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Regulation of Bim by TCR Signals in CD4/CD8 Double-Positive Thymocytes¹

Anna Bunin^*, Fatima W. Khwaja and Gilbert J. Kersh^2,*

^* Department of Pathology and Laboratory Medicine and Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA 30322

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3 injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim_EL is rapidly phosphorylated in thymocytes in response to CD3 cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x_L bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x_L in immature thymocytes.

	Introduction

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Developing T cells that are CD4/CD8 double positive (DP)³ or in the transition between DP and CD4 or CD8 single positive (SP) will undergo apoptosis if they receive a strong signal through the TCR (1, 2, 3). Strong signals are delivered to thymocytes when the TCR recognizes self peptide/MHC complexes with relatively high affinity. Thus, negative selection is one mechanism by which autoreactive T cells are eliminated (4). Failure to delete autoreactive cells in the thymus results in widespread autoimmunity (5, 6). In mature T cells, strong signals through the TCR can lead to cytokine production and proliferation, whereas the same stimulus in a DP thymocyte results in apoptosis. The mechanism by which TCR signals cause death in DP cells is poorly understood (7). Proximal TCR signals are required, but downstream of the initial phosphorylation events at the receptor much less is known.

A molecule that clearly plays a role in both thymocyte negative selection and death of peripheral T cells is Bim. Bim is a proapoptotic molecule that is related to the antiapoptotic protein Bcl-2 by the presence of a single BH3 (Bcl-2 homology) domain (8). Bim is part of a family of BH3-only proteins that promote apoptosis. Bim is thought to function by binding to Bcl-2 and/or Bcl-x_L on the mitochondrial membrane and inhibiting their antiapoptotic function (8, 9). Three isoforms of Bim exist that result from alternative splicing (8). These are Bim_EL, Bim_L, and Bim_S. Bim_EL is the most abundant isoform in thymocytes and T cells, whereas Bim_S is almost undetectable (10, 11). DP thymocytes in Bim-deficient mice do not undergo apoptosis in response to TCR signals, and Bim-deficient animals develop multiorgan autoimmunity (9, 12). Bim is also essential for the deletion of CD4⁺8^–CD24⁺ thymocytes in response to TCR ligation (13). Thus, Bim is required for normal negative selection.

The mechanism by which TCR signals regulate Bim function is not known. However, Bim is expressed in numerous cell types, and several modes of regulation have been described for Bim. These include induction of its expression (14, 15, 16, 17, 18), translocation to mitochondria after release from the dynein motor complex (19), and phosphorylation (20, 21, 22, 23). Although some studies have observed increases in Bim expression in thymocytes and T cells in response to TCR signals, a separate study using stimulation of murine peripheral T cells by superantigen in vivo found that Bim expression did not change after stimulation even though the superantigen stimulation in vivo led to Bim-dependent apoptosis (24). Whether other modes of Bim regulation come into play in thymocytes has not been reported, but in the superantigen model using peripheral T cells, Bim did not translocate to mitochondria (10). Thus, it is not clear how Bim is regulated in peripheral T cells or thymocytes.

In the present study we evaluated these potential mechanisms of Bim regulation in DP thymocytes by generating a strong signal through the TCR using anti-CD3 mAb stimulation. We found that Bim protein levels do not change in response to TCR cross-linking, and Bim also does not translocate from the cytoskeleton to the mitochondria in response to TCR signaling. We did find that most of the Bim in thymocytes is associated with mitochondria in the absence of TCR signals, and that it is rapidly phosphorylated in response to TCR cross-linking. Phospho-Bim is maintained for at least 24 h after TCR cross-linking. Also at later time points (24 h), we found that TCR cross-linking increases the proportion of Bim bound to Bcl-x_L. To control for glucocorticoid-mediated effects that may result from anti-CD3 treatment in vivo, we also show that neither Bim phosphorylation nor Bim association with Bcl-x_L is enhanced by dexamethasone treatment. Thus, our data are consistent with a model in which Bim apoptotic activity is regulated by phosphorylation, followed by increased Bim association with Bcl-x_L at the time of apoptosis.

	Materials and Methods

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Mice

C57BL/6 mice were purchased from the National Cancer Institute. Mice with a genetic deletion of the ₂-microglobulin gene (25) and mice that have all conventional MHC class II coding sequences deleted (26) were purchased from The Jackson Laboratory. The ₂-microglobulin-deficient and MHC class II-deficient mice were bred together to generate MHC^–/– mice. All procedures were approved by the Emory University institutional animal care and use committee.

