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J Biol Chem, Vol. 274, Issue 41, 29505-29509, October 8, 1999


Linkage of the BH4 Domain of Bcl-2 and the Nuclear Factor kappa B Signaling Pathway for Suppression of Apoptosis*

Danielle de Moissac, Hui Zheng, and Lorrie A. KirshenbaumDagger

From the Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Nuclear factor (NF) kappa B is a ubiquitously expressed transcription factor whose function is regulated by the cytoplasmic inhibitor protein, Ikappa Balpha . We have previously shown that Ikappa Balpha activity is diminished in ventricular myocytes expressing Bcl-2. (de Moissac, D., Mustapha, S., Greenberg, A. H., and Kirshenbaum, L. A. (1998) J. Biol. Chem. 273, 23946-23951). In view of the growing evidence that the conserved N-terminal BH4 domain of Bcl-2 plays a critical role in suppressing apoptosis, we ascertained whether this region accounts for the underlying effects of Bcl-2 on Ikappa Balpha activity. Transfection of human embryonic 293 cells with full length Bcl-2 resulted in a significant 1.9-fold reduction in Ikappa Balpha activity (p < 0.006) with a concomitant increase in DNA binding and 3.4-fold increase in NFkappa B-dependent gene transcription (p < 0.022) compared with vector transfected control cells. In contrast, no significant change in Ikappa Balpha activity was detected with either a BH4 domain deletion mutant (residues 10-30) or BH4 domain point substitution mutants, I14G, V15G, Y18G, K22G, and L23G (p = 2.77). However, a small 0.60-fold decrease (p < 0.04) in Ikappa Balpha activity was noted with the BH4 mutant I19G, suggesting that this residue may not be critical for Ikappa Balpha regulation. Furthermore, adenovirus-mediated delivery of an Ikappa Balpha mutant to prevent NFkappa B activation impaired the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha plus cycloheximide in ventricular myocytes. The data provide the first evidence for the regulation of Ikappa Balpha by Bcl-2 through a mechanism that requires the conserved BH4 domain that links Bcl-2 to the NFkappa B signaling pathway for suppression of apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Apoptosis or programmed cell death is a highly regulated event crucial for normal development and homeostasis. Deregulated cell death has been associated with disease entities such as cancer (1, 2), HIV (3), Huntington's disease (4), and more recently cardiovascular disease (5, 6). Although our understanding of the molecular mechanisms that underlie programmed cell death in mammalian cells is poorly defined, there is considerable evidence that the bcl-2 gene family may play a critical role in this process (reviewed in Refs. 7-9). Bcl-2 can delay or prevent apoptosis provoked by a variety of death- promoting signals, suggesting that it likely impinges on more that one component of the death signaling pathway.

Structural analysis studies of Bcl-2 have identified several key domains with putative anti-apoptotic properties (10-12). In particular, the N terminus of Bcl-2, which encompasses an amphipathic alpha -helical loop designated the BH4 domain, has been suggested to play a crucial role in the prevention of apoptosis. This is substantiated by studies in which the deletion or mutation of this region was shown to render Bcl-2 defective for suppression of apoptosis (10, 11, 13, 14), independent of its ability to dimerize with the pro-apoptotic factors Bax, Bak, or Bad (11). The mechanism by which the BH4 domain confers protection against apoptosis is unknown but may reside in its ability to modulate the activity of certain factors involved in the apoptotic process. The BH4 domain of Bcl-2 has been shown to bind to and sequester the calcium-activated phosphatase calcineurin (15) crucial for the nuclear import of NF-AT4 and signal-induced apoptosis in T-cells (16). Moreover, the BH4 domain has been deemed critical for the interaction with Raf-1 and the Caehorhabditis elegans CED 4 homologue, Apaf-1 (13, 17-19). In this regard, Apaf-1, in association with cytochrome c, dATP, and pro-caspase 9, has been implicated in a mitochondrial-dependent pathway for caspase activation and apoptosis (18, 19). Thus, the BH4 domain, through its ability to interact with potentially pro-apoptotic factors, represents a critical region within the Bcl-2 molecule for the prevention of apoptosis.

