and subcellular fractionation. Rafts were prepared by sucrose gradient centrifugation using a modification of Xavier et al. [8]. Briefly, cells ð2  108 Þ were washed in PBS, lysed in cold MES-buffered saline (MBS; (25 mM MES [pH 6.5], 150 mM NaCl), 0.5% Triton X-100, 1 mM sodium orthovanadate (Na3 VO4 ), 2 mM EDTA (Gibco), 1 mM PMSF (Sigma), and 1 lg/ml aprotinin (Sigma)) and homogenized [8]. Samples were mixed with an equal volume of 85% sucrose (w/v, Sigma) in MBS buffer, transferred to ultracentrifuge tubes, and overlaid with 6 ml of 35% sucrose and 4 ml of 5% sucrose prepared in MBS buffer [8]. Rafts were harvested from the 5–35% sucrose interface following a 14–16 h ultracentrifugation at 200,000g in a SW40Ti rotor. Detergent-insoluble raft aggregates were pelleted by microcentrifugation at 14,000g for 30–40 min at 4 °C and resuspended in 100–350 ll of MBS. Protein concentrations were measured using the detergentinsensitive BCA Protein Assay (Pierce, Rockford, IL). The post nuclear supernatant (PNS), PM, and cytosol (CYTO) fractions were prepared using modifications of other procedures [8,38,39]. Briefly, 1–2  108 cells were washed in PBS, resuspended in 1 ml of homogenization buffer (HB; 0.25 M sucrose, 1 mM EDTA, 20 mM tricine [pH 7.8], 1 mM Na3 VO4 , 1 mM PMSF, and 1 lg/ml of

