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The Journal of Immunology, 1998, 161: 1169-1175.
Copyright © 1998 by The American Association of Immunologists

Characterization of a Novel Bax-Associated Protein Expressed in Hemopoietic Tissues and Regulated During Thymocyte Apoptosis1

Huiling He2,*, Pamela A. Hershberger3,* and Susan A. McCarthy4,*,{dagger}

Departments of * Surgery and {dagger} Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Members of the Bcl-2 protein family have been implicated as critical intracellular regulators of apoptosis. Most studies of this protein family have utilized transformed and/or transfected cell lines expressing high levels of these proteins. In the current study, we have analyzed normal murine lymphoid cells and tissues and have detected a previously unreported protein of approximately 16 kDa recognized by an anti-Bax Ab. This 16-kDa protein is abundant in hemopoietic tissues of both wild-type and Bax knock-out mice, it can heterodimerize with Bax in normal lymphocytes, and it is dramatically down-modulated in thymocytes in response to apoptotic stimuli. These results suggest that this protein may have antiapoptotic activity and may participate in the regulation of apoptosis in normal lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of the T lymphocyte population to distinguish between "self" and "non-self" is one of the central characteristics of the immune system. This ability permits the immune system, in principle, to respond to virtually any foreign pathogen or mutated cell without concurrently injuring normal self cells and tissues. T lymphocyte lineage repertoire selection takes place primarily during the intrathymic development of T cells from their immature bone marrow-derived progenitors. Positive selection, which promotes the survival and differentiation of T cells in which the TCR have at least some avidity for complexes of self-MHC plus peptide, occurs in the CD4+CD8+ thymocytes. Cells that are not positively selected are deleted from the immune repertoire by apoptosis (1). Negative selection also occurs in CD4+CD8+ thymocytes. During negative selection, developing T cells in which the TCR have high avidity for complexes of self-MHC plus self-peptides are deleted, again by the process of apoptosis (1). Understanding the processes regulating the apoptotic death of these cells is therefore critical to future therapeutic manipulation of the T cell repertoire.

Apoptosis is a cell-autonomous process by which a cell mediates its own destruction. As opposed to necrosis, apoptosis is considered "physiologic," since cells dying by apoptosis are rapidly recognized and engulfed by phagocytic cells, which limits the potential for loss of intracellular contents and generation of an inflammatory response. The events that together comprise apoptosis have been well studied at the microscopic and ultrastructural levels in a variety of physiologic situations in vivo and in a variety of in vitro systems. These events include a rapid decrease in cell volume, a rapid alteration in the mitochondrial transmembrane potential, a loss of membrane asymmetry, membrane blebbing, and DNA degradation into nucleosome-sized fragments (reviewed in 2 .

Although apoptosis can be induced in lymphocytes by a diverse set of stimuli (including ionizing radiation, glucocorticoids, Ag receptor engagement, and cross-linking of cell surface death receptors including Fas and the TNFR), it is brought about by the activation of a common set of death effector molecules belonging to the caspase family of proteases (reviewed in 3 . Recent studies indicate that caspases may be activated in cells signaled to undergo apoptosis either as components of receptor-associated death-inducing signaling complexes (4, 5) or by proteins such as cytochrome c (6) that are released from mitochondria. Once activated, caspases mediate apoptosis through the proteolytic cleavage of key nuclear proteins including U1–70 kDa, DNA-PKcs, lamin B, poly(ADP-ribose) polymerase, and DNA topoisomerases I and II (7, 8). Despite their expression of caspases, cells may survive in the presence of apoptotic stimuli due to the function of apoptotic suppressor proteins belonging to the Bcl-2 family, which have been reported to block caspase activation (9, 10, 11, 12).