In mammalian cells, the transcription factor NFkappa B1 is comprised of 50-kDa and 65-kDa protein subunits (20-22) bound to the cytoplasmic inhibitor protein Ikappa Balpha (23-26). Activation of NFkappa B by agents such as TNFalpha or interleukin-1beta involves the N-terminal phosphorylation and degradation of Ikappa Balpha by the ubiquitin-proteasome pathway (27, 28). Ostensibly, degradation of Ikappa Balpha unmasks the NFkappa B nuclear localization motif, permitting NFkappa B to translocate to the nucleus and direct gene transcription (29).

Recently, an anti-apoptotic function for NFkappa B has been described (30-32). This is substantiated by studies in which cells defective for NFkappa B were found to be more sensitive to pro-apoptotic signals than NFkappa B expressing cells (31, 32). Although TNFalpha leads to NFkappa B activation, there is emerging evidence that TNFalpha predominately triggers apoptosis in cells that are either deficient or defective for NFkappa B (30, 31). This has led to the suggestion that TNFalpha is sufficient to activate both pro- and anti-apoptotic pathways with the anti-apoptotic signals, mediated through the NFkappa B, dominating to suppress death promoting signals and apoptosis (30-32).

We have recently shown that adenoviral-mediated gene delivery of Bcl-2 to ventricular myocytes increased NFkappa B activity and prevented apoptosis mediated by TNFalpha plus cycloheximide (33). This was attributed to Bcl-2-mediated phosphorylation of Ikappa Balpha at residues Ser-32 and Ser-36 followed by Ikappa Balpha degradation by the proteasome (33, 34). Because NFkappa B has been suggested to play a protective role in the suppression of apoptosis, we ascertained in the present study whether the N-terminal BH4 domain of Bcl-2 accounts for the underlying effects of Bcl-2 on NFkappa B activity. In this report, we show that activation of NFkappa B by Bcl-2 is mediated by the degradation of the cytoplasmic inhibitor protein Ikappa Balpha through a mechanism that involves the BH4 domain of Bcl-2. We further show that Bcl-2 utilizes an NFkappa B-dependent pathway to suppress apoptosis mediated by TNFalpha .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell Culture and Transfection-- Human embryonic kidney 293 cells (American Tissue Type Collection) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies, Inc.) as previously reported (33). For transfection experiments, cells were transfected for 3 h with Dulbecco's modified Eagle's medium containing Superfect (Qiagen) and 1-5 µg of CMV-driven eukaryotic expression vectors of either the wild type Bcl-2 (12), Bcl-2 BH4 domain deletion mutant (amino acids 10-30) designated Delta BH4, (kindly provided by John Reed), or point substitution mutants of the BH4 domain at the designated amino acid positions; I14G, V15G, Y18G, I19G, K22G, and L23G (generously provided by Tristram Parslow) (14); and epitope FLAG-tagged wild type Ikappa Balpha and the Ikappa Balpha mutant (Ikappa Balpha S32A,S36A) (kindly provided by D. Ballard) (27). Luciferase constructs containing NFkappa B response elements were previously described (33, 36). Cells were transfected with the CMV-driven eukaryotic expression vector, pcDNA 3 (In Vitrogen) lacking the cDNA insert for all transfection controls. To control for potential differences in transfection efficiency among cell cultures, luciferase reporter activity was normalized to beta -galactosidase activity and expressed as fold increase. Following transfection, cells were washed and maintained in 10% fetal bovine serum with Dulbecco's modified Eagle's medium for 24 h. Data were obtained from at least n = 3 independent cultures with three replicates for each condition. Results were compared by Scheffe's multiple comparison test for analysis of variance and the unpaired two-tailed Student's t test, using a significance level of p < 0.05.