Antibodies

Western blots were probed with mouse anti-Bcl-x_L, hamster anti-TCR (BD Pharmingen), rabbit anti-Bim (Sigma-Aldrich and Stressgen), mouse 60-kDa heat shock protein Ab (Stressgen), rabbit anti-Bcl-2 (N-19), mouse anti-HDAC-1 (H-11), rabbit anti-Bax (N-20; Santa Cruz Biotechnology), mouse anti--tubulin (Sigma-Aldrich), mouse anti-voltage-dependent anion channel-1 (Calbiochem), and rabbit anti-ERK1/2 (Promega). Immune complexes were revealed with HRP-conjugated goat anti-rabbit Fc-specific and sheep anti-mouse Fc-specific Abs at 1/10,000 (Jackson ImmunoResearch Laboratories) and ECL reagents (Amersham Biosciences).

In vitro and in vivo TCR stimulation of thymocytes

Six-well plates were coated with 10 µg/ml goat anti-Armenian hamster IgG (Jackson ImmunoResearch Laboratories), followed by PBS or 10 µg/ml hamster anti-CD3 (2C11-145). Thymocytes were harvested from C57BL/6 mice or MHC-deficient mice (6–8 wk of age), transferred onto Ab-coated plates in serum-free medium, and centrifuged at 394 x g for 1 min. Plates were then placed at 37°C for 30 min. Cells remaining in suspension were removed, whereas adherent cells were lysed in 1% Triton X-100 lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1 mM phenylmethylsulfate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate. Lysates were incubated on ice for 15 min and centrifuged at 16,000 x g, and supernatants were used for Western blot analysis.

Subcellular fractionation of thymocytes

Thymocytes (2.8 x 10⁸) from C57BL/6 mice were harvested and subjected to fractionation essentially as described previously (10), except that extraction buffer III and benzonaze from the Subcellular Proteome Extraction kit (Calbiochem) were used to separate nuclear and cytoskeletal components.

Purification of mitochondria

Thymocytes (7 x 10⁸ cells/condition), were washed in TD buffer (133 mM NaCl, 5 mM KCl, 25 mM Tris-HCl (pH 7.5), and 0.7 mM NaH₂PO₄). Pellets were resuspended in RSB buffer (10 mM NaCl, 1.5 mM CaCl₂, 10 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate) to give 10 times the original volume of the pellet. Cells were allowed to swell for 10 min before being disrupted with a Dounce homogenizer (Kontes). After disruption, 2.5x MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl (pH 7.5), and 5 mM EDTA) was added to make 1x MS buffer and mixed gently. Homogenates were centrifuged three times at 1260 x g at 4°C, and the pellets were discarded each time. Resulting supernatants were centrifuged at 20,000 x g for 15 min at 4°C. Postnuclear, postmitochondrial supernatants were stored on ice until analysis. Pellets (heavy membrane fraction) were resuspended in sucrose-TE buffer (20% sucrose, 50 mM Tris-HCl (pH 7.5), and 10 mM EDTA) and layered on top of a sucrose step gradient containing 1.5 M sucrose, 10 mM Tris-HCl (pH 7.5), and 5 mM EDTA (bottom layer) and 1.0 M sucrose, 10 mM Tris-HCl, and 5 mM EDTA (pH 7.5; top layer). Step gradients were centrifuged at 100,000 x g for 1 h at 4°C. The interface between the top and bottom sucrose layers was collected using a Pasteur pipette. Volumes were made up to 1 ml with sucrose TE buffer, and samples were centrifuged at 20,000 x g for 15 min at 4°C. Mitochondrial pellets were resuspended in lysis buffer or directly in SDS sample buffer.

Alkali treatment of membranes

The effect of alkali extraction on heavy membranes isolated from postnuclear supernatants from C57BL/6 thymocytes (3 x 10⁸ cells/condition) was determined essentially as described previously (27).

Immunoprecipitation

Thymocytes were lysed at a concentration of 3 x 10⁸ cells/ml in 0.2% Nonidet P-40 lysis buffer. Lysates were incubated on ice for 15 min, centrifuged at 16,000 x g for 5 min at 4°C, and precleared with protein A beads (Sigma-Aldrich). Bim was immunoprecipitated from lysates with 3 µg of rabbit anti-Bim Ab (Stressgen). Control lysates were incubated with 3 µg of normal rabbit IgG (Santa Cruz Biotechnology). Ab/lysate mixtures were gently rocked at 4°C for 1 h. Protein A beads (50 µl of a 50% (v/v) slurry) were added to each sample and rocked at 4°C for an additional hour. Protein A beads were washed with lysis buffer three times, then washed with PBS and transferred to fresh tubes; bound proteins were eluted with nonreducing 4x SDS sample buffer by boiling for 5 min and subjected to Western blot analysis.