Bcl-2 was first implicated in the regulation of lymphocyte survival when it was discovered that the t(14:18) chromosomal translocation found in the majority of non-Hodgkin’s lymphomas resulted in an up-regulation of Bcl-2 expression (13, 14, 15). Subsequent studies demonstrated that Bcl-2 overexpression promotes the survival of diverse cell types in the presence of a variety of apoptotic stimuli (reviewed in 16 . Efforts to elucidate the mechanism for Bcl-2 suppression of apoptosis (via the identification of Bcl-2 interacting proteins or Bcl-2 homologues) have resulted in the description of the Bcl-2 family of intracellular apoptotic regulators that includes proapoptotic members such as Bax (17), Bad (18), and Bak (19, 20, 21) as well as antiapoptotic members such as Bcl-XL (22). With the exception of Bad, which is a cytosolic protein, the identified Bcl-2 family members are membrane-associated proteins.

To date, there have been few correlations made between modulation of Bcl-2 family member expression and the onset of apoptosis. However, the activity of Bcl-2 family members has recently been shown to be directly regulated by survival signals (23). In those studies, it was demonstrated that removal of survival signals resulted in 1) Bad phosphorylation, 2) changes in Bad-binding partners and Bad subcellular localization, and 3) induction of apoptosis. Those findings raise the interesting possibility that additional cytosolic Bcl-2 family members analogous to Bad may exist and may serve to integrate extracellular signals with modulation of Bcl-2 and/or Bax activity.

To further investigate the role of Bcl-2 family members in regulating the survival of normal lymphocytes, we analyzed the effects of apoptosis-inducing stimuli on the expression of Bcl-2 and Bax in normal murine thymocytes and B lymphocytes. Although we detected no consistent apoptosis-associated modulation of the expression of these proteins, we did detect a previously unreported protein of ~16 kDa (referred to here as P16, based on the approximate size of the protein and not meant to imply any relationship to the cell cycle-associated p16) that is rapidly and reproducibly down-modulated in apoptotic thymocytes (this study) and B lymphocytes (accompanying paper (54)). P16 is recognized by an anti-Bax Ab, but is expressed in hemopoietic tissues derived from Bax-deficient mice, indicating that P16 is not an alternatively spliced variant of Bax or a Bax degradation product. P16 has a cytosolic localization and can heterodimerize with Bax in normal lymphocytes. Thus, P16 may represent a new cytosolic Bcl-2 family member, perhaps analogous to Bad but with antiapoptotic activity. In that case, down-modulation of P16 may increase the proapoptotic activity of Bax, perhaps by increasing Bax homodimerization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C57BL/6J (B6)5 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were used between 7 and 13 wk of age. Bax-/- (24) and Bax+/+ control mice on the 129 SV background strain were a generous gift from Dr. Stanley Korsmeyer (Washington University School of Medicine, St. Louis, MO).

Abs and chemicals

The rabbit polyclonal anti-mouse Bax Abs and the corresponding control peptides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-E2 Ab (designated N20 by Santa Cruz) is a specific anti-Bax Ab raised in rabbits against a 19-amino acid peptide (E2 peptide; designated N20 peptide by Santa Cruz) corresponding to residues 11–30 of the Bax protein, encoded by exon 2 of the Bax gene. Anti-E3 Ab (designated P19 by Santa Cruz) is a specific anti-Bax Ab raised in rabbits against a 19-amino acid peptide (E3 peptide; designated P19 peptide by Santa Cruz) corresponding to residues 43–61 of the Bax protein, encoded by exon 3 of the Bax gene. The hamster monoclonal anti-Bcl-2 Ab 3F11 and the monoclonal anti-Bax Ab G206-1276 were purchased from PharMingen (San Diego, CA). Goat anti-rabbit IgG conjugated with horseradish peroxidase, N-ethylmaleimide (NEM), and dexamethasone (Dex) were purchased from Sigma Chemical (St. Louis, MO).

Lymphocyte preparation and culture

Splenocytes and thymocytes were prepared by mechanical disruption of freshly isolated B6 thymi and spleens, respectively. Splenocytes were treated briefly with ACK (25) to lyse contaminating erythrocytes. Lymphocyte suspensions were then filtered through nylon mesh to remove cell aggregates and washed. Washed cells were either analyzed immediately or were resuspended in fresh RPMI medium supplemented with 5% FCS, 2-ME, glutamine, sodium pyruvate, nonessential amino acids, penicillin, and streptomycin and cultured at a density of 2.5 x 106 cells/ml at 37°C in 7.5% CO2.