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aprotinin) and PNS was prepared as described [8]. The CYTO fraction was isolated from the PNS fraction by diluting 0.5 ml PNS with 0.5 ml HB, ultracentrifugation for 1 h at 108,000g in a TLA-100.3 Beckman rotor at 4 °C, and supernatant was collected. The PM fraction was isolated from the PNS fraction by overlaying 8 ml of 30% Percoll (Amersham Pharmacia Biotech AB, Uppsala, Sweden) prepared in HB with 1.5 ml of PNS and subsequent ultracentrifugation at 84,700g for 35 min in a Beckman 80Ti rotor at 4 °C. The PM containing band was collected from the buffer/Percoll interface. Protein concentration of the PM and CYTO fractions were determined using the detergentinsensitive DC protein assay (BioRad, Hercules, CA). Western blot analysis of cytosol, plasma membrane, and raft fractions. Ten micrograms of each subcellular protein fraction (or 5 lg for p56lck analysis) was separated by 7.5% SDS–PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes and stained with Amido black dye to verify equal protein loading between lanes prior to probing. Membranes were blocked with Tris-buffered saline (TBS; 20 mM Tris [pH 7.6], 137 mM NaCl) containing 0.3% Tween 20 and 3% BSA (TTB) for 1 h at RT. Primary Abs were incubated on the membranes in TTB for 1 h. The primary Abs used included: mouse anti-Lck mAb (Santa Cruz Biotechnology), mouse anti-Zap-70 mAb, mouse anti-calpain-I mAb (Chemicon), and rabbit anti-calpain II polyclonal Ab. After extensive washing with TBS containing 0.3% Tween 20 (TBST; Sigma), anti-mouse IgG-horseradish peroxidase (HRP), and anti-rabbit IgG-HRP immunoconjugates (Santa Cruz Biotechnology) were incubated on the appropriate membranes in TTB for 30–60 min. After extensive washing with TBST, Western detection was performed using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, England) on Cronex film (Sterling Diagnostic Imaging, Newark, DE). Slot blot analysis of subcellular fractions for ganglioside GM1 . Five micrograms of each subcellular fraction (CYTO, PM and raft) was slot blotted onto nitrocellulose in TBS buffer and the membranes were blocked in TBS containing 5% BSA for 1 h. Ganglioside GM1 was detected on the membranes by incubation with CTxB-conjugated HRP (Sigma) in TBS–BSA for 1h. Membranes were washed with TBS and detection of the HRP-conjugated CTxB was performed using ECL on Cronex film. In vitro calpain activity assay. Raft fractions (30 lg per well) were transferred to a 96-well flat bottom plate (Costar, Corning, NY) in two sets of at least duplicate samples. One set of samples was incubated for 15 min at RT in the presence of 100 mM calpeptin (Calbiochem, La Jolla, CA). The calpain substrate (100 lM; N-suddinyl-Leu-Tyr-7amido-4-methylcoumarin, Sigma) was added to each well, the plates were incubated at 37 °C for 30 min and the mean fluorescence was measured on a Cytofluor plate reader (excitation: 360 nm, emission: 460 nm). After reading the plate, 20 mM CaCl2 (Fisher Scientific, Fair Lawn, NJ) was added to all wells at a final concentration of 5 mM and the plate was re-read. Talin cleavage following CD3 crosslinking. Jurkat cells or T-cells were stimulated in Eppendorf tubes (0.5 ml/tube at 1:5  107 cell/ml) with anti-CD3 mAb (4 lg) for 10 min on ice in RPMI media containing 2 mM CaCl2 . CD3 mAb was crosslinked on the cell surface with rabbit anti-mouse IgG Ab (8 lg; Cappel, West Chester, PA) for 30 min at 37 °C. Cells were pelleted by microcentrifugation and lysed in 250 ll of 1 Laemmli buffer (BioRad). Cell lysates were boiled for 5 min and 40/ ll per lane separated on 6 or 7.5% SDS–PAGE. Proteins were transferred to nitrocellulose for 1–2 h on ice in transfer buffer containing 192 mM glycine, 25 mM Tris, and 20% methanol. Membranes were probed with goat anti-talin Ab (Santa Cruz Biotechnology) followed by rabbit anti-goat IgG-HRPO secondary Ab (Santa Cruz Biotechnology) and exposed to film using ECL reagents. In some cases, cells were pretreated with either: 100 lM calpeptin or 5 mM EGTA (Sigma) on ice for 10 min and 5 min, respectively, prior to and during the antiCD3 mAb crosslinking steps. In addition, some cells were costimulated with 1 lg of anti-CD28 Ab (PharMingen, San Diego, CA) for

5 min just prior to adding the rabbit anti-mouse IgG crosslinking antibody.

Results and discussion Lipid rafts appear to be important in T-cell signaling [8–12]. Identification of all biologically relevant proteins associated with these membrane structures, however, is incomplete. To examine the potential association of calpain II with rafts in T-cells, dual color sequential scanning confocal microscopy was utilized. Raft aggregation was induced in Jurkat cells by Ab-crosslinking a GPI-linked protein, CD59. Colocalization of various proteins (bearing green fluorescent tags) with the raft islands (red tagged CD59) yields a yellow staining pattern in fluorescent overlay photographs (Fig. 1). After Ab-crosslinking of CD59, the src-related kinase, p59fyn , is localized within Triton X-100 insoluble raft islands (Fig. 1A). Consistent with the observations of [10], CD3 colocalizes with these raft islands (yellow areas in Fig. 1B). This association, however, appears to be weak and can be partially disrupted by detergent extraction when the TCR/CD3 complexes are not directly Ab-crosslinked prior to the Triton X-100 treatment (Fig. 1A versus B) [10]. Importantly, we demonstrate for the first time that calpain II is associated with detergent insoluble rafts as revealed by Ab-crosslinking of CD59 on Jurkat T-cells (Fig. 1A). Since a large pool of calpain resides in the T-cell cytoplasm, the identification of specifically membrane-associated calpain in unextracted cells is difficult (Fig. 1C). Triton X-100 extraction aids in the visualization of membrane-associated calpain, and more specifically raft-associated calpain (Fig. 1A). As demonstrated by Fig. 1C, p59fyn , CD3, and calpain II are located throughout the cells prior to any crosslinking steps (Fig. 1C). To our knowledge, these data are the first to show that a pool of calpain is constitutively associated with T-cell membrane rafts. Further experiments were done with Jurkat cells to determine whether calpain II was located in raft islands formed by crosslinking of the TCR/CD3 complex with anti-CD3 Ab. The results in Fig. 2A show that ganglioside GM1 (visualized by CTxB staining) and ZAP70, but not CD45, co-localize (yellow staining) in the aggregated rafts. This pattern of staining is consistent with the observations of others, hence reflecting successful raft isolation [10]. Importantly, we demonstrate that calpain II (green staining) colocalizes with rafts induced by anti-CD3 mAb (red staining) crosslinking (Fig. 2A, areas of colocalization in yellow). As a positive control for CD45 staining [10], anti-CD3 mAb stimulated Jurkat cells were not extracted with Triton X-100, and CD45 was detected with the anti-CD45 Ab (Fig. 2B). Consistent with the observation of [40], GM1 expression is diffuse and at low levels in unstimulated

