Bcl-2 has been described both as an inhibitor of programmed cell death and as an inhibitor of mitochondrial dysfunction during apoptosis. It is still not clear what biochemical activity of Bcl-2 is responsible for its function, but increasing evidence indicates that a functional activity of Bcl-2 on the endoplasmic reticulum (ER) protects mitochondria under diverse circumstances. Indeed, an emerging hypothesis is that, during apoptosis, the Bcl-2 family regulates ER-to-mitochondrion communication by BH3-only proteins and calcium ions and thereby triggers mitochondrial dysfunction and cell death.

Introduction

Apoptosis is a cellular process by which a cell initiates a preprogrammed set of signal transduction pathways that ultimately lead to the death of the cell, and is therefore crucial to development and tissue homeostasis. The process is highly conserved from Caenorhabditis elegans to humans. It is distinct from necrosis, which is characterized by loss of membrane integrity and cell homeostasis rather than a preprogrammed series of morphological and molecular events. One of the most notable effects of apoptosis in higher vertebrates is its role in tumor prevention: in normal cells, apoptosis will typically occur when a cell faces genomic instability and loss of cell-cycle control but, during progression to a cancerous form, cells may lose the ability to undergo apoptosis or sensitivity to apoptotic signals.

Apoptotic cells are characterized by the condensation of the nucleus, a decrease in cell size and plasma membrane blebbing (Kerr et al., 1972; Wyllie et al., 1980). At the molecular level, several common features are often associated with apoptosis: mitochondrial dysfunction and cytochrome c release, activation of a family of cysteine proteases called caspases, exposure of phosphatidyl serine to the extracellular space, and DNA cleavage at inter-nucleosomal regions. Mitochondria are of central importance in the apoptotic process. The release of cytochrome c and Smac/Diablo from mitochondria triggers caspase activiation. Cytochrome c binds to an adaptor molecule called Apaf-1, which results in the activation of caspases (reviewed by Green and Reed, 1998). Smac/Diablo induces apoptosis by de-repressing the inhibitor of apoptosis proteins (IAPs), leading to the activation of caspases, which regulate the induction and execution of apoptosis (reviewed by van Loo et al., 2002). Caspases exist as zymogens until they are cleaved into their active form. They can be divided into two different categories: the initiator caspases and the executioner caspases. The initiator caspases are activated by a variety of molecular cues, and the executioner caspases cleave substrates that catalyze the dismantling of the cell. Caspase 3, a typical executioner caspase, cleaves multiple downstream targets, among which are ICAD, an inhibitor of a nuclease that assists in nuclear condensation (Enari et al., 1998) and poly (ADP-ribose) polymerase (Nicholson et al., 1995; Tewari et al., 1995).

Bcl-2 family members function as regulators of almost all known forms of programmed cell death (for a review, see Cory and Adams, 2002). The bcl-2 gene was the first of its family to be discovered, and it was the first oncogene shown to be an inhibitor of apoptosis. Overexpressed Bcl-2 contributes to transformation of cells by preventing them from undergoing apoptosis, as seen in B-cell lymphomas in which Bcl-2 is upregulated following a chromosomal translocation. The criterion for membership of the Bcl-2 family is the presence of at least one Bcl-2-homology (BH) domain, of which there are four (BH1-BH4). There are three subfamilies of Bcl-2-related proteins. These are the anti-apoptotic subfamily (e.g. Bcl-2, Bcl-xL), the multi-domain pro-apoptotic subfamily (e.g. Bax, Bak) and the pro-apoptotic BH3-only subfamily (e.g. Bim, Bad). The anti-apoptotic subfamily has all four of the BH domains. The multi-domain pro-apoptotic proteins have all but the BH4 domain. The BH3-only subfamily members, as indicated by their name, contain only the BH3 domain.

The anti-apoptotic Bcl-2 proteins function, at least in part, by protecting the mitochondria during apoptosis, whereas the pro-apoptotic family members appear to disrupt mitochondria. Owing to the mitochondrial localization of many of the Bcl-2 family members, they are often assumed to have a direct biochemical effect on the mitochondria. However, Bcl-2 is also found on the endoplasmic reticulum (ER) and nuclear membranes (Akao et al., 1994; Krajewski et al., 1993). Yet, very little attention has been paid to the possible roles of Bcl-2 family members on these other intracellular membranes. Although the biochemical activity of Bcl-2 family members on intracellular membranes is not clear, it has been shown that membrane insertion of Bax and Bcl-2-type molecules is required for their function. Mutants of Bcl-2 that do not insert into membranes have reduced capacity to regulate apoptosis (Hockenbery et al., 1993; Nguyen et al., 1993; Tanaka et al., 1993). Similar mutants of Bax are also inactive, indicating that subcellular localization is important for their effects on apoptosis (Wolter et al., 1997). However, which intracellular membrane these proteins must insert into is still unknown. Here, we discuss studies describing the subcellular localization of Bcl-2, its effect on apoptosis regulation on various membranes, and the distribution of pro-apoptotic Bcl-2 family members. We propose a specific role for Bcl-2 on the ER in regulating ER-to-mitochondrion communication involving BH3-only proteins and calcium signaling.