Immunoblotting

Splenocytes and thymocytes were washed with cold PBS twice and then lysed at 4°C for 40 min in a lysis buffer containing 1% Triton X-100, 0.1% SDS, 50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. In certain experiments indicated within the text, the lysate buffer was also supplemented with 20 mM NEM. Lysates were spun in a microfuge at 13,200 rpm for 10 min, supernatants collected, and protein concentrations determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Thirty to 35 µg of each protein lysate was electrophoresed on 12.5 or 14% polyacrylamide gels under either nonreducing (no 2-ME or DTT added) or reducing conditions (5% 2-ME or varying concentrations of DTT added) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in 5% nonfat dry milk in T-TBS (18 mM Tris-Cl pH 7.6, 122 mM NaCl, 0.1% Tween 20) at room temperature for at least 2 h to minimize nonspecific binding of Ab. The membranes were incubated with the indicated primary Abs at 4°C overnight, then incubated with secondary Ab at room temperature for 1 h. Immune complexes were detected with the Renaissance chemiluminescence reagent (DuPont-NEN, Boston, MA) by treating the membranes according to the manufacturer’s protocol, followed by exposure to x-ray film (Sigma).

Immunoprecipitation

For immunoprecipitations, 1 x 107 splenocytes were lysed in an Nonidet P-40 buffer as previously described (17), except that the lysis buffer was supplemented with 20 mM NEM. The cell lysate (~150 µg protein) was precleared with protein A beads and immunoprecipitated with either 0.8 µg anti-E3 Bax Ab P19 or 1.0 µg anti-Bcl-2 Ab. The immunoprecipitates were electrophoresed on 14% SDS-polyacrylamide gels under reducing conditions and transferred to polyvinylidene difluoride membranes. The immunoprecipitated proteins were then detected by immunoblotting as described above.

Subcellular fractionation

The subcellular fractionation procedure was modified from an established protocol (25). Briefly, splenocytes were washed twice with cold PBS, pH 7.2, and resuspended in ice cold Dounce buffer with protease inhibitors (10 mM Tris-Cl, pH 7.6, 0.5 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 1.8 mg/ml iodoacetamide). After 5 min, the cells were subjected to Dounce homogenization (30 strokes). The nuclei were stabilized by adjusting the salt concentration to 0.15 M NaCl and collected by centrifugation at 500 x g for 5 min at 4°C. The postnuclear supernatant fraction was adjusted to 5 mM EDTA, then centrifuged at 100,000 x g for 45 min in a Beckman Ti-70 rotor at 4°C. The resulting membrane fraction (pellet) and soluble cytoplasmic fraction (supernatant) were collected. Protein lysates of nuclear and membrane fractions were prepared in Triton X-100 and SDS, as described above.

Elution of anti-E3 immunoreactive proteins from polyacrylamide gels

To characterize the 30- to 40-kDa anti-E3 immunoreactive protein(s), approximately 2 mg of spleen cell lysate was loaded in a wide well on a 14% polyacrylamide gel and electrophoresed under nonreducing conditions. The gel portion containing 30- to 0-kDa proteins was excised, and the proteins were eluted from the gel slice in a buffer containing 0.1% SDS, 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM DTT, 0.1 mg/ml BSA, and 0.1 mM EDTA, as previously described (26). The eluted proteins were precipitated by 4 volumes of ice-cold 100% acetone at -80°C overnight, resuspended in 2x sample buffer (100 mM Tris-Cl, pH 6.8, 10% 2-ME, 4% SDS, 0.2% bromophenol blue, and 20% glycerol), electrophoresed under reducing conditions, and analyzed by immunoblotting.