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Fig. 1. Localization of p59fyn , calpain II, and CD3 within the detergentinsoluble rafts formed by CD59-crosslinking on Jurkat cells. Cells were treated with mouse anti-human CD59 mAb followed by the crosslinking Ab, horse anti-mouse IgG-Texas red (TxR) immunoconjugate, to initiate and visualize raft formation (see Materials and methods). Three groups of cells were analyzed (panels A–C). Accordingly, Jurkat cells were either crosslinked with CD59 and extracted on ice for 5 min with 1% Triton X-100 prior to fixation (panel A), crosslinked with CD59 and then fixed with no detergent extraction (panel B) or fixed directly (i.e., control with no CD59 crosslinking and on extraction (panel C). All cells were permeablized and stained with anti-CD3-FITC mAb, anti-fyn polyclonal Ab, or anti-calpain II polyclonal Ab followed by goat anti-rabbit IgG-Oregon green (OG) secondary Ab, or secondary Ab alone. Fluorescence of FITC and OG (green) or TxR (red) was visualized on a confocal microscope utilizing sequential scanning methodology, with identical settings for the Triton X-100 extracted and non-extracted samples. Cells were examined using laser excitations at 488 and 568 nm with 100 objectives. The p59fyn , calpain II, and CD3 images (green) were overlaid to reveal regions of colocalization (appearing yellow) with CD59–TxR (red) stained cells.

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Fig. 2. Calpain II colocalizes with the TCR in Triton X-100 extracted Jurkat cells stimulated with anti-CD3 mAb. Cells were stimulated with mouse anti-human CD3 mAb (OKT3) followed by horse antimouse IgG-TxR immunoconjugate (see Materials and methods). Three groups of cells were analyzed. Accordingly, Jurkat cells were either crosslinked with CD3 and extracted on ice for 5 min with 1% Triton X-100 prior to fixation (panel A), crosslinked with CD3 and then fixed with no detergent extraction (panel B) or fixed directly (i.e., control with no CD3 crosslinking; panel C). All cells were permeablized and stained with either cholera toxin B (CTxB)-FITC, anti-CD45-FITC mAb, anti-Zap70, or anti-calpain II polyclonal Ab followed by goat anti-rabbit IgG-OG secondary Ab, or secondary Ab alone (data not shown). Fluorescence of FITC and OG (green) or TxR (red) was visualized on a confocal microscope utilizing sequential scanning methodology, with identical settings for the Triton X-100 extracted and non-extracted samples. Cells were examined using laser excitations at 488 and 568 nm with 100 objectives. CTxB, Zap-70, CD45, and calpain II images (green) were overlaid to reveal regions of colocalization (appearing yellow) with CD3-TxR (red).