Bcl-2 and Bcl-xL subcellular localization

Bcl-2 localizes to the ER, the outer mitochondrial membrane and the nuclear membrane, as shown by subcellular fractionation (Janiak et al., 1994; Krajewski et al., 1993) and electron microscopy (Akao et al., 1994; Krajewski et al., 1993; Monaghan et al., 1992). By contrast, the Bcl-2 family member Bcl-xL accumulates on the outer mitochondrial membrane (Kaufmann et al., 2003). Bcl-2 and Bcl-xL insert into membranes through a hydrophobic C-terminal transmembrane (TM) domain, the bulk of the protein lying on the cytoplasmic side of the membrane.

Recent work by Kaufmann et al. showed that targeting of Bcl-2 and Bcl-xL to intracellular membranes depends on the number and position of basic residues in the TM domain (Kaufmann et al., 2003). Bcl-xL has additional positively charged residues at the C-terminal end of the TM domain that are critical for specific mitochondrial targeting. This is in contrast to Bcl-2, and thus accounts for its ability to distribute between the ER, outer mitochondrial and nuclear membranes (Kaufmann et al., 2003).

Interestingly, full-length Bcl-2 induces apoptosis when transiently expressed in cells, whereas Bcl-xL does not (Uhlmann et al., 1998; Wang et al., 2001). The toxicity of transiently expressed Bcl-2 correlates with its expression on mitochondria, and is reproduced with a mutant form of Bcl-2 that is selectively targeted to mitochondria (Wang et al., 2001). Thus, mitochondria might not be the preferred site for pro-survival activities of Bcl-2.

ER-targeted Bcl-2

A great deal of information about the role of subcellular localization in Bcl-2 function has come from targeting mutants. The first such mutant is an ER-targeted form of Bcl-2 called Bcl-Cb5, in which the 21 C-terminal residues of Bcl-2 are replaced with the C-terminus of rat cytochrome B5 (Zhu et al., 1996). Cytochrome B5 is a TM protein that localizes to the ER (Mitoma and Ito, 1992). The Bcl-Cb5 fusion specifically targets the ER (Hacki et al., 2000; Zhu et al., 1996) and protects cells from apoptosis in some instances, but not in others (Zhu et al., 1996). In particular, Bcl-Cb5 protects cells against apoptosis induced by Myc overexpression in Rat-1 fibroblasts, but does not inhibit serum-deprivation-induced apoptosis in Madin-Darby canine kidney (MDCK) cells. This is in contrast to a mitochondrion-targeted mutant and wild-type Bcl-2, which can inhibit serum-deprivation-induced apoptosis. The `spatially distinct pathways' elucidated by this study were later expanded to include other factors. Lee et al. showed that the topoisomerase inhibitor etoposide induces apoptosis in cells overexpressing Bcl-Cb5, whereas Myc overexpression cannot (Lee et al., 1999). In addition, these cells were shown to be resistant to apoptosis induced by staurosporine, brefeldin A/cycloheximide, tunicamycin (Hacki et al., 2000), thapsigargin (Wang et al., 2001) and ceramide (Annis et al., 2001). Furthermore, Bcl-Cb5 inhibits apoptosis induced by Bax overexpression (Wang et al., 2001). Table 1 provides a summary of the various apoptotic inducers used to challenge the protective effect of Bcl-Cb5. In most cases, Bcl-Cb5 protects against apoptosis.

Table 1.

Inhibition of various apoptosis inducers by Bcl-Cb5

Inducer Cell type Protection Reference
Serum withdrawal   MDCK   No  Zhu et al., 1996  
Myc overexpression   Rat-1/Myc   Yes  Zhu et al., 1996  
Etoposide   Rat-1   No  Lee et al., 1999  
Staurosporine   Rat-6   Yes  Hacki et al., 2000  
Tunicamycin   Rat-6   Yes  Hacki et al., 2000  
Brefeldin A/cycloheximide   Rat-6   Yes  Hacki et al., 2000  
Ceramide   Rat-1   Yes  Annis et al., 2001  
Ionizing radiation   Jurkat   Yes  Rudner et al., 2001  
Thapsigargin   MDA-MB-468   Yes  Wang et al., 2001  
Bax overexpression   HEK-293   Yes  Wang et al., 2001  
Bad overexpression   MCF7   Yes  Thomenius et al., 2003  
Inducer Cell type Protection Reference
Serum withdrawal   MDCK   No  Zhu et al., 1996  
Myc overexpression   Rat-1/Myc   Yes  Zhu et al., 1996  
Etoposide   Rat-1   No  Lee et al., 1999  
Staurosporine   Rat-6   Yes  Hacki et al., 2000  
Tunicamycin   Rat-6   Yes  Hacki et al., 2000  
Brefeldin A/cycloheximide   Rat-6   Yes  Hacki et al., 2000  
Ceramide   Rat-1   Yes  Annis et al., 2001  
Ionizing radiation   Jurkat   Yes  Rudner et al., 2001  
Thapsigargin   MDA-MB-468   Yes  Wang et al., 2001  
Bax overexpression   HEK-293   Yes  Wang et al., 2001  
Bad overexpression   MCF7   Yes  Thomenius et al., 2003  

One conclusion that is clear from the studies of Bcl-Cb5 is that this mutant still protects the mitochondria. There are data suggesting that it can inhibit disruption of mitochondrial membrane potential (Annis et al., 2001), release of cytochrome c from mitochondria (Hacki et al., 2000) and oligomerization of Bax (Thomenius et al., 2003). Because all of these events occur on mitochondria, Bcl-Cb5 must have an indirect protective effect. In other words, there must be some factors that Bcl-2 affects that in turn affect mitochondria. On the basis of recent work from several laboratories, including our own, we propose that there are two primary pro-apoptotic factors regulated by Bcl-2 on the ER: BH3-only pro-apoptotic proteins and calcium ions. Evidence for both is summarized below.