Acid phosphatase treatment

Protein A-rabbit anti-Bim immune complexes were prepared and washed three times in 1% Nonidet P-40 lysis buffer without phosphatase inhibitors. Lysates were made from stimulated or unstimulated thymocytes, incubated on ice for 15 min, and cleared of insoluble material by centrifuging for 5 min at 16,000 x g. Rabbit anti-Bim-protein A complexes were added to the lysates, and the mixtures were rocked at 4°C for 30 min. Immune complexes were then washed three times with 50 mM PIPES and 1 mM DTT (pH 6.0), resuspended in 100 µl of this buffer, and incubated for 10 min at 30°C. Acid potato phosphatase (Sigma-Aldrich) was then added to appropriate samples (10 U/sample). Samples were incubated for 10 min at 30°C and for 1 min at 37°C, washed three times with 50 mM PIPES and 1 mM DTT (pH 6.0), resuspended in nonreducing SDS sample buffer, boiled, and analyzed by Western blot.

Two-dimensional (2D) gel electrophoresis

Bim was immunoprecipitated from 7 x 10⁸ thymocytes as described above. Protein A beads were washed with water three times, and pellets were dried in a Speed-Vac (Savant) with heat for 15 min. Proteins were eluted from protein A beads using 1 ml of isoelectric focusing lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris, 100 mM DTT, and a 1/1000 dilution of protease and phosphatase inhibitor mixtures; Sigma-Aldrich). The immunoprecipitated samples were concentrated to a final volume of 100 µl using Centricon-YM3 columns (Millipore). The first dimension was performed on an IPGphor system from Amersham Pharmacia using Immobiline dry strips (pH 3–10; 13 cm; Amersham Biosciences). For analytical gels, immobilized pH gradient dry strips were rehydrated overnight with 100 µg of total protein mixed with destreak rehydration buffer (8 M urea, 4% CHAPS, 100 mM DTT, 100 mM protease inhibitor, and 0.25% ampholytes; Amersham Biosciences) to a final volume of 250 µl. Next, prefocusing to remove ionic components of the samples was performed over 6 h by increasing the voltage linearly from 150 to 5,000 V. The prefocusing step was followed by focusing of proteins to a total of 100,000 Vh. Immobilized pH gradient strips were then equilibrated using equilibration buffer (6 M urea, 2% SDS, 0.05 M Tris base (pH 8.8), and 20% glycerol), first containing 10 mg/ml DTT and then 25 mg/ml iodoacetamide for 30 min each, to reduce and alkylate the focused proteins. Once equilibrated, the proteins were separated in the second dimension on 10% polyacrylamide gels with 2% SDS using the Protean II system from Bio-Rad. The gels were transferred to nitrocellulose membrane using the Criterion system from Bio-Rad and were analyzed by Western blot for Bim.

	Results

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The model system

To evaluate biochemical events in thymocytes responding to strong TCR signals, it is necessary to simultaneously activate the majority of thymocytes. In most of our experiments, we have done this by treating thymocytes with anti-CD3 Ab both in vivo and in vitro. Two potential problems that can occur when using this approach are the activation of peripheral T cells and SP thymocytes. Activation of peripheral T cells results in the secretion of large amounts of cytokines. These cytokines do not directly kill DP thymocytes, but induce an increase in levels of endogenous glucocorticoids that is lethal to the DP cells (28) (A. Bunin and G. J. Kersh, manuscript in preparation). In light of this problem, in many of the experiments we compared the results obtained using anti-CD3 treatment in vivo to those obtained using treatment with anti-CD3 in vitro and treatment with dexamethasone in vivo. Activation of SP thymocytes can also complicate the results, because biochemical events observed in whole thymocytes responding to anti-CD3 may be taking place primarily in the SP population. Therefore, many of our experiments were performed using MHC-deficient mice that do not have any mature SP thymocytes, thus limiting the responding population to immature DP cells.