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T-cells (Fig. 2C). As observed with p59fyn , CD3, and calpain II staining prior to any Ab-crosslinking steps (Fig. 1C), the ganglioside GM1 (CTxB staining), and ZAP-70 are also distributed broadly throughout the cells prior to Ab-crosslinking (Fig. 2C). These data indicate that calpain II is colocalized with TCR/CD3-associated rafts after anti-CD3 mAb stimulation of Jurkat cells. To confirm our morphologic findings that calpain II is associated rafts, subcellular fractionation of Jurkat cells was done to obtain raft, PM, and cytosol (CYTO) fractions which could be examined by Western analysis. As previously reported [10], the src-family kinase, p56lck , is found predominantly within raft fractions with only as small amount detected in the PM (Fig. 3, panel A). Interestingly, we detected the presence of calpain II, but not calpain I, in the raft fractions obtained from Jurkat cells (Fig. 3, panel A) which suggest that it is calpain II and not calpain I which is important in membrane-linked signaling. Similar results were obtained with freshly isolated human peripheral blood T-cells stimulated for 3 days in vitro with PHA (data not shown). Slot blot analysis of the isolated subcellular fractions confirmed the presence of ganglioside GM1 in the raft fraction as visualized by CTxB-HRP (Fig. 3, panel B). The association of calpain with the cell membrane may not be linked to calcium elevation and subsequent calpain activation since ionomycin treatment of Jurkat cells does not effect the amount of calpain associated with raft fractions (Morford, unpublished observation). Experiments were also done to quantitate calpain activity in raft fractions (Fig. 4). The results show that calpain activity is detected in the raft fractions isolated from Jurkat cells and it was enhanced with exogenous Ca2þ and could by inhibited with the calpain specific inhibitor, calpeptin (Fig. 4). It is unclear as to what differences, if any, exist between membrane-associated calpain and calpain located in the cytosol. While the precise membraneassociation which anchors calpain into lipid rafts re-

Fig. 3. Calpain II, but not calpain I, associates with raft fractions obtained from Jurkat cells. Cytosol (CYTO), plasma membrane (PM), and RAFT fractions were isolated from Jurkat cells as described in Materials and methods. (A) Fractions were analyzed by Western blot under reducing conditions. Ten micrograms of protein was examined per lane on each blot with the exception of the p56lck blot, which contains 5 lg of protein per lane. In (B), GM1 glycosphingolipids were detected on a slot blot with 5 lg of protein per well by reactivity to cholera toxin B (CTxB) conjugated to HRP. (A) and (B) show data from one of three representative experiments.

Fig. 4. The calpain activity associated with the raft fractions from Jurkat cells can be enhanced with calcium and inhibited with calpeptin. Raft fractions were isolated from Jurkat cells and in vitro calpain activity in the fractions was measured as described in Materials and methods. The results are expressed as arbitrary fluorescent units. The data are representative of one of four replicate experiments.

mains to be established, it is intriguing to hypothesize that calpain binds phosphatidylinositol-(4,5)-biphosphate ðPIP2 Þ. Support for this theory is gained from several observations. First, calpain can associate with acidic phospholipids [41,42]. Second, association of calpain with acidic phospholipids significantly reduces the ½Ca2þ  requirement necessary for calpain activation ðPIP2 > PIP PIÞ [41–46]. Third, PIP2 is found at higher concentrations in lipid rafts than in the plasma membrane [1,3]. Finally, we have demonstrated that neomycin, which firmly binds the polar head of PIP2 , inhibits ionomycin-induced calpain activation in T-cells (Overstreet, unpublished observation). It is possible that calpain could associate with membrane lipids via the Gly repeat found in the N-terminus of the molecule [47]. Finally, to demonstrate a functional role for calpain in early T-cell receptor-mediated signaling, experiments were conducted to identify calpain-mediated cytoskeletal remodeling events that are involved in immune synapse formation. We demonstrate that crosslinking CD3 on the surface of freshly isolated human T-cells results in the cleavage of talin after 30 min of stimulation (Fig. 5, lane 2), which could be inhibited with calpeptin (lane 3) or EGTA (lane 4). Crosslinking CD28 in parallel with CD3 did not enhance the cleavage of talin during this time interval (lane 5). Pretreatment with calpeptin (lane 6) or EGTA (lane 7) also inhibited talin cleavage in CD3 plus CD28-crosslinked T-cells. Similar talin cleavage patterns were observed in Jurkat cells stimulated for 30 min with