Bcl-2 and pro-apoptotic Bcl-2 family members

The predominant hypothesis for the mechanism of Bcl-2 function is that it inhibits the action of pro-apoptotic Bcl-2 family members. In particular, Bcl-2 inhibits the activation of Bax, thereby protecting mitochondria and preventing cytochrome c release. It was initially thought that Bcl-2 inhibits Bax through a direct interaction (Oltvai et al., 1993). However, it is not clear that this interaction is necessary for the anti-apoptotic activity of Bcl-2. Mutants of Bcl-xL that cannot bind to Bax can nevertheless protect against apoptosis (Cheng et al., 1996). In addition, it has been demonstrated that Bax and Bcl-Cb5 do not interact during apoptosis, although Bcl-Cb5 does inhibit apoptosis (Annis et al., 2001). Also, Bcl-Cb5 can inhibit the oligomerization of a mutant of Bax that is constitutively present on mitochondria (Thomenius et al., 2003). Since Bcl-Cb5 and mitochondrial Bax are spatially separated, it is unlikely that they interact.

Two models for how Bcl-2 inhibits apoptosis are prevalent in the literature (see Fig. 1). In the first, BH3-only proteins induce apoptosis by inhibiting the anti-apoptotic activity of Bcl-2 (Bouillet and Strasser, 2002). However, there is emerging data suggesting BH3-only proteins have a toxic effect that is independent of Bcl-2 inhibition (Wei et al., 2001). There is also evidence indicating that BH3-only proteins directly activate Bax (Desagher et al., 1999; Grinberg et al., 2002; Letai et al., 2002; Marani et al., 2002) or directly affect the mitochondria (Grinberg et al., 2002; Sugiyama et al., 2002). These data suggest an alternative model in which Bcl-2 lies upstream of BH3-only proteins and inhibits them from activating Bax and inducing apoptosis. This model is also supported by recent findings suggesting that Bcl-Cb5 prevents the mitochondrial localization of a constitutively active form of the BH3-only protein Bad (Bad3A) (Thomenius et al., 2003). Bad3A can bind to Bcl-2 on the ER, which suggests that Bcl-2 can inhibit BH3-only proteins at either the ER or the mitochondrial membrane. These findings could also explain the `spatially distinct pathways' described by Zhu et al. (Zhu et al., 1996): it might be necessary for Bcl-2 to be on mitochondria to bind to certain BH3-only proteins or to bind active Bax during responses to certain cell death stimuli. For instance, DNA-damaging agents such as etoposide and doxorubicin induce Bbc3/PUMA, which localizes to the mitochondria (Han et al., 2001). Bcl-Cb5 might be unable to inhibit a mitochondrial BH3-only protein, because it cannot access it in the cell.

Fig. 1.

Alternative models of Bcl-2 family interactions. Left, anti-apoptotic Bcl-2 family members bind multi-domain pro-apoptotic family members (e.g. Bax), preventing them from inducing cytochrome c release. BH3-only proteins relieve this inhibition, freeing the multi-domain pro-apoptotic family members. Right, anti-apoptotic Bcl-2 family members bind to BH3-only proteins, thus preventing them from inducing Bax activity and cytochrome c release.

Fig. 1.

Alternative models of Bcl-2 family interactions. Left, anti-apoptotic Bcl-2 family members bind multi-domain pro-apoptotic family members (e.g. Bax), preventing them from inducing cytochrome c release. BH3-only proteins relieve this inhibition, freeing the multi-domain pro-apoptotic family members. Right, anti-apoptotic Bcl-2 family members bind to BH3-only proteins, thus preventing them from inducing Bax activity and cytochrome c release.

The idea that Bcl-2 family members have a role on the ER is also strengthened by reports suggesting that pro-apoptotic Bcl-2 family members can also function on the ER. Two reports show that the BH3-only protein Bik can induce apoptosis from the ER (Germain et al., 2002; Mathai et al., 2002). Bax and Bak can also stimulate apoptosis from the ER (Nutt et al., 2002; Scorrano et al., 2003; Zong et al., 2003). Consistent with the proposed role of Bcl-2 on the ER are data supporting the notion that BH3 peptides can either sensitize cells to apoptosis by binding to anti-apoptotic proteins or induce apoptosis by activating multi-domain pro-apoptotic proteins (Letai et al., 2002). Fig. 2 depicts a model whereby the `sensitizer' BH3-only proteins (e.g. Bad) displace `inducer' BH3-only proteins (e.g. Bid) sequestered to the ER by Bcl-2, thus initiating apoptosis.

Fig. 2.

Potential model for the role of Bcl-2 on the ER. Bcl-2 on the ER sequesters activator BH3-only proteins, preventing them from interacting with Bax. Activator BH3-only proteins are displaced by sensitizer BH3-only proteins, freeing them to activate Bax and induce cytochrome c release.

Fig. 2.

Potential model for the role of Bcl-2 on the ER. Bcl-2 on the ER sequesters activator BH3-only proteins, preventing them from interacting with Bax. Activator BH3-only proteins are displaced by sensitizer BH3-only proteins, freeing them to activate Bax and induce cytochrome c release.