DNA fragmentation assay

DNA fragmentation assays were performed as previously described (27). Briefly, 5.0 x 106 thymocytes were incubated in 2 ml of tissue culture medium in the presence of the indicated stimuli for 21 h at 37°C. After culture, the cells (1 x 107 per experimental group) were collected, washed three times in PBS, and lysed for 30 min at 4°C in a buffer containing 5 mM Tris, pH 8.0, 1 mM EDTA, and 0.5% Triton X-100. Fragmented and intact DNA were separated by centrifugation at 13,200 rpm at 4°C. Fragmented DNA (supernatant) and intact DNA (pellet) fractions were adjusted to the same final volume in the lysis buffer. Each sample was then sonicated, and the samples were plated in triplicate 100-µl serial dilutions in Dynatech MicroFluor 96-well plates (Dynatech Labs, Alexandria, VA). One hundred microliters of a solution of 0.6 mg/ml of the fluorescent DNA dye, DAPI (4,6-diamidino-2-phenylindole), was then added to each well. The relative amount of DNA in each sample was derived from the fluorescence emission at 465 nm, as measured on a Dynatech MicroFluor plate reader. The percentage of DNA fragmentation was calculated as: % fragmentation = 100 x (DNA in supernatant/(DNA in supernatant + DNA in pellet)).

Densitometry

The relative protein expression levels of P16, Bax, and Bcl-2 in control and apoptotic thymocytes were quantitated by densitometry (Molecular Dynamics, Sunnyvale, CA) of enhanced chemiluminescence (ECL)-developed immunoblots, with the medium control (Med) group’s protein level set at 100. The protein levels in Rad and Dex groups were calculated relative to the Med group.

Statistics

Statistical analyses used the Student’s t test; p <= 0.05 is considered a statistically significant difference between values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of a novel 16-kDa protein by anti-Bax Ab

To investigate the regulation of Bcl-2 family members during apoptosis in normal lymphocytes, we undertook a series of studies to quantitate the expression of Bax and Bcl-2 in these cells. Protein extracts from normal fresh murine B6 thymocyte and splenocyte populations were separated on reducing gels and analyzed by immunoblotting with anti-Bax Abs (Fig. 1GoA). An Ab specific for an epitope encoded by Bax exon 2 (Anti-E2) identified the 21-kDa Bax protein in these cells, as expected. Surprisingly, an Ab specific for an epitope encoded by Bax exon 3 (Anti-E3) identified not only 21-kDa Bax, but also an additional protein of ~16 kDa, referred to here as P16. The binding of the anti-E3 Ab to both Bax and P16 was specific, since it was specifically blocked by soluble exon 3 peptide during immunoblotting (Fig. 1GoA). BALB/c, DBA/2J, and C3H/HeN strain cells also expressed the P16 protein (data not shown). P16 was also detected in splenocytes (Fig. 1GoB) and thymocytes (data not shown) from Bax knock-out mice, demonstrating that P16 is a distinct gene product and not a Bax degradation product or a Bax alternative splice product.



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  		FIGURE 1. Detection of a novel 16-kDa protein by anti-Bax Ab. A, Lysates from B6 splenocytes (SP) and thymocytes (TH) were separated by SDS PAGE under reducing conditions and immunoblotted with Abs generated against peptides encoded in Bax exon 2 (Anti-E2) or Bax exon 3 (Anti-E3). For peptide neutralization, anti-E2 or anti-E3 Ab was mixed with a 50-fold (w/w) excess of the indicated peptide and incubated at 4°C overnight before immunoblotting. B, Lysates from Bax+/+ (wt) and Bax-/- (ko) splenocytes were separated by SDS PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab.

 
The P16 protein was also identified by anti-E3 Bax Ab in other hemopoietic tissues taken from normal mice (Fig. 2Go), including bone marrow, lymph node, and the intestine (which contains intraepithelial lymphocytes, B lymphocytes, and macrophages) (28). Interestingly, the brain, which contains bone marrow-derived cells (29), also expressed the P16 protein. In contrast to fresh lymphoid tissues, a series of thymocyte cell lines and B lymphocyte cell lines expressed little or no detectable P16 protein (data not shown). Nonhemopoietic tissues also generally expressed little or no detectable P16 protein (Fig. 2Go).