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Fig. 5. CD3 or CD3 plus CD28 crosslinking of human T-cells for 30 min leads to calpeptin-inhibitable, talin cleavage. Freshly isolated na€ıve human T-cells were stimulated for 30 min utilizing CD3 antibody crosslinking techniques. T-cells were lysed with Laemmli buffer, proteins separated on SDS–PAGE and transferred to nitrocellulose. Western probing was performed as described in Materials and methods. Lane 1: unstimulated T-cells, lane 2: anti-CD3 Ab crosslinked T-cells, lane 3: anti-CD3 Ab crosslinked T-cells pretreated with 100 lM calpeptin, lane 4: anti-CD3 Ab crosslinked T-cells pretreated with 5 mM EGTA, lane 5: anti-CD3 plus anti-CD28 Ab crosslinked T-cells, Lane 6: anti-CD3 plus anti-CD28 Ab crosslinked Tcells pretreated with 100 lM calpeptin, and lane 7: anti-CD3 and anti-CD28 Ab crosslinked T-cells pretreated with 5 mM EGTA.

ionomycin in the presence and absence of either calpeptin or EGTA (data not shown). Others have previously demonstrated that calpain can modify integrin receptors and enhance integrin binding affinities [31,48]. For example, calpain may play a role in LFA-1 tethering of T-cells to APC during the early phase of the immune response. Interestingly, Shaw and colleagues have noted that LFA-1 is recruited to the central contact region early (2–25 min) in immature immune synapse formation and is reorganized into the p-SMAC region in the 15–30 min following stimulation of na€ıve T-cells [22]. Hence, the temporal and spatial activation of calpain resulting in the cleavage of talin suggests a role for this protease in the cytoskeletal rearrangement processes required for the formation of the mature immune synapse generated during the T-cell activation process. Based on this work and that of others [28,31,48], calpain-induced reorganization of the cytoskeleton could facilitate T-cell shape changes that facilitate efficient interactions with the APC. In addition, calpain cleavage of talin could enhance the movement of LFA-1 within the immature immune synapse to generate the mature synapse profile with LFA-1 residing in the p-SMAC region. Our results demonstrate that calpain II colocalizes with the lipid rafts formed during TCR/CD3 stimulation and is capable of cleaving the cytoskeletal protein, talin. In summary, this finding extends our knowledge of the subcellular localization of calpain to include rafts as well as the cytosol, actin cytoskeleton, and PM. This constitutive association of calpain II with T-cell lipid rafts strategically places this protease in the membrane to assist in immune synapse formation. This location affords calpain the opportunity to have an active role in cytoskeletal remodeling, e.g., cleaving such proteins as talin [31], ezrin [34], and a-actinin [28], as well as enhancing LFA-1-mediated T-cell adhesion [41], processes needed for efficient c- and p-SMAC formation within the immune synapse. Thus, although, the signaling cascades associated with cytoskeletal reorganization on which SMAC formation and T-cell activation are contingent remain to be elucidated, our results suggest the calpain II may have an important and previously unrecognized role in T-cell function.

Acknowledgments Special thanks to the University of Kentucky Medical Center Electron Microscopy and Imaging Facility Staff, especially Dr. Bruce Maley, Richard Watson, and Mary Gail Engle.

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