There is also considerable work describing the interaction of Bcl-2 and the ER-resident protein Bap31 and the ability of this interaction to regulate the activation of caspases. Bap31 associates with procaspase 8, which results in cleavage of Bap31 and release of a pro-apoptotic fragment as well as active caspase 8. Bcl-2 inhibits this process by binding to Bap31 (Breckenridge et al., 2002; Ng and Shore, 1998; Nguyen et al., 2000). This important area of Bcl-2 function has been reviewed extensively elsewhere (Rudner et al., 2002) and is therefore not discussed further here. Nevertheless, it is important to mention that recent work has revealed a novel ER-localized BH3-only protein called Spike, which regulates the interaction between Bcl-xL and Bap31 (Mund et al., 2003). This lends further support to the role of Bcl-2 family members on the ER.

Bcl-2 and pro-apoptotic calcium signals

Another candidate for a factor that relays signals from ER-localized Bcl-2 to mitochondria is the calcium ion. Calcium has been implicated in the control of apoptosis through both the extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) death pathways (reviewed by Orrenius et al., 2003). Moreover, conditions that reduce the ER lumenal calcium concentration (i.e. low extracellular calcium or deficiency of the pro-apoptotic Bcl-2 family members Bax and Bak) protect mitochondria by impeding the transfer of calcium ions from the ER lumen to mitochondria (Pinton et al., 2001; Scorrano et al., 2003).

Calcium ions may communicate death signals from the ER to mitochondria either directly, by increasing mitochondrial calcium concentration, or indirectly, by triggering cell-intrinsic death pathways through calcineurin activation. In either case, calcium signals generated by inositol 1,4,5-trisphosphate (InsP3)-mediated release of calcium from the ER lumen are of central importance.

One of the primary functions of the ER is as a source of calcium signals that are released through InsP3 receptors (reviewed by Petersen, 2002; Putney et al., 2001; Berridge et al., 2003). The modulation of intracellular calcium concentration is a common signaling mechanism used in many biological systems, including T and B lymphocytes, which rely on calcium signaling to initiate development, activation and death (reviewed by Lewis, 2001; Winslow et al., 2003). Many agonists that induce increases in cytosolic calcium levels do so by stimulating the production of InsP3, which then binds to InsP3 receptors in the ER (reviewed by Patel et al., 1999; Thrower et al., 2001). These InsP3 receptors release calcium from the ER, generating spikes and waves whose frequencies and amplitudes transmit information that is sensed, for example, by calcium-sensitive phosphatases and kinases.

InsP3-linked calcium signals activate intrinsic death pathways by direct and indirect effects on mitochondria (Fig. 3). Mitochondria play crucial roles in both cell life and cell death. Increases in the level of mitochondrial matrix calcium evoked by calcium-mobilizing agonists play a fundamental role in cellular energy metabolism (reviewed by Smaili et al., 2000; Hajnoczky et al., 2000). Mitochondria also play a central role in cell death by releasing apoptotic factors (e.g. cytochrome c) that activate caspases (reviewed by Desagher and Martinou, 2000; Ferri and Kroemer, 2001; Wang, 2001; Nieminen, 2003). Apoptotic stimuli induce a switch in mitochondrial calcium signaling at the beginning of the apoptotic process by facilitating calcium-induced opening of the mitochondrial permeability transition pore (Szalai et al., 1999; Pacher and Hajnoczky, 2001). Thus, InsP3-linked calcium spikes can, under certain circumstances, trigger the mitochondrial permeability transition and, in turn, cytochrome c release. The apoptotic switch in mitochondrial calcium signaling can be mediated by the pro-apoptotic protein tcBid, which increases the magnitude of mitochondrial calcium signals by selectively permeabilizing the outer mitochondrial membrane (Csordas et al., 2002). Calcium also activates cell-intrinsic death pathways indirectly by activating calcineurin, which dephosphorylates and thereby activates the proapoptotic Bcl-2 family member Bad (Wang et al., 1999; Saito et al., 2000) or mediates induction of another Bcl-2 family member, Bik (Jiang and Clark, 2001).

Fig. 3.

The role of calcium ions in communicating death signals to mitochondria, and its regulation by Bcl-2. The ER lumen serves as a source of calcium ions that are released via InsP3 receptors. The resulting elevation of cytosolic calcium either directly mediates loss of mitochondrial permeability transition through increased uptake of calcium into the mitochondrial matrix, a process that is enhanced by permeabilization of the outer mitochondrial membrane by tcBid, or indirectly by activating calcineurin, which in turn activates Bad. Bcl-2 located on the ER membrane interferes with calcium-mediated apoptotic signals, either by decreasing ER lumenal calcium concentration (left) or by docking calcineurin to InsP3 receptors, thereby inhibiting InsP3-mediated calcium release and calcineurin-mediated dephosphorylation of Bad-P (right).

Fig. 3.

The role of calcium ions in communicating death signals to mitochondria, and its regulation by Bcl-2. The ER lumen serves as a source of calcium ions that are released via InsP3 receptors. The resulting elevation of cytosolic calcium either directly mediates loss of mitochondrial permeability transition through increased uptake of calcium into the mitochondrial matrix, a process that is enhanced by permeabilization of the outer mitochondrial membrane by tcBid, or indirectly by activating calcineurin, which in turn activates Bad. Bcl-2 located on the ER membrane interferes with calcium-mediated apoptotic signals, either by decreasing ER lumenal calcium concentration (left) or by docking calcineurin to InsP3 receptors, thereby inhibiting InsP3-mediated calcium release and calcineurin-mediated dephosphorylation of Bad-P (right).