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  		FIGURE 2. Tissue distribution of the P16 protein. B6 tissues were collected and rinsed with cold PBS twice. Spleen, thymus, and bone marrow were prepared as single-cell suspensions and then lysed. Other tissues were homogenized in lysis buffer. Four sequential linear segments of the intestine were prepared. The lysates were separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab.

 
Subcellular fractionation of splenocytes (Fig. 3Go) demonstrated that P16 was located within the soluble cytoplasmic fraction; in contrast, Bax and Bcl-2 were associated with membrane and nuclear fractions. The membrane association of Bcl-2 appears to be well-established, whereas the intracellular localization of Bax remains controversial (17, 30, 31, 32). The lack of membrane association of P16 may reflect a lack of the hydrophobic domain of many Bcl-2 family members. P16 was not recognized by anti-Bcl-2 (Fig. 3Go) or anti-Bcl-X Abs (data not shown). Together, these results suggest that normal murine leukocytes express a previously unreported protein that shares an antigenic epitope with, but is distinct from, Bax.



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  		FIGURE 3. Intracellular localization of the P16 protein. B6 splenocytes were homogenized and fractionated into soluble cytoplasmic, membrane, and nuclear fractions. The fractions were separated by SDS-PAGE under reducing conditions, immunoblotted with anti-E3 Bax Ab and then reimmunoblotted with anti-Bcl-2 Ab.

 
P16 associates with Bax in normal lymphocytes

The immunoblots shown in Figures 2Go and 3Go also indicate a set of anti-E3 immunoreactive bands at ~32 to 35 kDa. The intensity of these bands varied among protein preparations, suggesting that they might represent semistable complexes of Bax and P16 and/or of P16 with itself; only the latter complexes could form in cells from Bax knock-out mice. Treatment of cell pellets from wild-type mice with NEM-containing lysis buffer reproducibly converted the 32- to 35-kDa band to ~37 kDa in splenocytes (Fig. 4GoA, lanes 1 and 2) and thymocytes (data not shown). NEM prevents the creation of artificial disulfide bonds in vitro (including intramolecular bonds that tend to increase migration rate in the gel) and better preserves the natural state of proteins and protein complexes (33). This result suggests that 37 kDa reflects the actual protein/complex size in vivo. Direct comparison of the same NEM-treated protein preparation run on reducing and nonreducing gels (Fig. 4GoA, lanes 2 and 4) demonstrated that the 37-kDa band decreased in intensity and the 16-kDa band increased in intensity under reducing conditions. In additional experiments using stronger reducing conditions, complete dissociation of the 37-kDa complex was achieved (Fig. 4GoB). These results indicate that the 37-kDa band represents a disulfide-dependent complex containing P16. This disulfide dependence could reflect an intramolecular bond within P16 needed to preserve the integrity of the complex, given that the migration of P16 itself is affected by NEM (lanes 1 and 2). Such disulfide linkages involving intracellular proteins, including Bax, are unusual but not unprecedented (34, 35).



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  		FIGURE 4. P16 protein is present in a disulfide-dependent 37-kDa complex. A, Lysates from B6 splenocytes were prepared in standard lysis buffer or in lysis buffer supplemented with NEM, to prevent formation or rearrangement of disulfide bonds in proteins. The lysates were separated by SDS PAGE under reducing or nonreducing conditions, and immunoblotted with anti-E3 Bax Ab. B, Lysates from B6 splenocytes prepared in NEM-containing lysis buffer, with or without the indicated concentrations of the reducing agent DTT, were separated by SDS PAGE and immunoblotted with anti-E3 Bax Ab.