Levels of Bim do not change in response to TCR cross-linking on thymocytes

The analysis of Bim protein expression in several cell types has demonstrated that some cells express Bim at low levels, but in response to an apoptotic stimulus, Bim expression is markedly induced (14, 15, 16). However, in other cases Bim levels are unchanged after apoptotic stimulus (10, 24). To evaluate the possibility that Bim is regulated by a change in expression level in thymocytes after TCR cross-linking, we injected PBS or anti-CD3 Ab into wild-type mice i.v.; harvested thymocytes at 6, 12, and 24 h after the injection; and analyzed thymocyte lysates by Western blot. As shown in Fig. 1A, Bim expression in wild-type thymocytes did not change in response to a strong signal through the TCR. By injecting anti-CD3 Ab i.v., we consistently observed evidence of TCR signaling in thymocytes as early as 20 min after injection (data not shown) and evidence of apoptosis as early as 12 h after injection (Fig. 1B). In this experiment there are probably signals derived from anti-CD3 acting on the DP cells directly as well as glucocorticoid-derived signals, but this combination of signals does not alter the total levels of Bim in the 24 h after stimulation.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. TCR cross-linking on thymocytes does not induce expression of BimEL. A, C57BL/6 mice were i.v. injected with 50 µg of anti-CD3. At the indicated times after injection, thymocytes were lysed, and proteins were analyzed by Western blot. B, C57BL/6 mice were injected with anti-CD3 or with PBS. At the indicated time points, the number of live thymocytes was determined using trypan blue exclusion.

In various cell lines, Bim has been shown to be sequestered at the microtubule-associated dynein motor complex by binding tightly to the LC8 cytoplasmic dynein L chain (19). In response to UV irradiation, the Bim/LC8 complex was released from the microtubules and relocated to the mitochondria, where it interacted with Bcl-2 (19). The release of Bim from the cytoskeleton has been shown to enable its proapoptotic activity. To determine whether translocation from the dynein motor complex to the mitochondria played a role in thymocyte apoptosis, we isolated cytosolic, membrane, nuclear, and cytoskeletal fractions from thymocytes 24 h after i.v. injection of anti-CD3 or PBS and determined the relative amount of Bim in each fraction (Fig. 2). We found that nearly all Bim protein can be detected in fractions containing intracellular membranes (membrane and nuclear fractions) both before and after anti-CD3 treatment. Only a small subset of total Bim is associated with the cytoskeleton in thymocytes, and Bim bound to the cytoskeleton does not get released after anti-CD3 injection. In fact, we consistently observed a slight increase in Bim associated with the cytoskeleton after anti-CD3 treatment (Fig. 2). It is clear, however, that Bim translocation from cytoskeleton to mitochondria does not play a role in its regulation in thymocytes.

View larger version (57K): [in this window] [in a new window]   FIGURE 2. Bim does not change its subcellular localization in response to a strong signal through the TCR. C57BL/6 mice were i.v. injected with PBS or 50 µg of anti-CD3. Twenty-four hours later, thymocytes were isolated and subjected to subcellular fractionation. Proteins from each resulting fraction were resolved by SDS-PAGE and analyzed by Western blot. The fractions were probed with four different Abs to verify the fractionation. As expected, Bax was detected in the cytoplasmic and membrane fractions, the TCR -chain was detected in the membrane fraction, histone deacetylase-1 (HDAC-1) was detected primarily in the nuclear fraction, and -tubulin was detected in all fractions, including the cytoskeleton. The fractions are labeled as follows: CYT, cytoplasm; M, membrane; N, nucleus; CSK, cytoskeleton.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Bim remains associated with mitochondria after TCR cross-linking on thymocytes in vivo. C57BL/6 mice were injected i.v. with anti-CD3 or PBS. Twenty-four hours later, thymocytes were homogenized, and the mitochondrial fraction was separated from the nuclei and from the remaining material using differential centrifugation and a sucrose step gradient. Proteins from the mitochondrial fractions and from the postnuclear, postmitochondrial supernatants were analyzed by Western blot.

Bim is rapidly phosphorylated in vivo and in vitro in response to CD3 cross-linking

Previous studies have shown that Bim can also be regulated by phosphorylation; however, Bim phosphorylation in response to a signal through the TCR has not been reported. Phosphorylation of Bim can result in either up- or down-regulation of the Bim proapoptotic function. For example, several studies have shown that Bim can be phosphorylated by the MAPK ERK, which results in a reduction of proapoptotic function (17). In some cases this has been attributed to the targeting of phospho-Bim for proteosomal degradation (22, 23). Thus, phosphorylation of Bim by ERK can promote cell survival. In contrast, in neurons, phosphorylation of Bim by the MAPK JNK on serine 65 has been shown to result in the up-regulation of proapoptotic function (20). In 293 T human embryonic kidney cells that overexpress Bim_L, JNK phosphorylates Bim_L within the LC8 binding motif (threonine 56), causing the release of Bim from the cytoskeleton (21).

