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Recent Progress on the Regulation of Apoptosis by Bcl-2 Family Members

ANDY 1. MINN,',f RACHEL E. SWAIN,* AVERIL MA,?,§ AND CRAIG 6. THOMPSON',t,*,§,(,II

'Gwen Knapp Center for Lupus ond Immunology Research, tCommi&e on Immunology, #Committee on Cancer Biology, heparhnent of Medicine, THoword Hughes, Medical Instihk,

I IDephent of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637

1. Significance of Programmed Cell Death

The development of multicellular organisms and the renewal of differen- tiated cell types in adult organisms rely on an expansion in cell numbers, making the control of cell division an important process in metazoan development and homeostasis. However, properly regulated development and homeostasis also require cell death (Ellis et al., 1991; Jacobson et al., 1997). Developmentally regulated cell death has been studied in both invertebrate and vertebrate animals and has been referred to as programmed cell death (PCD). Originally, this term was used to emphasize that this type of death is part of developmental programs. With the finding that regulated forms of cell death also occur in adult multicellular organisms, the term PCD has been adopted to describe all forms of cell death that are mediated by an intracelliilar program as opposed to those that occur through necrosis. Despite the long-standing knowledge that cells can undergo PCD, its importance in metazoan development and homeostasis has only recentIy been widely appreciated.

PCD has many important roles in development. For example, PCD is utilized for the many morphologcal changes that take place during embryogenesis. This can be seen early in the embryo when PCD sculpts the digits by eliminating the unwanted cells between them (Saunders, 1966) or when PCD causes the Mullerian duct to regress in the male in order to establish sexual dimorphism (Jost, 1971). PCD is also important for the elimination of excess cells or cells that have developed improperly. In the developing vertebrate nervous system, many neuronal cells are produced in excess. The number of neurons that is needed is dictated by the size of the organ that the neurons enervate (Hamburger, 1975, 1992). The neurons that survive are the ones that have successfully competed for trophic factors andlor synaptic connections. Therefore, the developmental program successfully uses mechanisms of PCD in order to ensure that the supply of cells is exactly equivalent to the demand that is established by the developing organ.

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In addition to the developing nervous system, the vertebrate immune system also illustrates the importance of cell death in eliminating cells that have developed improperly (Abbas, 1996; Nossal, 1994; Surli and Sprent, 1994). As with neurond cells, lymphocyte development begns with the generation of a large excess of cells. The purpose of such overproliferation is to allow for lymphocytes to stochastically generate antigen receptors of unique specificity. The minority of lymphocytes that produce a functional antigen receptor through V( D)J recombination are signaled to survive through a process of positive selection while the rest are eliminated by PCD. However, the random generation of antigen receptors also produces lymphocytes that are self-reactive. Therefore, a mechanism must be in place to eliminate those cells that pose a threat to the welfare of the organism. Potentially autoreactive lymphocytes are eliniinated by PCD through a process of negative selection. The developmental processes of positive and negative selection in the T lymphocyte immune system lead to the removal of greater than 95% of all thymocytes by PCD, an extreme but illustrative example of the role that PCD plays in the development of inulticellular organism.

The important roles that PCD play in the maintenance of inulticellular organisms are not limited to development. Once development has been completed, PCD reinains an active process to ensure proper organismal homeostasis. It has been proposed that nearly all cells in an adult organism are programmed to die and that this fate must be actively suppressed by environmental survival cues (Raff, 1992; Weil et al., 1996). For example, a tissue-specific cell dies if it is not residing in the proper tissue. Such a cell is committed to die because it is not receiving appropriate environmental survivd cues. In this way, the requirement for proper environmentd survival signals not only serves to dictate the tissue specificity of various cells but also creates a niche of limited size to support them.

The homeostasis of a particular organ system can be maintained not only by constant survival signals that inhibit cell death but also by signals that activate the PCD machinery. During an immune response, lymphocytes that are reactive to a particular foreign antigen undergo a dramatic expansion in order to combat the foreign antigen. Once the pathogen is contained, the expanded cells must be removed in order to return cell numbers to a homeostatic level (Osborne, 1996). PCD rids the host of cells that have completed their function through a series of “death receptors.” The failure of PCD mechanisms to remove lymphocytes after the elimination of antigen can lead to disorders in which lymphocytes accumu- late inappropriately. However, the triggering of excessive PCD by viruses such as human immunodeficiency virus (HIV) can lead to the tremendous

REGULATION OF APOPTOSIS BY Bcl-2 FAMILY MEMBERS 247

loss of lymphocytes and result in acquired immune deficiency syndrome known as AIDS.

Since PCD plays a pivotal role in the development and in the maintenance of homeostasis within inulticellular organisms, disruptions in the pathways by which the cell death program operates are thought to contribute to the pathology of various human diseases (Thompson, 1995). Muta- tions in genes known to control PCD have been associated with pathologies such as cancer, autoimmunity, and neurodegenerative disorders. Thus, much attention has focused on trying to define the molecular mechanism by which PCD is regulated. This review concentrates on a family of critical cell death regulators known as the Bcl-2 family and summarizes some of the most recent advances in our understanding of their properties, structure, and function.