Western Blot Analysis-- For immunodetection of Ikappa Balpha protein, 293 cells were harvested in 1.0% Triton X-100, 1.0% sodium dodecyl sulfate, 0.1% sodium deoxycholate, 140 mM NaCl, 10 mM Tris-HCl, pH 8.0 (RIPA buffer). Cell lysates (50 µg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel at 140 V for 4 h and electrophoretically transferred to polyvinylidiene difluoride membrane (Roche Diagnostics). For detection of Ikappa Balpha protein, the polyvinylidiene difluoride filter was incubated for 3 h with a mouse monoclonal antibody directed toward human Ikappa Balpha /MAD-3 protein clone C21 (1 µg/ml Santa Cruz Biotechnology) in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.3% Tween-20, 0.1% bovine serum albumin. Expression of wild type and Delta BH4 deletion mutant forms of Bcl-2 proteins were detected using a hamster monoclonal antibody directed toward Bcl-2 clone 6C8 (kindly provided by S. Korsmeyer). For detection of transfected Ikappa Balpha -FLAG-tagged proteins, cell lysates were incubated with 1 µg of murine anti-FLAG M2 antibody (Kodak) and immunoprecipitated with 25 µl of protein G agarose beads (Amersham Pharmacia Biotech) at 4 °C for 4 h (27, 33). Immunoprecipitates were washed twice and mixed with 2× SDS Laemmeli loading buffer, boiled, and subjected to gel electrophoresis as described above. Bound proteins were detected by chemiluminescence reaction with horseradish peroxidase-conjugated antibody against mouse or hamster IgG using ECL reagents (Amersham Pharmacia Biotech).

Electromobility Gel Shift Assay-- Nuclear extracts of cells were prepared as described previously by McKinsey et al. (28). A 32P-radiolabeled duplex oliogonucleotide probe with NFkappa B consensus binding sites 5'-AGTTGAGGGGACTTTCGCAGGC-3' was used as a template for the gel shift experiments (27). DNA binding reaction mixtures (20 µl) were carried out on ice and contained 10 µg of nuclear extract, 2 µg of double-stranded probe, poly(dI-dC), (Amersham Pharmacia Biotech), and 10 µg of bovine serum albumin in 20 mM HEPES, pH 7.9, 5% glycerol, 1 mM EDTA, 5 mM dithiothreitol. NFkappa B super shift experiments were conducted with a murine antibody directed toward the p65 subunit of NFkappa B clone C20 (1 µg/ml Santa Cruz). Nuclear-protein complexes were resolved on a native 5% polyacrylamide gel in 1× Tris-Borate EDTA, pH 8.0, and detected by autoradiography (33, 37).

Detection of Apoptosis-- To visualize apoptotic nuclei in cardiac myocytes in situ, ventricular myocytes were fixed in 4% paraformaldehyde, pH 7.4, and subjected to terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay (6, 38). In brief, myocytes were incubated for 1 h at 37 °C in TdT buffer containing 140 mM sodium cocodylate, 1 mM cobalt chloride, 30 mM Tris-HCl, pH 7.2, 50 units of terminal deoxynucleotide transferase and 1 nmol of fluorescein-conjugated dUTP (Roche Molecular Biochemicals). Following the TdT reaction, myocytes were washed three times in phosphate-buffered saline and mounted on glass slides as previously reported (6). Data were obtained from at least three independent cell cultures with three replicates for each condition using >= 200 cells for each condition. Results were compared by Scheffe's multiple comparison test for analysis of variance and the unpaired two-tailed Student's t test, using a significance level of p < 0.05. Genomic DNA was isolated from ventricular myocytes for nucleosomal DNA fragmentation by gel electrophoresis as described previously (33).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To establish whether Bcl-2 could lead to activation of NFkappa B, 293 cells were transfected with a luciferase reporter gene containing putative binding sites for NFkappa B (27, 39) in the presence and absence of Bcl-2. A 3.4-fold increase (p < 0.022) in luciferase reporter gene activity was observed in cells expressing Bcl-2 compared with control cells transfected with the eukaryotic expression vector pcDNA3 alone (Fig. 1). Similar effects were observed in TNFalpha -stimulated cells that served as a positive control for induction of NFkappa B gene activation. In contrast, however, cells transfected with a Bcl-2 cDNA lacking the N-terminal BH4 domain failed to activate NFkappa B-dependent gene transcription (p = 0.31) compared with cells transfected with wild type Bcl-2.