The full extent of involvement of InsP3-mediated calcium signals in apoptosis may not yet be realized. A deficiency of InsP3 receptors inhibits apoptosis induction by glucocorticosteroids and ionizing radiation in T cells (Jayaraman and Marks, 1997; Khan et al., 1996) and by surface IgM ligation in B cells (Sugawara et al., 1997). Moreover, calcineurin overexpression sensitizes cells to apoptosis induction following growth factor withdrawal (Shibasaki and McKeon, 1995). Thus, calcium signals appear to be involved in mediating apoptosis in response to a wide range of apoptotic stimuli.

A focal point of Bcl-2 action may be to impede calcium release from the ER and thereby inhibit activation of mitochondrial death pathways. Reports published almost a decade ago indicated that Bcl-2 inhibits calcium release from the ER, which dampens calcium oscillations and prevents redistribution of calcium from the ER to mitochondria following growth factor withdrawal (Baffy et al., 1993; Lam et al., 1994; Magnelli et al., 1994). Subsequently, there have been conflicting theories about the mechanism. We have provided evidence that Bcl-2 inhibits calcium release from the ER and thereby preserves the ER calcium pool (Distelhorst et al., 1996; He et al., 1997), whereas others have suggested that Bcl-2 suppresses ER calcium release by decreasing ER lumenal calcium concentration (Pinton et al., 2000; Foyouzi-Youssefi et al., 2000). The latter reports indicating that Bcl-2 decreases ER lumenal calcium concentration relied heavily on transient expression of Bcl-2. This may be a problem because, as noted above, transient expression of wild-type Bcl-2 is toxic to a broad range of cell types, and actually induces apoptosis rather than inhibits it (Uhlmann et al., 1998; Wang et al., 2001).

Bcl-2 on the ER can also interfere with calcium-mediated death signals through its interaction with the calcium-activated phosphatase calcineurin. Bcl-2 is known to form a tight complex with calcineurin, and calcineurin associated with Bcl-2 retains phosphatase activity (Shibasaki et al., 1997; Srivastava et al., 1999). Recent findings confirm the association of Bcl-2 with calcineurin in brain cells and provide evidence that Bcl-2 docks calcineurin to InsP3 receptors (Erin et al., 2003a; Erin et al., 2003b). Significantly, Linette et al. (Linette et al., 1996) demonstrated that Bcl-2 inhibits anti-CD3/T-cell receptor (TCR)-mediated activation of NFATc and induction of interleukin 2 (IL-2) expression, which thereby inhibits cell-cycle entry by delaying G0/G1 transition into S phase and also inhibits TCR-activation-induced apoptosis. Active NFATc is generated by calcineurin, which binds to and dephosphorylates NFATc in the cytoplasm, permitting NFATc to enter the nucleus. It has been suggested that Bcl-2 inhibits NFATc activation by sequestering calcineurin to intracellular membranes (Shibasaki et al., 1997).

In T cells, calcium/calcineurin-mediated activation of NFATc increases expression of IL-2, which in turn stimulates dual pathways, one leading to cell death and the other leading to cell survival. IL-2 induces cell death through Stat2-mediated induction of the death receptor ligand Fas, but promotes cell survival through Akt-mediated induction of Bcl-2 expression (Parijs et al., 1999). This raises the possibility that increased expression of Bcl-2 may be part of a feedback loop that dampens InsP3-mediated calcium signals and thereby controls T-cell proliferation while maintaining cell survival. Indeed, we have found that Bcl-2 interacts with InsP3 receptors and inhibits InsP3-mediated calcium release from the ER (C.W.D., unpublished).

Conclusions

The Bcl-2 family has been studied extensively and yet no biochemical function has been proposed that definitively explains its effect on apoptosis. Studies of Bcl-Cb5 indicate that this effect can be performed on the ER. Thus, it is likely that there are functions of Bcl-2 that can operate on either mitochondrial or ER membranes.

The subcellular distribution of Bcl-2 proteins no doubt plays a crucial role in their ability to regulate apoptosis. The sequestration of BH3-only proteins by Bcl-2 on the ER appears to be an attractive protection mechanism because sequestration of these proteins solely to the mitochondria might be `dangerous' for healthy cells. Keeping pro-apoptotic proteins at their sites of action might provide a rapid response, but it also might result in random and undesired apoptosis. It is more likely that Bcl-2 on the ER is a first line of defense against apoptotic signals and serves to keep pro-apoptotic proteins away from their site of action, whereas Bcl-2 and Bcl-xL on the mitochondria inhibit BH3-only proteins and multi-domain proteins that have already accumulated on the mitochondria. Evidence describing the role of ER-to-mitochondrion calcium signaling in apoptosis regulation is also growing, but how Bcl-2 family members regulate this process is unknown. Since Bcl-2 reduces InsP3 receptor activity by direct interaction (C.W.D., unpublished), and Bax and Bak increase calcium release from the ER (Nutt et al., 2002; Scorrano et al., 2003; Zong et al., 2003), calcium is probably an important apoptotic signal from the ER.

Acknowledgements

The authors appreciate the helpful comments and suggestions of Martin Bootman. This work was supported by NIH grants RO1 CA85804 (C.W.D.) and T32 CA73515 (M.J.T.).