II. The Genetics of Programmed Cell Death

The components of the programmed cell death pathway are highly conserved throughout metazoan evolution. It is likely that all signals that initiate programmed cell death ultimately trigger a central execution mechanism. Some of these signals include growth factor withdrawal, viral infection, inappropriate oncogene activation, cytotoxic T-cell killing, drug treatment, DNA damage, and other events that perturb normal cellular function (Yang and Korsmeyer, 1996). Based on an analysis of several examples of cell death in mammals, Wyllie and colleagues grossly characterized the morphological changes that result from this central execution mechanism and named it apoptosis, a Greek term meaning “falling off’ which describes leaves or petals (Kerr et al., 1972). Morphologically, apoptosis results in the loss of cell volume, membrane blebbing, and chromatin condensation (Cohen, 1993). DNA fragmentation into oligonucleosomd size fragments occurs through a calcium-dependent endonuclease, and the cell fragments into apoptosis bodies to facilitate consumption by phagocytes. Because this process does not result in the leakage of cellular contents, dead cells can be cleanly removed. Although some biochemical and morphological differences are apparent between certain cells undergoing programmed cell death and the classical description of apoptosis (Ellis et al., 1991; Schwartz et al., 1993), apoptosis is generally equated with PCD.

It is difficult to ascertain whether a phenomenon associated with a dying cell is critically regulated by the central cell death machinery or is merely a consequence of the activation of this machinery. The morphological change that were found to be associated with apoptosis guided investiga- tions into the nature of the central cell death machinery. The prominence and deleterious consequences of the nuclear changes that occur during

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apoptosis led to the early hypothesis that the central death machinery focused around the control of DNA condensation and DNA fragmentation. However, cells with their nuclei removed are still able to undergo apoptosis (Jacobson et al., 1994). Thus, “nuclear death” is a consequence of apoptosis, and the irreversible step controlled by the cell death machinery is likely to involve cytoplasmic factors. Insight into the nature of these cytoplasmic factors has been obtained with the identification of genes that control programmed cell death.

During the development of the nematode Caenorhabditis elegans, 1090 somatic cells are generated, and of these cells, 131 are programmed to die, Genetic analysis revealed that three genes, CED-3, CED-4, and CED- 9, control all 131 of these developmental programmed cell deaths (Hen- gartner and Horvitz, 1994b). PCD requires CED-3 and CED-4, as loss of function mutations in either of these genes prevents the death of nearly all cells that normally are programmed to die (Ellis and Horvitz, 1986). Genetic mosaic experiments demonstrate that these gene products function cell autonomously (Yuan and Horvitz, 1990). Dominant gain of function CED-9 mutations prevent all cells from dying and result in viable but functionally compromised worms. Loss of function CED-9 mutations are lethal, presumably because cells that normally should survive undergo programmed cell death (Hengartner et al., 1992). Extended genetic analysis of these genes in C. elegans show that CED-9 acts as an inhibitor of CED- 4 and prevents CED-4 from activating the death-inducing properties of CED-3 (Shaham and Horvitz, 1996). Thus, the programmed cell death occurs during the development of C. elegans is genetically encoded and functions in a fashion in which CED-9 inhibits CED-4, which in turn activates CED-3.

A mammalian homolog of CED-9 was identified as bcl-2, a gene that was initially cloned from human B-cell lymphomas with a characteristic t(14;18) chromosomal translocation (Bakhshi et al., 1985; Cleary et al., 1986; Hengartner and Horvitz, 1994a). Studies revealed that Bcl-2 is a novel protooncogene that does not promote cell proliferation but rather promotes cell survival. When cells are subjected to a wide variety of cytotoxic stimuli that induce mammalian cell death, such as growth factor withdrawal, chemotherapeutic drugs, metabolic toxins, viral infections, and inappropriate oncogene expression, expression of Bcl-2 is able to inhibit death in each case. Mammalian Bcl-2 is able to complement CED-9 in C. elegans (Hengartner and Horvitz, 1994a; Vaux et al., 1992). Thus, in mammalian cells, a tremendous variety of cell death signals all converge on a central cell death machinery that is controlled by Bcl-2. Furthermore, this central cell death machinery is conserved from the developmental cell death program of C. elegans.

CED-3 was cloned and shown to be ho~nologous to a previously cloned maininalian gene interleubn-lp ( IL-lp) converting enzyme, or ICE (Mi- Lira et d., 1993). ICE was initially identified as a cysteine protease that converts the 31-kDa pro form of IL-lp into a 17.5-kDa mature form. A role for this protein in cell death was confirmed when ICE was transiently introduced into a mammalian cell line and found to cause agoptosis. Many more ICE-like proteases have been cloned from inaininalian systems, and these proteases have been renamed caspases (Faucheu et nl., 1995; Kainens et d., 1995; Kumar et nl., 1994; Wang et nl., 1994). Transfection of many of these caspases into mammalian cells causes apoptosis, and elimination of some of these genes in mice by gene targeting causes defects in apoptosis (Kuida et d , 1995, 1996). All of these caspases are synthesized in a pro form and must be activated by proteolytic cleavage (Kuinar, 1995). The proteolytic activity of these proteases is directed to rare cleavage sites containing Asp-X, where X is any ainino acid. Some targets for caspases include poly(ADP)-ribose polymerase (Lazebnik et nl., 1994), nuclear lain- ins (Takahashi et nl., 1996), fodrin (Cryns et nl., 1996), pel-activated kinase 2 (PAK2) (Rude1 and Bokoch, 1997), and DNA fragmentation factor (DFF) (Liu et d, 1997). Cleavage of some of these substrates has been proposed to irreversibly cominit a cell to PCD and induce at least some of the morphological changes associated with apoptosis. It has also been proposed that one reason for the exi5tence of multiple caspases is that some are involved in the ainplification of the response through cleavage of the pro forins of other caspases. Such early acting caspases inay include caspase 1 (ICE) (Enari et nl., 1995) and caspase 2 (Nedd2) (Harvey et nl., 1997). Other caspases nre inore distally involved in the execution of the cell death program, such as caspase 3 (CPP32) (Faleiro et a1 , 1997). Thus, much evidence points toward inembers of the caspase family as tlie executioners of tlie central cell death machinery.