Phosphorylated Bim species have been observed to migrate slower on SDS-polyacrylamide gels (17, 20, 21). Because TCR signaling during negative selection of thymocytes results in activation of MAPK pathways, we considered the possibility that phosphorylation of Bim may play a role in its regulation during negative selection. To address this, we examined the electrophoretic mobility of Bim after anti-CD3 stimulation. Although we did not observe a shift in Bim mobility in the data shown in Fig. 1, these gels were run for a relatively short amount of time to observe Bim_EL and Bim_L isoforms. As shown in Fig. 4A, when gels were run long enough to observe small mobility changes, thymocytes harvested 30 min after injection of PBS or anti-CD3 had a considerable portion of Bim that migrated with reduced mobility in the anti-CD3-treated animals, and previous studies have attributed this shift to phosphorylation (15, 18, 19).

View larger version (61K): [in this window] [in a new window]   FIGURE 4. BimEL is phosphorylated in response to TCR cross-linking on thymocytes in vitro and in vivo. A, In response to TCR cross-linking in vivo, BimEL shows retarded electrophoretic mobility. Mice were injected with anti-CD3 or PBS. Thirty minutes later, thymocytes were harvested, lysates were made, and proteins were analyzed by Western blot. B, Slowed migration of Bim in response to TCR cross-linking can be inhibited by phosphatase treatment. Thymocytes from C57BL/6 mice were isolated, stimulated for 30 min with plate-bound anti-CD3 Ab, and lysed. Bim was immunoprecipitated from the lysates, and immune complexes were treated with potato acid phosphatase. C57BL/6 (C) or MHC-deficient (D) thymocytes were stimulated with plate-bound anti-CD3 Ab for 30 min. Thymocytes were lysed, and proteins were analyzed by Western blot. A shift in BimEL electrophoretic mobility is indicated by arrows. E, Time course of Bim phosphorylation in vivo. C57BL/6 mice were injected with saline or anti-CD3. Thymocytes were harvested at the indicated times after injection and lysed. Lysates were analyzed by Western blot. F, BimEL is phosphorylated in response to stimulation with anti-CD3 in vitro. BimEL is slightly phosphorylated after in vivo dexamethasone treatment. Thymocytes were stimulated with plate-bound anti-CD3 or dexamethasone (10 mg/kg) for 2 h in vivo or were left untreated. Thirty minutes after stimulation, thymocytes were lysed, and Bim was immunoprecipitated using Bim-specific Ab. Immune complexes were resolved by 2D gel electrophoresis, and membranes were probed with an Ab specific for Bim. The arrows point at Bim species that change their phosphorylation status.

The induction of phosphorylation in response to anti-CD3 injection could be due to direct TCR cross-linking on thymocytes or to glucocorticoid production in response to peripheral T cell activation. To rule out a role for peripheral T cells in the change in Bim phosphorylation, we stimulated thymocytes in vitro with plate-bound anti-CD3. In vitro stimulation of thymocytes also resulted in a shift in electrophoretic mobility of Bim_EL (Fig. 4C). The stimulation of thymocytes in culture includes activation of both DP and SP thymocytes. It is possible that the increased Bim phosphorylation that we have observed is predominantly in the SP population or is dependent on products derived from the SP population. We therefore wanted to determine whether increased Bim phosphorylation could be observed in the DP population in the absence of SP stimulation. To this end we stimulated MHC-deficient thymocytes in vitro with plate-bound anti-CD3 and analyzed Bim expression by Western blot. MHC-deficient thymocytes are >90% DP and do not contain any SP cells due to the absence of TCR ligands. Similar to normal thymocytes, we observed that slower migrating forms of Bim are induced in MHC-deficient thymocytes stimulated in vitro (Fig. 4D). To examine the time course of Bim phosphorylation, we stimulated wild-type thymocytes with plate-bound anti-CD3 and analyzed thymocyte lysates at different time points after stimulation. Fig. 4E shows that Bim phosphorylation is sustained for at least 24 h after in vivo anti-CD3 stimulation.