References

Akao, Y., Otsuki, Y., Kataoka, S., Ito, Y. and Tsujimoto, Y. (
1994
). Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes.
Cancer Res.
54
,
2468
-2471.
Annis, M. G., Zamzami, N., Zhu, W., Penn, L. Z., Kroemer, G., Leber, B. and Andrews, D. W. (
2001
). Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.
Oncogene
20
,
1939
-1952.
Baffy, G., Miyashita, T., Williamson, J. R. and Reed, J. C. (
1993
). Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production.
J. Biol. Chem.
268
,
6511
-6519.
Berridge, M. J., Bootman, M. D. and Roderick, H. L. (
2003
). Calcium signaling: dynamics, homeostasis and remodelling.
Nat. Rev. Mol. Cell. Biol.
4
,
517
-529.
Bouillet, P. and Strasser, A. (
2002
). BH3-only proteins – evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death.
J. Cell. Sci.
115
,
1567
-1574.
Breckenridge, D. G., Nguyen, M., Kuppig, S., Reth, M. and Shore, G. C. (
2002
). The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA
99
,
4331
-4336.
Cheng, E. H., Levine, B., Boise, L. H., Thompson, C. B. and Hardwick, J. M. (
1996
). Bax-independent inhibition of apoptosis by Bcl-XL.
Nature
379
,
554
-556.
Cory, S. and Adams, J. M. (
2002
). The Bcl2 family: regulators of the cellular life-or-death switch.
Nat. Rev. Cancer
2
,
647
-656.
Csordas, G., Madesh, M., Antonsson, B. and Hajnoczky, G. (
2002
). tcBid promotes Ca2+ signal propagation to the mitochondria: control of Ca2+ permeation through the outer mitochondrial membrane.
EMBO J.
21
,
2198
-2206.
Desagher, S. and Martinou, J. C. (
2000
). Mitochondria as the central control point of apoptosis.
Trends Cell Biol.
10
,
369
-377.
Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B. and Martinou, J. C. (
1999
). Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.
J. Cell Biol.
144
,
891
-901.
Distelhorst, C. W., Lam, M. and McCormick, T. S. (
1996
). Bcl-2 inhibits hydrogen peroxide-induced ER Ca2+ pool depletion.
Oncogene
12
,
2051
-2055.
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (
1998
). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature
391
,
43
-50.
Erin, N., Bronson, S. K. and Billingsley, M. L. (
2003a
). Calcium-dependent interaction of calcineurin with Bcl-2 in neuronal tissue.
Neuroscience
117
,
541
-555.
Erin, N., Lehman, R. A. W., Boyer, P. J. and Billingsley, M. L. (
2003b
). In vitro hypoxia and excitotoxicity in human brain induce calcineurin-Bcl-2 interactions.
Neuroscience
117
,
557
-565.
Ferri, K. F. and Kroemer, G. (
2001
). Organelle-specific initiation of cell death pathways.
Nat. Cell Biol.
3
,
E255
-E263.
Foyouzi-Youssefi, R., Arnaudeau, S., Borner, C., Kelley, W. L., Tschopp, J., Lew, D. P., Demaurex, N. and Krause, K. H. (
2000
). Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA
97
,
5723
-5728.
Germain, M., Mathai, J. P. and Shore, G. C. (
2002
). BH-3-only BIK functions at the endoplasmic reticulum to stimulate cytochrome c release from mitochondria.
J. Biol. Chem.
277
,
18053
-18060.
Green, D. R. and Reed, J. C. (
1998
). Mitochondria and apoptosis.
Science
281
,
1309
-1312.
Grinberg, M., Sarig, R., Zaltsman, Y., Frumkin, D., Grammatikakis, N., Reuveny, E. and Gross, A. (
2002
). tBID homooligomerizes in the mitochondrial membrane to induce apoptosis.
J. Biol. Chem.
277
,
12237
-12245.
Hacki, J., Egger, L., Monney, L., Conus, S., Rosse, T., Fellay, I. and Borner, C. (
2000
). Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2.
Oncogene
19
,
2286
-2295.
Hajnoczky, G., Csordas, G., Krishnamurthy, R. and Szalai, G. (
2000
). Mitochondrial calcium signaling driven by the IP3 receptor.
J. Bioenerg. Biomembr.
32
,
15
-25.
Han, J., Flemington, C., Houghton, A. B., Gu, Z., Zambetti, G. P., Lutz, R. J., Zhu, L. and Chittenden, T. (
2001
). Expression of bbc3, a proapoptotic BH3-only gene, is regulated by diverse cell death and survival signals.
Proc. Natl. Acad. Sci. USA
98
,
11318
-11323.
He, H., Lam, M., McCormick, T. S. and Distelhorst, C. W. (
1997
). Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2.
J. Cell Biol.
138
,
1219
-1228.
Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L. and Korsmeyer, S. J. (
1993
). Bcl-2 functions in an antioxidant pathway to prevent apoptosis.
Cell
75
,
241
-251.
Janiak, F., Leber, B. and Andrews, D. W. (
1994
). Assembly of Bcl-2 into microsomal and outer mitochondrial membranes.
J. Biol. Chem.
269
,
9842
-9849.
Jayaraman, T. and Marks, A. R. (
1997
). T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.
Mol. Cell. Biol.
17
,
3005
-3012.
Jiang, A. and Clark, E. A. (
2001
). Involvement of Bik, a proapoptotic member of the Bcl-2 family, in surface IgM-mediated B cell apoptosis.
J. Immunol.
166
,
6025
-6033.