The critical role of caspases in the cell death program has been further highlighted by studies on various maininalian cell death receptors (Yuan, 1997). The best characterized of the death receptors belong to the TNF family of receptors and include TNF-Rl and Fas/APO- 1. These receptors seem to tie involved in preserving lymphocyte homeostasis by eliminating previously activated or inappropriately activated lymphocytes during an immune response (Abbas, 1996). The cytoplasmic domain of these receptors contains an amino acid region known a s the death domain. This domain is involved in the recruitment of caspase 8 (FLICE, Mach, Mch5), which is activated after ligation of the Fas receptor (Boldin et a1 , 1996; Muzio et nl., 1996). This finding suggests that death receptors can function through a direct link to the protease machinery that is responsible for causing cell death.

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Although the mechanism that death receptors utilize to cause apoptosis seems elegantly simple, the pathways that most apoptotic signals use do not seem to be so direct. Similar to the way that CED-9 acts upstream of CED-3 in C. elegans, most apoptotic stimuli in mammalian cells eventually converge on the Bcl-2 family members to regulate a step prior to caspase activation (Chinnaiyan et al., 1996). What is the mechanism by which Bcl- 2 functions to prevent caspase activation and thus apoptosis? The difficulty in answering this question is partly due to the fact that many cytoplasmic changes occur in cells undergoing apoptosis, including alterations in the cellular redox state (Hockenberry et al., 1993; Kane et nl., 1993), changes in the subcellular distribution of ions (Barry et al., 1993; Lam et al., 1994), and the disruption of mitochondrial function (Zamzami et al., 1995a). By preventing apoptosis, BcI-2 may indirectly prevent all of these changes. Many proposals regarding the mechanism by which Bcl-2 functions, such as by regulating calcium fluxes or by acting as an antioxidant, have been shown likely to be incorrect, illustrating the difficulty in distinguishing between cause and effect (Jacobson and Raff, 1995; Reynolds and Eastman, 1996; Shimizu et nl., 1995). Moreover, these proposals have proved inade- quate in explaining the fundamental question of how Bcl-2 inhibits caspase activation. Thus, the biochemical mechanisms utilized by Bcl-2 to regulate cell survival have remained elusive. An outline of the current understanding of the PCD pathway is presented in Fig. 1.

111. The &I-2 Family

CED-9 appears to be the only protein of its land in C. elegans. In contrast, several mammalian proteins exist that are homologs of Bcl-2 perhaps because of the complexity of mammalian organisms as compared to the invertebrate nematode. Furthermore, some of these homologs promotes rather than inhibit cell death. These death agonists and antagonists comprise the Bcl-2 family and share homology in as many as four amino acid regions denoted as Bcl-2 homology (BH)1, BH2, BH3, or BH4 (Boyd et al., 1995; Yin et al., 1994; Zha et al., 1996a). The BH1 and BH2 domains are approximately 21 and 16 amino acids long, respectively, and are generally separated by 30-40 amino acids. The BH4 domain, which is located at the N terminus, is generally found in the protein that inhibit cell death. The BH3 domain is found in all family members, but appears to be especially important in family members that promote cell death because some death agonists lack all of the homology domains except the BH3. Figure 2 diagrams the architecture of some of the Bcl-2 family members discussed in this review.

REGULATION OF i\POPTOSIS BY 13~1-2 FAMILY MEMBERS 25 1

The Genetics of Programmed Cell Death in C. elegans A

CED-9 -1 CED-4 4 CED-3 4 Cell Death

B The Pathway of Programmed Cell Death in Mammals

Death Receptors Growth Factor Withdrawal Cell Cycle Perturbations DNNMetabolie Damage

Endonuelease Activation

f Central Cell - Apaf-1 (CED-4) - Caspase (CED-3) - cell Surface - Cell Death and Death Signal Activation Activation Alterstions PhagoWosis

Cytoskeietal Reorganization

BcI-2 Bax BcI-x, Bak

1 Bcl-2 Family Members (CED-9)

FIG. 1. Suniinary of the pathways of prograinmed cell death in C elegans and in main- i d s . See text for details.