To further analyze Bim phosphorylation in the absence of peripheral T cell activation, we stimulated wild-type thymocytes in vitro with plate-bound anti-CD3 for 30 min. Bim immunoprecipitates were subjected to 2D gel electrophoresis, followed by blotting with Bim-specific Ab. Fig. 4F shows that, consistent with our phosphatase treatment experiments (Fig. 4B), Bim_EL as well as Bim_L exist as a set of species with different isoelectric points, consistent with varying amounts of phosphorylation. Upon stimulation with anti-CD3 in vitro, Bim_EL, but not Bim_L, becomes hyperphosphorylated. This is reflected in the emergence of more acidic Bim_EL isoforms (indicated by arrows on the acidic side) and the disappearance of the most alkalic unphosphorylated Bim_EL species (indicated by the arrow on the alkalic side). These data indicate that Bim is rapidly phosphorylated in thymocytes in response to TCR signals. To rule out a role for peripheral T cell-dependent glucocorticoids in the induction of Bim phosphorylation, we treated mice with dexamethasone for 2 h in vivo and analyzed Bim on a 2D gel. Treatment of thymocytes with dexamethasone resulted in minimal induction of phosphorylation on Bim_EL.

Bim binds increased amounts of Bcl-x_L in response to anti-CD3 injection

It has been observed numerous times that BH3-only proteins bind prosurvival Bcl-2 family members (30). In mice with targeted deletion of the bcl-2 gene, the impairment of cell survival caused by the absence of bcl-2 was rescued when Bcl-2-deficient mice were crossed to Bim-deficient mice (31). This indicates that Bim is a critical promoter of cell death, and that Bcl-2 and Bim act in opposition to each other. Therefore, we analyzed the ability of Bim to bind Bcl-x_L and Bcl-2 in thymocytes after anti-CD3 treatment. Total levels of Bcl-2 in wild-type thymocytes were slightly increased with anti-CD3 stimulation, whereas levels of Bcl-x_L expression were attenuated (Fig. 5A). To assess Bim binding to Bcl-2 and Bcl-x_L, we immunoprecipitated Bim from in vivo-stimulated thymocytes 24 h after stimulation. We then probed immune complexes resolved on SDS-PAGE for the presence of Bcl-x_L and Bcl-2. We observed an increased amount of Bcl-2 associated with Bim in stimulated thymocytes compared with control unstimulated cells (Fig. 5B). The amount of Bcl-x_L associated with Bim was the same in control and stimulated thymocytes, even as the total levels of Bcl-x_L in thymocytes decreased. This suggests that a higher proportion of the Bcl-x_L in stimulated cells is bound to Bim.

View larger version (54K): [in this window] [in a new window]   FIGURE 5. TCR ligation results in enhanced binding of Bim to Bcl-2 and Bcl-xL in C57BL/6 and MHC–/– thymocytes. A, Total levels of Bcl-2 and Bcl-xL in response to TCR triggering in vivo. C57BL/6 mice were injected i.v. with 50 µg of anti-CD3. Thymocytes were lysed at the indicated time points after injection. Total cell lysates were resolved by gel electrophoresis and analyzed by Western blot. B, Bim binds increased amounts of Bcl-2 and Bcl-xL in thymocytes undergoing apoptosis in response to a strong signal through the TCR. C57BL/6 mice were injected i.v. with 50 µg of anti-CD3 or PBS. Twenty-four hours later, thymocytes were isolated and lysed, and Bim was immunoprecipitated using anti-Bim Ab. Immune complexes resolved on a gel and transferred onto a membrane were probed with Abs to Bcl-2 and Bcl-xL. C, Total levels of Bim, Bcl-xL, and Bcl-2 in MHC–/– thymocytes were determined as described in A. D, In the absence of mature SP thymocytes, Bim binds increased amounts of Bcl-xL and Bcl-2. Bim binding to Bcl-xL and Bcl-2 in MHC–/– mice was analyzed as described in B.