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  		Fig. 1.   NFkappa B-dependent gene transcription in 293 cells. Cells were transfected with luciferase reporter plasmid containing NFkappa B binding elements and transfected with eukaryotic expression plasmids encoding the wild type Bcl-2 (Bcl-2) or deletion mutant of Bcl-2 (Delta BH4). Cells stimulated with TNFalpha (10 ng/ml) as a positive control for NFkappa B gene transcription resulted in a greater than 10-fold induction (p < 0.0001) of NFkappa B transcription compared with vehicle-treated cells. A 3.4-fold increase (p < 0.022) in NFkappa B luciferase reporter activity was observed in cells expressing the wild type but not the BH4 deletion mutant of Bcl-2 (p = 0.31) compared with controls transfected with the eukaryotic expression vector pcDNA3. Data are expressed as the means ± S.E. (p < 0.05). Experiments were repeated at least three times with independent culture conditions with three replicates for each condition.

Electromobility shift analysis of nuclear extract prepared from 293 cells revealed a significant increase in DNA binding activity of NFkappa B in cells expressing the wild type Bcl-2 but not the Delta BH4 deletion mutant of Bcl-2 (Fig. 2, lane 3 versus lane 4) compared with control cells (lanes 1 and 6). A similar increase in the nuclear binding activity of NFkappa B was also observed in cells stimulated by TNFalpha (lane 5). Competition binding assays with 100-fold excess cold probe (lane 7) as well as supershift experiments with antibodies directed toward the p65 subunit (lane 8) confirmed that the migrating complex contained NFkappa B/p65.


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  		Fig. 2.   Electromobility gel shift analysis for NFkappa B. Equivalent amounts of nuclear extract from 293 cells were prepared following interventions and analyzed for NFkappa B binding activity with a 32P-labeled oligonucleotide probe containing NFkappa B binding sites. Lane 1, free probe; lane 2, nontransfected control cells (CNTL); lane 3, 293 expressing wild type Bcl-2 (Bcl-2); lane 4, 293 cells expressing the Bcl-2 Delta BH4 deletion mutant (BH4MT); lane 5, TNFalpha (10 ng/ml)-stimulated cells; lane 6, pcDNA3 vector transfected 293 cells. Competition (comp) binding analysis of nuclear extract with 100-fold excess cold probe is shown in lane 7. Supershift analysis is shown in lane 8; nuclear extract from cells was incubated with rabbit antibody directed toward the p65 subunit of NFkappa B (see "Experimental Procedures" for details). Arrow indicates NFkappa B complex.

Because NFkappa B activity is largely influenced by Ikappa Balpha , which sequesters NFkappa B in the cytoplasm, we determined whether the observed increase in nuclear NFkappa B binding activity and gene expression in the presence of Bcl-2 was due to a reduction in Ikappa Balpha protein levels. Protein extracts of 293 cells expressing Bcl-2 and FLAG-tagged Ikappa Balpha proteins were subjected to Western blot analysis and probed with a murine antibody directed toward Ikappa Balpha . As shown in Fig. 3, Ikappa Balpha levels were significantly suppressed in cells expressing the wild type Bcl-2 but not the Delta BH4 mutant of Bcl-2 compared with vector transfected control cells.