A. PROTEINS THAT INHIBIT APOPTOSIS

The first hoinolog of Bcl-2 was cloned using low stringency hybridization from chicken libraries. This approach yielded a homologous gene named bcl-x (Boise et al., 1993), bcl-x was subsequently cloned from human libraries where it was discovered that bcl-x has two alternatively spliced forms, bcl-xL and bcZ-xs. Bcl-xL has the largest open reading frame of 233

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FIG. 2. Schematic representation of the general architecture of Bcl-2-family members. Bcl-2 homology (BH) domains and the carboxy-terminal transmembrane (TM) domain are labeled. On the right of each structure are representative Bcl-2 families that share the depicted structure. Diagram is not drawn to scale.

amino acids and, like Bcl-2, protects cells against multiple apoptotic stimuli. Although the predicted molecular mass of Bcl-xL is 26 kDa, the protein migrates aberrantly slowly on an SDS-PAGE gel at 30 kDa. In Bcl-xs, 63 amino acids containing the BH1 and BH2 regions are removed by alternative splicing and, in contrast to Bcl-xL, result in a protein that promotes cell death. Bcl-xs will be discussed in more detail later. Bcl-x also has a third form called Bcl-xs, which is a 209 amino acid protein that results from an unspliced transcript (Gonzalez-Garcia et al., 1994). Bcl-xp contains a unique stretch of 21 amino acids at the C terminus and lacks the 19 hydrophobic amino acids that anchor BcI-xL to membranes. A similar product that results from an unspliced transcript has also been described for Bcl-2 (Tsujimoto and Croce, 1986). Other members of the Bcl-2 family that have been shown to inhibit apoptosis include Mcl-1 (Zhou et al., 1997), A1 (Lin et al., 1996), and Bcl-w (Gibson et al., 1996).

The existence of multiple Bcl-2 family members in mammals is likely due to the need to regulate cell death in a tissue-specific manner or to allow cells to rapidly alter their apoptotic threshold in response to changing environmental signals. Consistent with this, it has been found that Bcl- 2, BcI-xL, Bcl-w, Mcl-1, and A1 exhibit differences in tissue expression, developmental expression, and inducibility in response to extrinsic stimuli (Boise et al., 1995; Gonzalez-Garcia et al., 1995; Krajewski et al., 1994, 1995; Lin et al., 1993; Ma et d., 1995; Yang et al., 1996). Germline elimination of bcl-x: results in lethality at embryonic day 13 associated with massive neuronal and hematapoietic cell death (Motoyama et al., 1995).


bcl-x knockout chimeric mice also show survival defects for immature but not for peripheral lymphocytes (Maet al., 1995). In contrast, bcl-2 knockout mice complete embryonic development but demonstrate several abnormal- ities postnatally (Nakayainaet al., 1994; Veis et at., 1993). These abnormali- ties include the death of resting peripheral lymphocytes but not of immature or activated lymphocytes. The different results from the bcl-x and bcl-2 knockout mice are consistent with the developmental expression patterns of each gene in the nervous system and in lymphocytes. Although elimination of bcl-x results in the loss of both Bcl-xL and Bcl-xs, the pheno- type is thought to result predominantly from the absence of BcI-xL because only defects in cell survival are observed and because Bcl-x, expression is extremely low in mouse tissue (Gonzalez-Garcia et al., 1994).

Finally, homologs of the death-inhibiting Bcl-2 family have also been found in proteins encoded by DNA viruses. The best characterized is the weakly homologous adenovirus protein E1B-19k (Han et al., 1996). Other examples include BHRFl from EBV (Henderson et al., 1993), LMWF- HL from the African swine fever virus (Neilan et al., 1993), and KSbcl-2 from Kaposi sarcoma-associated herpesvirus (HHV8) (Cheng et nl., 1997). The purpose of these Bcl-2 homologs may be to counter the propensity of host cells to undergo apoptosis while infection is being established.

B. PROTEINS THAT PROMOTE APOPTOSIS

One of the first pro-apoptotic Bcl-2 family members to be characterized was Bax (Oltvai et al., 1993). Bax is a 21-kDa protein that was cloned based on its ability to coirninunoprecipitate with Bcl-2. Bax, in harboring a BH1, BH2, and BH3 domain, shares extensive amino acid homology with other Bcl-2 family members. Despite this extensive homology, Bax either accelerates apoptosis in response to a death stimulus or is able to kill cells directly when transfected into mammalian cells. When Bcl-2 is coexpressed with Bax, cells become resistant to this pro-apoptotic effect. In the absence of Bcl-2, Bax forins homodimers, but in the presence of Bcl-2, Bax and Bcl- 2 forin heterodimers with each other. Bax also interacts with and counters the protective properties of Bcl-xL. When mutations were introduced into either the BH1 or the BH2 domain of Bcl-2 or BcI-xL, heterodiiner formation was prevented, suggesting that Bcl-2 and BcI-xL both interact with Bax through the BH1 and BH2 regions (Sedlak et al., 1995; Yin et al., 1994). One model, referred to as the rheostat model, to explain the role of heterodimerization proposes that the amount of Bax that is not hetero- diinerized to Bcl-2 (or Bcl-xL) sets the apoptotic threshold of a cell (Yang and Korsmeyer, 1996). The greater the amount of uncoinplexed Bax, the lower the apoptotic threshold.

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Interaction cloning with death antagonists and degenerate polymerase chain reaction cloning to Bcl-2 homology domains resulted in the isolation of Bak, another Bcl-2 family member that promotes apoptosis (Chittenden et al., 1995b; Farrow et al., 1995; Kiefer et al., 1995). Bak is a 26-kDa protein that also contain BH1, BH2, and BH3 domains. In general, it is functionally similar to Bax. Bak interacts with death antagonists such as Bcl-2, Bcl-xl,, and E1B-19k and inhibits their survival properties. In addition, Bak is able to kill cells directly when overexpressed in certain systems. Interestingly, Bak is also able to inhibit cell death under some circum- stances, suggesting that its effect on cell survival is context dependent.