Kaufmann, T., Schlipf, S., Sanz, J., Neubert, K., Stein, R. and Borner, C. (
2003
). Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane.
J. Cell Biol.
160
,
53
-64.
Kerr, J. F., Wyllie, A. H. and Currie, A. R. (
1972
). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br. J. Cancer
.
26
,
239
-257.
Khan, A. A., Soloski, M. J., Sharp, A. H., Schilling, G., Sabatini, D. M., Li, S.-H., Ross, C. A. and Snyder, S. H. (
1996
). Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5,-trisphosphate receptor.
Science
273
,
503
-507.
Krajewski, S., Tanaka, S., Takayama, S., Schibler, M. J., Fenton, W. and Reed, J. C. (
1993
). Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.
Cancer Res.
53
,
4701
-4714.
Lam, M., Dubyak, G., Chen, L., Nuñez, G., Miesfeld, R. L. and Distelhorst, C. W. (
1994
). Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes.
Proc. Natl. Acad. Sci. USA
91
,
6569
-6573.
Lee, S. T., Hoeflich, K. P., Wasfy, G. W., Woodgett, J. R., Leber, B., Andrews, D. W., Hedley, D. W. and Penn, L. Z. (
1999
). Bcl-2 targeted to the endoplasmic reticulum can inhibit apoptosis induced by Myc but not etoposide in Rat-1 fibroblasts.
Oncogene
18
,
3520
-3528.
Letai, A., Bassik, M., Walensky, L., Sorcinelli, M., Weiler, S. and Korsmeyer, S. (
2002
). Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics.
Cancer Cell
2
,
183
-192.
Lewis, R. S. (
2001
). Calcium signaling mechanisms in T lymphocytes.
Annu. Rev. Immunol.
19
,
497
-521.
Linette, G. P., Li, Y., Roth, K. and Korsmeyer, S. J. (
1996
). Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.
Proc. Natl. Acad. Sci. USA
93
,
9545
-9552.
Magnelli, L., Cinelli, M., Turchetti, A. and Chiarugi, V. P. (
1994
). Bcl-2 overexpression abolishes early calcium waving preceding apoptosis in NIH-3T3 murine fibroblasts.
Biochem. Biophys. Res. Commun.
204
,
84
-90.
Marani, M., Tenev, T., Hancock, D., Downward, J. and Lemoine, N. R. (
2002
). Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis.
Mol. Cell. Biol.
22
,
3577
-3589.
Mathai, J. P., Germain, M., Marcellus, R. C. and Shore, G. C. (
2002
). Induction and endoplasmic reticulum location of BIK/NBK in response to apoptotic signaling by E1A and p53.
Oncogene
21
,
2534
-2544.
Mitoma, J. and Ito, A. (
1992
). The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.
EMBO J.
11
,
4197
-4203.
Monaghan, P., Robertson, D., Amos, T. A., Dyer, M. J., Mason, D. Y. and Greaves, M. F. (
1992
). Ultrastructural localization of bcl-2 protein.
J. Histochem. Cytochem.
40
,
1819
-1825.
Mund, T., Gewies, A., Schoenfeld, N., Bauer, M. K. and Grimm, S. (
2003
). Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.
FASEB J.
17
,
696
-698.
Ng, F. W. and Shore, G. C. (
1998
). Bcl-XL cooperatively associates with the Bap31 complex in the endoplasmic reticulum, dependent on procaspase-8 and Ced-4 adaptor.
J. Biol. Chem.
273
,
3140
-3143.
Nguyen, M., Millar, D. G., Yong, V. W., Korsmeyer, S. J. and Shore, G. C. (
1993
). Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence.
J. Biol. Chem.
268
,
25265
-25268.
Nguyen, M., Breckenridge, D. G., Ducret, A. and Shore, G. C. (
2000
). Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.
Mol. Cell. Biol.
20
,
6731
-6740.
Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A. et al. (
1995
). Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.
Nature
376
,
37
-43.
Nieminen, A.-L. (
2003
). Apoptosis and necrosis in health and disease: role of mitochondria.
Int. Rev. Cytol.
224
,
29
-55.
Nutt, L. K., Pataer, A., Pahler, J., Fang, B., Roth, J., McConkey, D. J. and Swisher, S. G. (
2002
). Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.
J. Biol. Chem.
277
,
9219
-9225.
Oltvai, Z. N., Milliman, C. L. and Korsmeyer, S. J. (
1993
). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.
Cell
74
,
609
-619.
Orrenius, S., Zhivotovsky, B. and Nicotera, P. (
2003
). Regulation of cell death: the calcium-apoptosis link.
Nat. Rev. Mol. Cell. Biol.
4
,
552
-565.
Pacher, P. and Hajnoczky, G. (
2001
). Propagation of the apoptotic signal by mitochondrial waves.
EMBO J.
20
,
4107
-4121.
Parijs, L. V., Refaeli, Y., Lord, J. D., Nelson, B. H., Abbas, A. K. and Baltimore, D. (
1999
). Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death.
Immunity
11
,
281
-288.
Patel, S., Joseph, S. K. and Thomas, A. P. (
1999
). Molecular properties of inositol 1,4,5-trisphosphate receptors.
Cell Calcium
25
,
247
-264.
Petersen, O. H. (
2002
). Calcium signal compartmentalization.
Biol. Res.
35
,
177
-182.
Pinton, P., Ferrari, D., Magalhaes, P., Schulze-Osthoff, K., di Virgilio, F., Pozzan, T. and Rizzuto, R. (
2000
). Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells.
J. Cell Biol.
148
,
857
-862.