 
Figure 4Go did not convincingly demonstrate an increase in monomeric Bax upon reduction of the 37-kDa band; thus, the possibility remained that the 37-kDa complex was composed exclusively of P16 homodimers or of heterodimers of P16 with an unknown protein. To directly characterize the 37-kDa protein complex, the 30- to 40-kDa portion of nonreducing gels were therefore excised, and the proteins were eluted and rerun under nonreducing and reducing conditions and immunoblotted with anti-Bax Abs (Fig. 5Go). Anti-E3 Ab, which binds both Bax and P16, revealed both proteins in the eluate under reducing conditions, indicating that the 37-kDa complex contains both Bax and P16. Two other anti-Bax Abs, which do not bind P16, revealed only Bax in the eluate; note that the anti-E2 Bax Ab does not bind well to the 37-kDa complex, perhaps due to masking of the E2 epitope in the complex. The failure to fully dissociate the eluted 37-kDa complex even under optimal reducing conditions (determined from Fig. 4GoB) may indicate that electrophoresis of the complex through the gel renders it difficult to subsequently disrupt.



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  		FIGURE 5. The 37-kDa complex contains both Bax and P16. A lysate of B6 splenocytes prepared in NEM-containing lysis buffer were separated by SDS PAGE under nonreducing conditions. The 30–40-kDa protein fraction was recovered from the gel, re-run under nonreducing and reducing conditions, and immunoblotted with Anti-E3, Anti-E2, or G206–1276 anti-Bax Ab, as indicated.

 
Immunoprecipitation of NEM-treated protein preparations with anti-E3 Bax Ab precipitated P16, Bax, and the 37-kDa complex (Fig. 6Go, left panel, lane 4). Coimmunoprecipitation studies were performed to investigate potential interactions between P16 and Bcl-2, as a complement to the studies described above demonstrating interactions between P16 and Bax. Immunoprecipitation of splenocyte lysates with anti-E3 Bax Ab coprecipitated very little Bcl-2 (Fig. 6Go, right panel, lane 4), and immunoprecipitation with anti-Bcl-2 Ab coprecipitated very little Bax and no detectable P16 (Fig. 6Go, left panel, lane 5); equivalent results were obtained with thymocyte lysates (data not shown). These results are consistent with other reports demonstrating that Bcl-2/Bax complexes are most readily detected in transfected cells overexpressing one or both of these proteins (17, 35, 36). This result also demonstrates that P16 associates preferentially with Bax, and not with Bcl-2, in normal lymphocytes.



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  		FIGURE 6. Immunoprecipitation of P16 and Bax proteins. A lysate from B6 splenocytes prepared in an NEM-containing Nonidet P-40 lysis buffer was precleared with protein A beads and immunoprecipitated with anti-E3 Bax Ab or anti-Bcl-2 Ab. ABs plus beads, or lysate plus beads, were used as negative controls. The immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions, immunoblotted with anti-E3 Bax Ab, and then reimmunoblotted with anti-Bcl-2 Ab. Light chains of anti-E3 Bax Ab (one light chain) and anti-Bcl-2 Ab (two light chains, perhaps due to expression of the endogenous light chain of the hybridoma fusion partner tumor cell) in the immunoprecipitates are marked as Ig-L chain.

 
Down-modulation of P16 during thymocyte apoptosis

The relative abundance of the P16/Bax protein complex in normal untransfected cells suggests that this complex may reflect an important physiologic interaction related to the regulation of apoptosis. We therefore investigated the effect of apoptotic stimuli on P16, Bax, and Bcl-2 expression in fresh thymocytes (Fig. 7Go). Radiation (a p53-dependent stimulus (37, 38)) and dexamethasone (a p53-independent stimulus (37, 38)) were tested in overnight in vitro stimulation cultures. Each stimulus induced extensive DNA fragmentation in the thymocytes. These stimuli did not alter expression of Bcl-2. Dexamethasone, but not radiation, induced a partial decrease in Bax expression. In contrast, both apoptotic stimuli induced dramatic decreases in P16 expression in the thymocytes. Apoptotic blebs were also collected from the thymocyte cultures, but showed no compensatory concentration of P16 protein (data not shown), indicating that total cellular P16 was decreased in apoptosing cells. No compensatory increase in the 37-kDa complex was seen in apoptotic cells with reduced P16 expression (data not shown). We have observed a similar decrease in P16, before extensive DNA fragmentation, upon induction of apoptosis in splenic B lymphocytes (see accompanying paper (54)). These results raise the possibility that P16 protein serves an antiapoptotic function in normal lymphocytes via its intracellular interaction with Bax. Stimuli that reduce expression of P16 may therefore act to increase the proapoptotic activity of Bax, perhaps by increasing Bax homodimerization.