Although Bax and Bak share the BH1, BH2, and BH3 doinains with death antagonists, the BH3 region has been suggested to be critical in conferring pro-apoptotic properties. This is because the BH3 domain of Bak was found to be sufficient to both cause apoptosis and heterodimerize with Bc~-xL (Chittenden et al., 1995a). In addition, when the BH3 domain of Bax was added to Bcl-2 by mutagenesis, the resulting hybrid molecule promoted apoptosis (Hunter and Parslow, 1996).

Consistent with the importance of the BH3 domain in the death-promoting Bcl-2 family members, several pro-apoptotic Bcl-2 family members have been cloned that share no amino acid homology to other Bcl-2 proteins except for the BH3 domain. These include Bik (Boyd et al., 1995), Bid (Wang et al., 1996b), and Hrk (Inohara et al., 1997). Bad is a death agonist that was originally identified to have weakly homologous BH1 and BH2 domains (Yang et aE., 1995), but recent data demonstrate that it is likely also a BH3-only protein (Kelekar et al., 1997; Zha et al., 1997). The pro- apoptotic function of these proteins generally correlates with the ability to heterodimerize with Bcl-2 and/or BcI-xL. Thus, heterodimerization with the death agonists seems to involve their BH3 domains. However, it is unclear how this domain promotes cell death. One idea is that BH3 domains of death agonists can act as competitive diinerization substrates with Baxl Bak and bind to BcI-~/Bc~-xL to displace prebound Bax/Bak. According to the rheostat model, this would result in an overall increase in free Baxl Bak, leading to cell death. In support of this idea, Bad is only able to promote cell death if it can interact with Bcl-xL.

The alternatively spliced form of Bcl-x, Bcl-xs, was originally characterized as being a pro-apoptotic protein (Boise et al., 1993). When cointro- duced with Bcl-2, Bcl-xs was able to inhibit the antiapoptotic effects of Bcl-2 and BcI-xL. Bcl-xs lacks the BH1 and BH2 domains due to alternative splicing; however, it retains the BH3 domain and is the only pro-apoptotic Bcl-2 protein with a BH4 domain. Yeast two-hybrid studies showed that Bcl-xs is able to heterodimerize with Bcl-2 or BcI-xL (Sato et al., 1994;

REGULATION OF APOPTOSIS BY Bcl-2 FAMILY MEMBERS 25s

Sedlak et al., 1995). However, these results may be artifactual as this interaction is not seen in mammalian cells (Minn et al., 1996).

C SUBCELLULAR LOCALIZATIOK OF Bcl-2 FAMILY MEMBERS

Bcl-2 and Bcl-xL, localize to similar subcellular compartments. Electron microscopy has revealed that these proteins can be found in the outer membrane of the mitochondria, the outer nuclear envelope, and the endoplasmic reticulum (Akao et al., 1994; Krajewsh et al., 1993; Lithgow et al., 1994). Furthermore, the staining pattern of Bcl-2 on mitochondrial membranes is patchy, suggesting that it can localize to contact sites, which are regions where the mitochondrial outer and inner membranes meet. Targeting to the outer mitochondrial membrane seems to depend on the carboxy-terminal transmembrane tail. In Bcl-2, this 19 amino acid sequence is necessary and sufficient to direct the protein to outer membranes of isolated mitochondria through an ATP-dependent, temperature-sensitive process (Nguyen et al., 1993). The C-terminal transmembrane sequence of Bcl-2 can be replaced by a similar sequence from the yeast outer mitochondrial membrane protein Mas70p. This chimeric protein retains full antiapoptotic function and suggests that the C terminus of Bcl-2 may simply function to target Bcl-2 to correct intracellular locations. Deletion of the C terminus of Bcl-2 either abrogates or diminishes its antiapoptotic properties, depending on the experimental system (Hockenberry et al., 1993; Nguyen et al., 1994). Eliniination of the C terminus of Bcl-xL also interferes with its antiapoptotic effects (A. J. Minn, B. S. Chang, and C. B. Thompson, unpublished data). These results argue that targeting of Bcl-2 and Bcl-xL to the mitochondria is important for their abilities to regulate programmed cell death.

Other reports suggest that there may not be an absolute requirement for membrane attachment of Bcl-2 and Bcl-xL for antiapoptotic function. Deletion of a C-terminal region from Bcl-2 that includes the membrane anchor domain was reported to have no effect on survival function when assayed using cell death induced by nerve growth factor (NGF) withdrawal or tumor necrosis factor (TNF) treatment (Borner et al., 1994). Similarly, microinjection of Bcl-x, into symphathetic neurons followed by NGF depri- vation resulted in protection against cell death that was comparable to that exhibited by Bcl-x,. (Gonzalez-Garcia et al., 1995). When Bcl-2 was retargeted to the endoplasinic reticular (ER) membrane using the ActA insertion sequence it was still able to prevent some forms of cell death but not others (Zhu et al., 1996). However, it is difficult to interpret experiments that attempt to target Bcl-2 away from the mitochondria because Bcl-2 family members heterodimerize with each other. Therefore, mutants or variants of Bcl-2 that lack the transmembrane tail may still

256 ANDY J. MINN et al.

retain significant mitochondrial association through interaction with other mitochondria-associated Bcl-2 proteins. Alternatively, nonmitochondrially localized Bcl-2 may be able to inhibit cell death by sequestering death- promoting Bcl-2 family members away from the mitochondria. Yet another possibility is the Bcl-2 has antiapoptotic properties that are independent of mitochondrial association. Bcl-2 has been reported to influence calcium flux from the endoplasmic reticulum and the nucleus, as will be discussed later. In general, however, most studies indicate that mitochondrial localization is important for the full antiapopotic effect of the protein.