Pinton, P., Ferrari, D., Rapizzi, E., de Virgilio, F., Pozzan, T. and Rizzuto, R. (
2001
). The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action.
EMBO J.
20
,
2690
-2701.
Putney, J. W., Jr, Broad, L. M., Braun, F. J., Lievremont, J. P. and Bird, G. S. (
2001
). Mechanisms of capacitative calcium entry.
J. Cell Sci.
114
,
2223
-2229.
Rudner, J., Lepple-Wienhues, A., Budach, W., Berschauer, J., Friedrich, B., Wesselborg, S., Schulze-Osthoff, K. and Belka, C. (
2001
). Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.
J. Cell Sci.
114
,
4161
-4172.
Rudner, J., Jendrossek, V. and Belka, C. (
2002
). New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum.
Apoptosis
7
,
441
-447.
Saito, S., Hiroi, Y., Zou, Y., Aikawa, R., Toko, H., Shibasaki, F., Yazaki, Y., Nagai, R. and Komuro, I. (
2000
). β-adrenergic pathway induces apoptosis through calcineurin activation in cardiac myocytes.
J. Biol. Chem.
275
,
34528
-34533.
Scorrano, L., Oakes, S. A., Opferman, J. T., Cheng, E. H., Sorcinelli, M. D., Pozzan, T. and Korsmeyer, S. J. (
2003
). BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis.
Science
300
,
135
-139.
Shibasaki, F. and McKeon, F. (
1995
). Calcineurin functions in Ca2+-activated cell death in mammalian cells.
J. Cell Biol.
131
,
735
-743.
Shibasaki, F., Kondo, E., Akagi, T. and McKeon, F. (
1997
). Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2.
Nature
386
,
728
-731.
Smaili, S. S., Hsu, Y. T., Youle, R. J. and Russell, J. T. (
2000
). Mitochondria in Ca2+ signaling and apoptosis.
J. Bioenerg. Biomembr.
32
,
35
-46.
Srivastava, R. K., Sasaki, C. Y., Hardwick, J. M. and Longo, D. L. (
1999
). Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fax ligand transcription.
J. Exp. Med.
190
,
253
-265.
Sugawara, H., Kurosaki, M., Takata, M. and Kurosaki, T. (
1997
). Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.
EMBO J.
16
,
3078
-3088.
Sugiyama, T., Shimizu, S., Matsuoka, Y., Yoneda, Y. and Tsujimoto, Y. (
2002
). Activation of mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim.
Oncogene
21
,
4944
-4956.
Szalai, G., Krishnamurthy, R. and Hajnoczky, G. (
1999
). Apoptosis driven by IP(3)-linked mitochondrial calcium signals.
EMBO J.
18
,
6349
-6361.
Tanaka, S., Saito, K. and Reed, J. C. (
1993
). Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival.
J. Biol. Chem.
268
,
10920
-10926.
Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S. and Dixit, V. M. (
1995
). Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase.
Cell
81
,
801
-809.
Thomenius, M. J., Wang, N. S., Reineks, E. Z., Wang, Z. and Distelhorst, C. W. (
2003
). Bcl-2 on the endoplasmic reticulum regulates Bax activity by binding to BH3-only proteins.
J. Biol. Chem.
278
,
6243
-6250.
Thrower, E. C., Hagar, R. E. and Ehrlich, B. E. (
2001
). Regulation of Ins(1,4,5)P3 receptor isoforms by endogenous modulators.
Trends Pharmacol. Sci.
22
,
580
-586.
Uhlmann, E. J., Subramanian, T., Vater, C. A., Lutz, R. and Chinnadurai, G. (
1998
). A potent cell death activity associated with transient high level expression of BCL-2.
J. Biol. Chem.
273
,
17926
-17932.
van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J. and Vandenabeele, P. (
2002
). The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet.
Cell Death Differ.
9
,
1031
-1042.
Wang, H. G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T. F. and Reed, J. C. (
1999
). Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD.
Science
284
,
339
-343.
Wang, N. S., Unkila, M. T., Reineks, E. Z. and Distelhorst, C. W. (
2001
). Transient expression of wild-type or mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death.
J. Biol. Chem.
276
,
44117
-44128.
Wang, X. (
2001
). The expanding role of mitochondria in apoptosis.
Genes Dev.
15
,
2922
-2933.
Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B. and Korsmeyer, S. J. (
2001
). Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
Science
292
,
727
-730.
Winslow, M. M., Neilson, J. R. and Crabtree, G. R. (
2003
). Calcium signaling in lymphocytes.
Curr. Opin. Immunol.
15
,
299
-307.
Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G. and Youle, R. J. (
1997
). Movement of Bax from the cytosol to mitochondria during apoptosis.
J. Cell Biol.
139
,
1281
-1292.
Wyllie, A. H., Kerr, J. F. and Currie, A. R. (
1980
). Cell death: the significance of apoptosis.
Int. Rev. Cytol.
68
,
251
-306.
Zhu, W., Cowie, A., Wasfy, G. W., Penn, L. Z., Leber, B. and Andrews, D. W. (
1996
). Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.
EMBO J.
15
,
4130
-4141.
Zong, W. X., Li, C., Hatzivassiliou, G., Lindsten, T., Yu, Q. C., Yuan, J. and Thompson, C. B. (
2003
). Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.
J. Cell Biol.
162
,
59
-69.