Bim binds increased amounts of Bcl-2 in response to glucocorticoid treatment

We attempted to distinguish events that occur during glucocorticoid-induced apoptosis of thymocytes and apoptosis triggered by TCR cross-linking with regard to Bim binding to antiapoptotic Bcl-2 and Bcl-x_L. Fig. 6A shows that expression levels of Bim and Bcl-2 in thymocytes treated with dexamethasone in vivo remain constant, whereas levels of Bcl-x_L decrease. To estimate the amounts of Bcl-2 and Bcl-x_L associated with Bim, we immunoprecipitated Bim and analyzed immunoprecipitates by Western blot using Bcl-2- and Bcl-x_L-specific Abs. Fig. 6B shows that in thymocytes that have been treated with dexamethasone in vivo, Bim associates with a greater percentage of Bcl-2, whereas its interaction with Bcl-x_L is completely abolished. Overall, these results show that increased binding of Bcl-2 by Bim can be induced by glucocorticoid. Thus, after anti-CD3 treatment of wild-type mice, the increased Bim/Bcl-2 association may not be a direct result of TCR cross-linking on thymocytes, but may be secondary to peripheral T cell activation. This conclusion is supported by the reduced Bim/Bcl-2 association in MHC-deficient mice. However, the increased association of Bim with Bcl-x_L appears to be a direct consequence of TCR stimulation in DP thymocytes and may be an important component of negative selection.

View larger version (48K): [in this window] [in a new window]   FIGURE 6. Bim binds increased amounts of Bcl-2 and no Bcl-xL after 15 h of dexamethasone treatment in vivo. A, Total levels of Bim, Bcl-2, and Bcl-xL after 15 h of in vivo dexamethasone treatment. B, Mice were injected i.p. with vehicle (CTRL) or 10 mg/kg dexamethasone (DEX). Fifteen hours later, Bim was immunoprecipitated from thymocyte lysates. Immune complexes were analyzed for the presence of Bcl-2 and Bcl-xL by Western blot.

Some proapoptotic Bcl-2 family proteins change their association with intracellular membranes when activated (32, 33). To test whether Bim also changes its association with the membranes, we purified the heavy membrane fraction from thymocytes treated with anti-CD3 or PBS in vivo and subjected the heavy membrane fractions to alkali treatment. This treatment strips away the proteins that are peripherally associated with membranes while leaving the integral membrane proteins intact (34). Fig. 7 illustrates that Bim is a transmembrane protein both before and after anti-CD3 injection. Bim is found exclusively in the heavy membrane fraction, and alkali treatment does not change its association with the membranes. This is in contrast to tubulin, a protein peripherally associated with membranes (35) that was completely stripped by alkali treatment in our experiments (Fig. 7). Thus, in this experiment, Bim behaves like a voltage-dependent anion channel, a transmembrane mitochondrial anion channel that resides in the outer mitochondrial membrane and is not stripped by alkali treatment (Fig. 7) (36).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. BimEL is an integral membrane protein and does not change its association with membranes in response to TCR cross-linking in vivo. C57BL/6 mice were injected i.v. with 50 µg of anti-CD3 or PBS. Two hours later, thymocytes were isolated and homogenized, and heavy membrane fractions were obtained using differential centrifugation. Heavy membrane fractions were extracted with neutral buffer or 0.2 M Na2CO3, pH 11.5, to extract proteins peripherally associated with the membranes. Cytosol and microsome fractions were obtained from additional centrifugation of the post-heavy membrane supernatant. Proteins from each fraction were resolved by gel electrophoresis and analyzed by Western blot. C, cytosol; M, microsome; P+I, all proteins found in the heavy membrane fraction; I, integral membrane proteins from the heavy membrane fraction.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In cells that express Bim_EL and Bim_L constitutively, Bim can be found either at the mitochondria or associated with the microtubules. In the latter case, apoptotic stimuli cause the release of Bim from its sequestration at the microtubules (19). Once released, Bim relocates to the mitochondria. Studies of the translocation of Bim from microtubules to mitochondria have focused on the Bim_L isoform, which is not the major isoform expressed in thymocytes. It has recently been reported that in nonapoptotic T cells, Bim localizes to the mitochondria and does not relocate in response to apoptotic stimuli (10). Similarly, we have found that the subcellular distribution of Bim in healthy and apoptotic thymocytes is the same. Moreover, we found that Bim is primarily associated with the mitochondria both before and after stimulation of thymocytes, and thus, its apoptotic activity cannot be regulated by redistribution to this organelle in the thymus.

Another potential mode of Bim regulation is phosphorylation. Four serines in Bim have been shown to be the targets of phosphorylation by both ERK and JNK (15, 18). ERK-mediated phosphorylation has been consistently correlated with a decrease in the apoptotic activity of Bim, either by targeting of Bim for degradation or by other mechanism (15, 22, 23). JNK activity has been found to both increase the expression of Bim and increase its cytotoxicity (20, 37). Surprisingly, phosphorylation of serine 65 (serine 69 in human Bim) by ERK or JNK has been found to both increase and decrease the proapoptotic activity of Bim (20, 23). This suggests that the phosphorylation can have different effects depending on the context.