The death agonists that contain BH1, BH2, and BH3 domains, such as Bax and Bak, also contain a carboxy terminus transmembrane domain that targets these proteins to the outer mitochondrial, outer nuclear, and ER membranes (Krajewski et al., 1996; Yang and Korsmeyer, 1996). However, unlike Bcl-2 and Bc~-xL, which seem to reside predominantly at these intracellular membranes, evidence shows that Bax can be a predominantly cytosolic protein and is capable of being targeted to the intracellular membranes after an apoptotic signal (Hsu et al., 1997). How this process is controlled is unclear. As in the case of Bcl-2 and BcI-xL, the absence of a transmembrane tail from Bax compromises but does not completely eliminate function (Antonsson et al., 1997; Zha et al., 1996b).

Many of the death agonists, particularly the BH3-only versions, do not have a transmembrane anchor and are thought to be cytosolic. These proteins include Bid and Bad. However, the localization of these proteins, and thus their pro-apoptotic function, may be under the regulation of cell survival signals. Bad has been shown to undergo phosphorylation in the presence of growth factors such as IL-3 (Zha et al., 1996~). The phosphory- lated form of Bad is subsequently sequestered in the cytosol through binding to 14-3-3 proteins, molecules that recognize phosphoserine residues. Upon removal of growth factor, Bad is dephosphorylated and changes partners from 14-3-3 to Bcl-xL. Binding to BcI-xL not only redistributes Bad to the mitochondrial membrane, but is also inactivates the cell survival function of Bcl-xL and/or displaces another death agonist such as Bax, leading to the rapid death of the cell. The proto-oncogene Akt, a serine- threonine kinase that phosphorylates PI 3-kinase and is involved in the cell survival signal delivered by growtWsurvival factor receptors (Franke et al., 1997), has been shown to phosphorylate Bad and may be one explanation for the antiapoptotic effects of PI 3-kinase/Akt (Datta et al., 1997; del Peso et al., 1997).

D. WHICH Is THE EFFECTOR AND WHICH Is THE REGULATOR?

Although the extent of heterodimerization has been proposed to be important in setting the cellular apoptotic threshold, the question remains how Bcl-2 family members function to regulate cell survival. Does Bax


have a unique biochemical property that allows it to kill cells, and does Bcl-xL inhibit cell death simply by acting as a regulatory protein that ties up Bax? Another equally possible, although not mutually exclusive, model is that Bcl-x, has a unique biochemical property that directly promotes cell survival, and Bax simply inactivates this property through heterodimerization. Unfortunately, not only are the critical regulatory steps of cell death difficult to study due to the pleiotropic changes that occur in dying cells, but also the precise biochemical function of Bcl-2 family members in controlling this ill-defined step is elusive due to the extensive dimerization that occurs with family members and nonfamily members. Despite this, recent data may be unraveling the biochemical mechanism( s) by which Bcl-2 family members control apoptosis.

N. Mitochondria Can Control Apoptosis

The localization of Bcl-2 to the outer mitochondria membrane suggests that Bcl-2 can regulate cell death through a mitochondria-dependent mechanism. While initial findings that cells without mitochondrial DNA are able to undergo apoptosis and are protected by Bcl-2 led to the interpreta- tion that mitochondria are not essential in the control of PCD (Jacobson et al., 1993), more recent data, however, suggest that this organelle may play a central role in the cell death pathway.

Although many changes are observed in cells undergoing apoptosis, such as DNA fragmentation, generation of reactive oxygen species, and alterations in plasma membrane lipid composition, one of the earliest identifiable events in the loss of mitochondrial transmembrane potential (Kroemer, 1997). This electrochemical grahent across the inner mitochondrial membrane results mainly from the pumping of protons out of the matrix. This transmembrane potential is essential for mitochondrial bioen- ergetics, such as the production of ATP by oxidative phosphorylation. Lipophilic cationic dyes partition into the mitochondrial matrix according to the Nernst equation and, therefore, can be used as a measurement of the transmembrane potential. Cells undergoing apoptosis show very early signs of a reduced mitochondrial transmembrane potential (Zamzami et al., 199513). Cells that have lost their transmembrane potential are irreversibly committed to an apoptotic demise, which has led to the suggestion that apoptosis-induced alterations to the mitochondria involve the “point of no return.”