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  		Fig. 3.   The N-terminal BH4 domain of Bcl-2 is required for degradation of Ikappa Balpha . Cell lysates from 293 cells transfected with FLAG-tagged Ikappa Balpha protein in the presence and absence of wild type Bcl-2 (Bcl-2) or Delta BH4 deletion mutant (Delta BH4). Proteins were immunoprecipitated with anti-FLAG antibody followed by Western blot analysis for Ikappa Balpha using a rabbit antibody directed toward Ikappa Balpha , followed by horseradish peroxidase-conjugated anti-rabbit IgG. CNTL, cells transfected with equivalent amounts of the CMV-driven eukaryotic expression vector pcDNA3, lacking Bcl-2 cDNA insert.

To confirm the notion that the conserved N-terminal BH4 domain of Bcl-2 is responsible for the underlying effects on Ikappa Balpha activity, we utilized point substitution mutations of the BH4 domain that had been previously shown to disrupt the anti-apoptotic function of Bcl-2 (14). In contrast to cells transfected with wild type Bcl-2 that displayed a 1.9-fold reduction (p < 0.006) in Ikappa Balpha activity compared with vector transfected control cells, cells transfected with the BH4 domain point mutants, with the exception of the I19G mutant, were not statistically different from vector transfected control cells (p = 2.77; Fig. 4, A and D). Interestingly, a small 0.60-fold reduction (p < 0.04) in Ikappa Balpha activity was observed with the I19G BH4 mutant.


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  		Fig. 4.   The effect of BH4 domain point mutants on Ikappa Balpha activity. A, cell lysates from 293 cells transfected with wild type Bcl-2 (Bcl-2) or Delta BH4 point substitution mutants were prepared for Western blot analysis for detection of Ikappa Balpha protein as shown in Fig. 3. The six BH4 domain point mutants utilized are indicated by the amino acid substitution at the designated positions: I14G, V15G, Y18G, I19G, K22G, and L23G. pcDNA3, control cells transfected with equivalent amounts of the CMV-driven eukaryotic expression vector lacking Bcl-2 cDNA inset. B, Ponceau S stained filter to demonstrate equivalent loading of protein samples. C, cell lysate from A was subjected to Western blot analysis for detection of Bcl-2 proteins using a hamster monoclonal antibody directed toward human Bcl-2. The figure verifies that Bcl-2 proteins were expressed to comparable levels. D, histogram represents densitometric analysis of three Western blots for Ikappa Balpha activity in the presence of wild type (WT) Bcl-2 and BH4 domain mutants. Data are expressed as fold reduction relative to vector transfected control cells. Ikappa Balpha activity is repressed by 1.9-fold (p < 0.006) by wild type Bcl-2. The BH4 domain point mutants I14G, V15G, Y18G, K22G, and L23G had no apparent effect on Ikappa Balpha activity (p = 2.77) with the exception of the I19G, which repressed Ikappa Balpha by 0.60-fold (p < 0.04) compared with vector control cells.

To verify that the observed differences between wild type Bcl-2 and BH4 domain mutants on Ikappa Balpha activity were not due to discrepancies in protein loading, the filter was stained with Ponceau S dye, which confirmed equivalent protein loading (Fig. 4B). In addition, Western blot analysis of these samples revealed that the wild type and BH4 domain mutants were expressed to comparable levels (Fig. 4C), ruling out the possibility that the noted differences in Ikappa Balpha activity were due to discrepancies in Bcl-2 protein expression.

Furthermore, consistent with our Western blot data for Ikappa Balpha , electromobility shift analysis for NFkappa B revealed that each of the point mutants tested with the exception of the I19G mutant were defective for directing NFkappa B-dependent DNA binding and were not significantly different from vector transfected control cells (Fig. 5). The fact that the I19G BH4 mutant had an intermediate effect on NFkappa B DNA binding compared with wild type Bcl-2 suggests that this residue may not be critical for directing Ikappa Balpha degradation.