We have found that Bim_EL is a phosphoprotein in resting thymocytes. This is in agreement with a previous study demonstrating that in resting thymocytes Bim_EL exists as four distinct phosphorylated species (11). Our results show that Bim_EL is rapidly hyperphosphorylated in response to TCR cross-linking on thymocytes both in vivo and in vitro. The hyperphosphorylation is sustained for at least 24 h after stimulation, a time at which a significant amount of apoptosis is occurring. We have considered the possibility that Bim phosphorylation results in an increase in Bim apoptotic activity. Activity of the MAPK JNK is thought to be critical for normal negative selection of thymocytes (40) and has also been shown to phosphorylate Bim_EL (20). It has been reported that Bim_EL and Bim_L can be phosphorylated by JNK within the dynein L chain binding domain, resulting in Bim translocation to mitochondria and an increase in apoptotic activity (19). This domain is shared by both Bim_EL and Bim_L isoforms. Our 2D electrophoresis experiments indicate that phosphorylation of dynein L chain binding domain, followed by the release of Bim from the microtubules, are not operational in thymocytes stimulated with anti-CD3. The dynein L chain binding domain is present in both Bim_EL and Bim_L, but only Bim_EL becomes phosphorylated in response to a strong signal through the TCR. Furthermore, we do not observe any translocation of Bim from the cytoskeleton to the mitochondria in response to anti-CD3 stimulation. However, there has been one report showing that Bim_EL phosphorylation by JNK can increase the apoptotic activity of Bim_EL independently of the dynein L chain binding domain (20). Thus, it is possible that TCR signaling in thymocytes enhances Bim apoptotic activity via JNK-dependent phosphorylation.

We have also demonstrated that in thymocytes undergoing apoptosis, Bim_EL binds increasing amounts of Bcl-2 and Bcl-x_L. The increased binding of Bcl-2 occurs in both dexamethasone-treated and anti-CD3-stimulated thymocytes. The increased association with Bcl-x_L takes place only in thymocytes that received a TCR signal. In addition, in MHC-deficient mice, Bim binds less Bcl-2 than in wild-type thymocytes. Taken together, these data suggest that Bcl-2 binding by Bim occurs during glucocorticoid-induced apoptosis, whereas increased binding of Bcl-x_L is characteristic of apoptosis induced by TCR signaling. It is possible that Bim_EL phosphorylation results in an ability to bind these prosurvival molecules with greater affinity; however, the mechanism by which TCR signaling enhances Bim association with Bcl-x_L remains unknown.

By binding an increasing proportion of Bcl-x_L, Bim may shift the cell fate decision toward apoptosis by limiting the amount of free Bcl-x_L. This is similar to a model proposed by Zhu et al. (10), who suggested that in mature T cells, Bim increases its association with Bcl-x_L after an apoptotic stimulus. In their system, the levels of Bcl-2 were declining rather than slightly increasing as we observed in thymocytes. Something common to both thymocytes and mature T cells is that Bim increases its association with Bcl-x_L after an apoptotic stimulus. Thus, in the T lineage, the amount of free Bcl-x_L may be a critical survival factor.

A study by Bouillet et al. (9) also found that the association between Bcl-x_L and Bim was increased after anti-CD3 stimulation of thymocytes, but differed from our results, in that they found that Bim expression was increased in response to TCR signals. The authors also found that Bcl-x_L expression did not change. The reason for this discrepancy is not clear, but a common theme that can be derived from our study and those by Zhu and Bouillet is that in T-lineage cells undergoing apoptosis, the amount of available Bcl-x_L bound by Bim is increased. Binding of Bcl-x_L by Bim could impair the ability of Bcl-x_L to counteract the apoptotic activity of Bax and Bak, thus promoting cell death.

	Acknowledgments

 
We thank Dr. Erwin G. Van Meir and Frederick J. Schnell for critically reading the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant AI48784.

² Address correspondence and reprint requests to Dr. Gilbert J. Kersh, Department of Pathology and Laboratory Medicine, Emory University, 101 Woodruff Circle, Room 7311 Woodruff Memorial Building, Atlanta, GA 30322. E-mail address: gkersh{at}emory.edu

³ Abbreviations used in this paper: DP, double positive; 2D, two-dimensional; SP, single positive.

Received for publication June 14, 2004. Accepted for publication May 15, 2005.

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