One way mitochondria lose their transmembrane potential is through a phenomenon known as permeability transition (PT) (Zoratti and Szabo, 1995). This process involves the sudden opening of a large permeability pathway across the inner membrane, leading to the loss of the transmem-

258 ANDY J . M I N N et (I/.

brane potential and swelling of the mitochondria. The permeability pathway results from the opening of the permeability transition pore. The exact composition of this “megachannel” is unclear; however, it is thought to exist at contact sites, where the inner and outer mitochondrial membranes, adjoin, and to consist of both inner membrane channels, such as the adenine nucleotide translocator, and outer membrane channels, such as the voltage-dependent anion channel (VDAC) and the peripheral benzodi- azapine receptor (Kroemer, 1997). Much data suggest that the opening of the PT pore is sensitive to the redox status of certain proteins, the redox status of pyridine nucleotides, the presence of calcium, the matrix pH, and the concentration of adenine nucleotides (Zoratti and Szabo, 1995). Thus, the PT pathway has been suggested to be a wide-spectrum sensor for the cellular environment, perhaps making it a suitable monitor for apoptotic signals. Additionally, PT is a process that exhibits positive feed- back since the disruption of mitochondrial function leads to the generation of many of the signals that contribute to PT in the first place, such as reactive oxygen species and increased cytosolic calcium.

The potential role that PT plays in programmed cell death was demon- strated by the use of a cell-free system that recapitulates the essential features of apoptosis. When isolated mitochondria are induced to undergo PT, added nuclei undergo morphological changes characteris tic of apoptosis (Zamzami et al., 1996). Isolated mitochondria containing Bcl-2 are inhibited from undergoing PT induced by some (but not all) agents, and known PT inhibitors, such as cyclosporin A and bongkrekic acid, can inhibit apoptosis when added to whole cells. One mechanism that explains how PT leads to apoptosis involves the release of a mitochondrial protein called apoptosis initiating factor (AIF) (Susin et al., 1996). AIF is able to activate the downstream caspase, caspase 3 (CPP32), and cause apoptotic changes to isolated nuclei. The inhibition of PT by pharmacological PT inhibitors or by Bcl-2 prevents the release of AIF. In addition, AIF may be another Gactor contributing to the self-amplification of PT, as AIF is also a potent PT inducer. Thus, in response to apoptotic signals, PT may result in the release of AIF and lead to the activation of downstream apoptotic effector functions. Bcl-2 may act directly, or indirectly, to prevent PT. Consistent with a role for Bcl-2 family members in controlling PT, overexpression of Bax also seems to cause PT in vivo (Xiang et al., 1996).

Further support that disruptions in mitochondrial homeostasis are critical to the initiation of apoptosis derive from the demonstration that the release of cytochrome c by mitochondria activates caspase 3 and results in subse- quent apoptotic changes to nuclei (Liu et al., 1996). Cytochrome c normally resides between the outer and the inner mitochondrial membranes to carry electrons between complex I11 and complex IV of the electron transport

KEGULATION OF .4POPTOSIS BY Bcl-2 FAMILY MEMBERS 259

chain. Cytochrome c is imported into the intermembrane space as apocyto- chrome c, but is then subsequently modified to the heme-containing holo- cytochrome c, a form that normally does not traverse back to the cytosol (Dumont et al., 1991). Unlike AIF, cytosolic cytochroine c seems to require additional cofactors, like dATP, in order to activate downstream caspases and cause apoptotic clianges. Bcl-2 can act in situ on the mitochondrial membrane to prevent the release of cytochrome c (Kluck et al., 1997; Yang et al., 1997). Furthermore, the addition of exogenous cytochrome c bypassed the inhibitory effects of Bcl-2. Thus, these data suggest that the release of cytochrome c from the mitochondria is another mechanism to activate the downstream apoptotic machinery, and Bcl-2 inhibits this activation by preventing the release of cytochrome c.

What mechanism is responsible for the release of cytochroine c? In contrast to the release of AIF, which seems to result after the PT-induced loss of mitochondrial transmembrane potential, cytoclirome c can be released from mitochondria that retain a transmembrane potential as mea- sured by the uptake of cationic lipophilic dyes. Cells undergoing apoptosis exhibit swelling of the mitochondria prior to the loss of mitochondria1 transmembrane potential (Vander Heiden et al., 1997). This swelling, as determined by electron microscopy, outer membrane permeability studies, and direct measurement of mitochondrial size, leads to an increase in the matrix volume and a physical disruption of the outer mitochondrial membrane. The release of cytochrome c from the intermembrane space is also observed coincidental with the alterations in initochondrial structure. Mitochondria1 swelling and the release of cytochrome c are followed by the loss of initochondrial transmembrane potential. In cells expressing Bcl- xl. mitochondria do not swell and cytochrorne c is not redistributed to the cytosol. These data suggest that apoptotic stimuli lead to the loss of' mitochondrial volume control which, in turn leads to the rupture of the outer mitochondrial membrane and the release of cytochrome c froin the intermembrane space. Presently, it is unclear how these mitochondrial events leading to cytochrome c release relate to PT and AIF. The ability of pharmacological PT inhibitors to inhibit apoptosis in cell culture argues for the primary importance of at least the PT pore; therefore, it would be interesting to determine if PT inhibitors, in addition to preventing the release of AIF, are able to prevent the release of cytochrome c. One possibility is that the release of cytochrorne c and the release of AIF are on a sequential pathway whereby AIF is released after cytochrome c. Regardless, a consistent observation is that Bcl-2 and Bcl-x,, are able to prevent alterations in mitochondrial function that would otherwise result in the release of mitochondria1 proteins that activate downstream caspases.