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  		FIGURE 7. Down-modulation of P16 protein levels in apoptotic thymocytes. B6 thymocytes were cultured for 21 h in medium (Med) with or without 1 µM Dex or were exposed to 500 rad of gamma radiation (Rad) before culture in medium. After culture, each thymocyte sample was collected and divided into two aliquots. One aliquot was used to determine the percentage of DNA fragmentation (DNA fragment). The second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab and anti-Bcl-2 Abs. The relative protein expression levels of P16, Bax, and Bcl-2 proteins were quantitated by densitometry, with the medium control (Med) group’s protein level set at 100 for each protein. The expression level for each protein in the Rad and Dex groups was calculated relative to the Med group for that protein (mean ± SD). Statistically significant reductions in P16 expression were observed for the Dex and Rad treatment groups relative to the Med control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the process of examining the expression of Bcl-2 family members in normal murine lymphoid cells and tissues, we detected a previously unreported 16-kDa protein (P16) recognized by a polyclonal anti-Bax Ab. Subsequent Western blot analyses demonstrated that P16 is expressed in hemopoietic tissues derived from Bax-deficient mice, indicating that P16 is not an alternatively spliced variant of Bax or a Bax degradation product. We have determined that P16 has a cytosolic localization and can heterodimerize with Bax but not Bcl-2 in normal lymphocytes. Unlike Bax and Bcl-2, P16 is dramatically down-modulated in thymocytes induced to undergo apoptosis by both p53-dependent (Rad) and p53-independent (Dex) stimuli. Given these data, we propose that P16 represents a new cytosolic Bcl-2 family member important in the regulation of lymphocyte survival.

Homology among Bcl-2 family members

Sequence alignments have revealed four clusters of highly conserved amino acids among Bcl-2 family members, which have been designated Bcl-2 homology domains 1 through 4 (BH1, BH2, BH3, and BH4). Given that the Ab recognizing P16 was raised against an amino acid sequence (residues 43–61) contiguous with the Bax BH3 domain (39, 40), we propose that P16 is also likely to encode a BH3 domain and may thus represent a new, and perhaps distantly related, Bcl-2 family member. This hypothesis is further supported by our observation that P16 can associate with Bax (Figs. 4Go and 5Go), a property shared by other Bcl-2 family members (17, 36). Although we do not know which, if any, of the other Bcl-2 homology domains are shared by P16, recent studies indicate that a BH3 domain alone would be sufficient to account for the Bax-binding function of P16 (41).

The cytoplasmic location of P16 suggests that P16, like Bad (18), may lack a C-terminal hydrophobic domain shared by other family members. In that case, P16 could associate in vivo with cellular membranes indirectly, via its dimerization with Bax (and possibly with other family members). The association of the 37-kDa complex with the membrane and nuclear fractions is consistent with this idea, since the Bax component could tether the complex to these membranes.

Heterodimerization between Bax and antiapoptotic Bcl-2 family members is thought to inhibit the death-promoting activity of Bax. With a few noted exceptions, mutations that disrupt the BH1, BH2, or BH3 domains of Bcl-2 or Bcl-XL reduce or eliminate their ability to dimerize with Bax and their ability to suppress apoptosis (36, 42, 43). Given these findings, and our observation that P16 is a Bax-associated protein that is rapidly down-modulated during lymphocyte apoptosis, we propose that P16 normally functions to suppress apoptosis via its dimerization with Bax.