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  		Fig. 5.   Effects of BH4 domain mutants on NFkappa B nuclear DNA binding. Equivalent amounts of nuclear extract from 293 cells were prepared following transfection with either wild type or BH4 domain mutants detailed in Fig. 4 and analyzed for NFkappa B DNA binding activity. Lane 1, free probe; lane 2, control cells (CNTL); lane 3, wild type Bcl-2 (Bcl-2); lanes 4-9, BH4 point substitution mutants; lane 10, pcDNA3 transfected cells. Wild type Bcl-2 but not the BH4 domain point mutants increase NFkappa B nuclear DNA binding, compared with controls. Arrow indicates NFkappa B complex.

Previously, we demonstrated that Bcl-2 activated NFkappa B and suppressed apoptosis of ventricular myocytes provoked by TNFalpha plus cycloheximide (33). Because NFkappa B has been reported to be important for suppressing apoptosis in mammalian cells, we tested functional significance of our observations by determining whether a block to NFkappa B activation, would impair the ability of Bcl-2 to rescue TNFalpha -mediated apoptosis. For these studies we generated a recombinant adenovirus that encodes a mutant version of the Ikappa Balpha molecule that contains serine to alanine point substitutions at amino acids 32 and 36, respectively. This renders Ikappa Balpha defective for phosphorylation and degradation, thereby preventing NFkappa B activation (27). As shown by gel shift analysis (Fig. 6), the Ikappa Balpha mutant prevented the increase in NFkappa B nuclear DNA binding activity mediated by Bcl-2, confirming that the Ikappa Balpha mutant was functionally active in these cells. Furthermore, expression of the Ikappa Balpha mutant impaired the ability of Bcl-2 to suppress apoptosis triggered by TNFalpha plus cycloheximide, demonstrated by the increased TUNEL positive nuclei (Fig. 7A) and nucleosomal DNA laddering (Fig. 7B).


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  		Fig. 6.   Bcl-2-mediated NFkappa B DNA binding in ventricular myocytes is inhibited by mutant Ikappa Balpha . Equivalent amounts of nuclear extract from 2-day-old post-natal ventricular myocytes were prepared for NFkappa B DNA binding activity following adenovirus-mediated delivery of Bcl-2 and mutant Ikappa Balpha S32/36A proteins (see text for details). Bcl-2 increases NFkappa B binding activity compared with uninfected control (CNTL) cells or those infected with a control adenovirus designated AdCMV. Ikappa Balpha S32/36A prevents Bcl-2-mediated increase in NFkappa B DNA binding.


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  		Fig. 7.   NFkappa B activation is required for Bcl-2-mediated suppression of apoptosis in ventricular myocytes. A, histogram represents the percentage of apoptosis by terminal deoxynucleotide nick-end-labeling TUNEL assay. 24 h after adenovirus-mediated gene transfer of Bcl-2 and Ikappa Balpha mutant S32/36A (Ikappa BMT) proteins to ventricular myocytes, cells were stimulated with TNFalpha (10 ng/ml) in the presence and absence of cycloheximide (CHX, 5 µg/ml) and assessed for evidence of nucleosomal DNA fragmentation by TUNEL assay. In the absence of cycloheximide, TNFalpha does not provoke apoptosis of ventricular myocytes, (percent apoptosis; control (CNTL) versus TNFalpha ; 5.6 ± 0.84% versus 7.5 ± 1.1%, p = 0.36). However, the combination of TNFalpha plus cycloheximide provokes apoptosis compared with vehicle-treated control cells or those stimulated with TNFalpha , (percent apoptosis; control versus TNF + cycloheximide, 5.6 ± 0.85% versus 38 ± 5.7%, p < 0.0002; TNFalpha versus TNFalpha  + cycloheximide, 7.5 ± 1.1% versus 38 ± 5.7%, p < 0.0004). Bcl-2 reduced the incidence of apoptosis provoked by TNFalpha plus CHX, (percent apoptosis; TNFalpha  + cycloheximide versus TNFalpha  + cycloheximide + Bcl-2, 38 ± 5.7% versus 16 ± 2.3%, p < 0.002), consistent with our earlier data (33). Inhibition of NFkappa B activation by Ikappa Balpha S32/36A (Ikappa BMT) impairs the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha plus cycloheximide in ventricular myocytes (percent apoptosis; TNFalpha  + cycloheximide + Bcl-2 versus TNFalpha  + cycloheximide + Bcl-2 + Ikappa BMT, 16 ± 2.3% versus 65 ± 9.8%, p < 0.001). B, nucleosomal DNA laddering of genomic DNA from ventricular myocytes following interventions described above. Inhibition of NFkappa B activation by Ikappa Balpha MT impairs the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha  + cycloheximide.