Bcl-2 family members and the regulation of lymphocyte survival

Bcl-2 and Bcl-XL are differentially expressed during T cell development and are thought to play similar, but nonoverlapping roles in promoting T cell survival. Bcl-2 is expressed at high levels in the CD4-CD8-, CD4+CD8-, and CD4-CD8+ thymic populations and in naive resting peripheral lymphocytes (44). In contrast, Bcl-XL is expressed at high levels in CD4+CD8+ thymocytes (i.e., cells at the appropriate stage for thymic selection) (45) and in recently activated peripheral T cells (46). Given this differential distribution, it has been proposed that Bcl-2 is important in the maintenance of resting T cells, whereas Bcl-XL may be more important in regulating postactivation T cell survival. Lymphocyte survival is also regulated in vivo by proapoptotic Bcl-2 family members as evidenced by the fact that the size of the B and T lymphocyte compartments is increased in Bax-deficient mice (24). Our data, demonstrating that P16 is rapidly down-modulated upon the induction of apoptosis in normal thymocytes (this study) and B lymphocytes (accompanying paper (54)), suggest that P16 may also play a critical role in regulating lymphocyte survival. The precise developmental stages at which P16 is expressed in T and B lymphocytes is currently under investigation; preliminary studies indicate that P16 is preferentially expressed in mature cells of these lineages (unpublished observation).

Biochemical properties of Bcl-2 family members

New insights into the mechanism by which Bcl-2 family members regulate apoptosis have been gained by the resolution of the crystal structure for Bcl-XL (47), the demonstration that Bcl-XL forms an ion-conducting channel when inserted into either planar lipid bilayers or lipid vesicles (48), and the demonstration that Bcl-2 prevents the release of proapoptotic proteins such as cytochrome c (49, 50) and apoptosis-inducing factor (AIF) (51) from the mitochondria. Taken together, these findings support a model in which Bcl-2 family members form mitochondrial ion-conducting channels or pores, which directly or indirectly control the mitochondrial permeability transition and the release of apoptotic mediators (reviewed in Refs. 52 and 53). It is therefore tempting to speculate that Bcl-2-related soluble proteins, such as Bad (18), Bid (40), and potentially P16, regulate apoptois via their dimerization with and influence on such membrane-associated ion channels.

We are beginning further physical characterization of the P16 protein and cloning of the gene encoding P16. The functional intracellular activity of P16, like the functional intracellular activity of other Bcl-2 family members, is still unknown. However, as the identification and characterization of P16 proceed, an understanding of its role in regulating lymphocyte apoptosis should proceed in parallel.


   Acknowledgments
 
We thank D. Dougall for technical assistance; Drs. M. Tector, R. Salter, T. Wright, and D. Johnson for reagents, discussions, and critiques of the manuscript; and Dr. Stanley Korsmeyer for providing Bax-/- and Bax+/+ control mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R29AI32554 (S.McC.), a grant from the Arthritis Foundation, Western Pennsylvania Chapter (S.McC.), American Cancer Society Grant IRG-58-35 (H.H.) and National Institutes of Health/NRSA Grant F32AI09634 (P.A.H.). Back

2 Current address: Department of Medicine, School of Medicine (BRB), Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106. Back

3 Current address: Department of Pharmacology, W1002 Biomedical Science Tower, Terrace and Lothrop Streets, University of Pittsburgh, Pittsburgh, PA 15213. Back

4 Address correspondence and reprint requests to Dr. Susan A. McCarthy, Department of Surgery, W1554 Biomedical Science Tower, Terrace and Lothrop Streets, University of Pittsburgh, Pittsburgh, PA 15213. E-mail address: Back

5 Abbreviations used in this paper: B6, C57BL/6J; Dex, dexamethasone; NEM, N-ethylmaleimide; Rad, gamma radiation. Back

Received for publication January 21, 1998. Accepted for publication March 31, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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