The mechanism by which Bcl-2 mediates NFkappa B activation is unknown but may involve the inactivation of Ikappa Balpha . In the present study, we provide evidence for the regulation of Ikappa Balpha activity by Bcl-2 through a mechanism that requires the BH4 domain of Bcl-2. Precedence for cellular factors other than NFkappa B to be regulated by Bcl-2 has been documented (40). Although the mode by which the BH4 domain modulates Ikappa Balpha activity is unknown, we have previously demonstrated that Bcl-2 leads to the phosphorylation of Ikappa Balpha and degradation by the proteasome (33). However, our studies indicate that Bcl-2 does not directly interact with Ikappa Balpha .2 Therefore, it is tempting to speculate that Bcl-2 modulates Ikappa Balpha activity by interacting with one or more cellular factors that directly or indirectly activate NFkappa B. Alternatively, the BH4 domain could influence the activity of Ikappa Balpha by interacting with one of the Ikappa B kinases (41, 42). Nevertheless, in view of the growing evidence that the BH4 domain of Bcl-2 is critical for the prevention of apoptosis, our finding that inhibition of NFkappa B activation impairs the anti-apoptotic properties of Bcl-2 provides compelling evidence that links Bcl-2 to the NFkappa B signaling pathway for the suppression of apoptosis. A current model for the operation of Bcl-2 proposes that the BH4 domain binds to and sequesters factors leading to caspase activation and apoptosis. In this regard, the physical interaction of BH4 domain with mitochondrial Apaf-1 has reportedly been shown to inhibit association of Apaf-1 with cytochrome c and caspase 9, preventing the subsequent processing of caspase 3 (18, 19). Furthermore, the relationship between Bcl-2 and the NFkappa B signaling pathway becomes even more profound, given that activated caspase 3 can directly cleave the N-terminal segment of Ikappa Balpha , resulting in a peptide fragment that inhibits NFkappa B activation (31, 35). Thus, Bcl-2 may in part operate through a mechanism that intersects the activation of caspases and of NFkappa B for the suppression of apoptosis.

Our data provide the first direct evidence for the regulation of Ikappa Balpha by Bcl-2 through a mechanism that requires the conserved BH4 domain and links Bcl-2 to the NFkappa B signaling pathway for the suppression of apoptosis.

    ACKNOWLEDGEMENTS

We are grateful to D. Ballard, J. Reed, and Tristram Parslow for the generous gift of reagents cited and to Drs. Arnold Greenberg and H. Weisman for critical comments on the manuscript.

    FOOTNOTES

* This work was supported by grants from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Heart and Stroke Foundation of Canada Scholar. To whom correspondence should be addressed: Inst. of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Rm. 3016, 351 Taché Ave., Winnipeg, Manitoba R2H 2A6, Canada. Tel.: 204-235-3661; Fax: 204-233-6723; E-mail: Lorrie@sbrc.umanitoba.ca.

2 D. de Moissac and L. A. Kirshenbaum, unpublished data.

    ABBREVIATIONS

The abbreviations used are: NFkappa B, nuclear factor kappa B; TNFalpha , tumor necrosis factor alpha ; CMV, cytomegalovirus; TUNEL, terminal deoxynucleotidyl transferase nick end labeling.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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