260 ANDY J. MINN et nl.

A. IDENTIFICATION OF A MAMMALIAN CED-4 HOMOLOG

Genetic evidence from C. elegans indicates that CED-9 is an upstream inhibitor of a pathway in which CED-4 activates CED-3 to cause PCD. The ability of Bcl-2 (CED-9 homolog) to prevent the release of mitochondria1 proteins that activate caspase 3 (CED-3 homolog) provides one mechanism to explain the genetic evidence from C. elegans. Several studies have provided insight on how CED-4 might act between CED-9 and CED-3 as a positive regulator of PCD (Chinnaiyan et al., 1997a,b; Seshagiri and Miller, 1997; Wu et al., 1997a). CED-3, like its mammalian homolog, is a cysteine protease that is synthesized in a pro form that can be activated proteolytically (Xue et al., 1996). When CED-3 is transfected into mammalian or insect cells, it can promote apoptosis, albeit weakly, and although one group reported that CED-4 is able to cause apoptosis when transfected alone, the consensus is that CED-4 does not cause apoptosis when introduced by itself. However, when CED-4 and CED-3 are coexpressed, a synergistic effect on cell death is observed. These results can be explained by the findings that CED-4 is able to interact with and enhance the proteolytic activation of CED-3, resulting in an increase in CED-3- mediated apoptosis. The ability of CED-4 to enhance CED-3 proteolysis and activation requires a nucleotide-binding niotif in CED-4. Furthermore, CED-4 complex formation with CED-3 involves the CED-3 prodomain. Mutations or deletions of the CED-3 prodomain prevent interaction with CED-4 and inhibit the effects of CED-4 on CED-3.

CED-9 has also been shown to interact with CED-4 (Chinnaiyan et al., 1997b; James et al., 1997; Spector et nl., 1997; Wu et al., 1997a,b). When CED-4 is coexpressed with CED-9 and CED-3, it forms a trimolecular complex with CED-9 and CED-3, but is unabIe to enhance CED-3 proteolytic activation, suggesting that CED-9 binding interferes with the activity of CED-4 and CED-3 (Seshagiri and Miller, 1997; Wu et al., 1997a). Furthermore, when CED-9 is expressed together with CED-4 and CED- 3 in either insect cells or mammalian cells, CED-9 inhibits the apoptosis that results from the synergism between CED-4 and CED-3.

The ability of CED-4 to regulate the proteolytic activation of CED-3 and to synergize with CED-3 to cause PCD indicates that a mainmalian CED-4 homolog might be involved in the cytochroine c-mediated activation of caspase 3. This prediction was confirmed with the biochemical purification of Apaf-1 and the demonstration that it is one of the additional cofactors required by cytochrome c to activate caspase 3 and to cause apoptosis in a cell-free system (Zou et al., 1997). Apaf-1 is a 130-kDa protein that contains a 320 amino acid region that is homologous to CED- 4. Interestingly, the amino-terminal 85 amino acids also show homology


to the prodomain of CED-3. The carboy terminus contains 12 WD repeats, which are thought to mediate protein-protein interactions. When transfected into mammalian cells, Apaf-1 does not cause cell death; however, when extracts are made from these cells, the addition of dATP, another cofactor required for cytochrome c-dependent caspase 3 activation, leads to the rapid activation of caspase 3. Apaf-1 binds directly to cytochrome c and also contains a nucleotide-binding motif, perhaps explaining the requirement for dATP. It has yet to be determined whether Apaf-1 interacts with any of the death-inhibiting Bcl-2 family members.

B. OTHER EFFECTS OF Bcl-2 ON CELL PHYSIOLOGY

One explanation for how Bcl-2 is able to regulate mitochondrial function and the distribution of mitochondrial proteins is through the regulation of membrane permeability. This putative function is also suggested by studies in which Bcl-2 was able to decrease the efflux of calcium from the endoplasmic reticulum after treatment with thapsigargin, a specific inhibitor of the ER calcium pump (Lam et al., 1994). Because Bcl-2 is also localized on the outer nuclear envelope and the nucleus is another site for calcium storage, the effect of Bcl-2 on nuclear calcium flux was also examined. In response to various stimuli, both nuclei in .situ and isolated nuclei were found to exclude calcium in a Bcl-2-dependent manner (Marin et al., 1996). Together, these results suggest that Bcl-2 is able to influence the membrane permeability of the intracellular membranes to which it dis- tributes.

V. Structure/Function Studies of &I-&

To begin to understand how Bcl-2 family members interact and what their potential biochemical functions might be, the three-dimensional structure of BcI-XL was solved by a combination of nuclear magnetic reso- nance (NMR) and X-ray crystallography techniques (Muchmore et d., 1996). BcI-xL is a predominantly (Y helical protein that consists of two central hydrophobic helices surrounded by five amphipathic hFlices (Fig. 3A). The central hydrophobic helices are approximately 30 A long and correspond to parts of the BH1 and BH2 domains. BH3, BH4, and parts of BH1 and BH2 contribute to the five surrounding amphipathic helices. An elongated hydrophobic cleft is formed on the surface of Bcl-xL by the juxtapositioning of BH1, BH2, and BH3 (Fig. 3B). The structure of Bcl- xL also reveals a flexible, unstructured region between BH4 and BH3 that is dispensable for antiapoptotic function and seems to act as a negative regulatory domain (Chang et al., 1997; Uhlmann et al., 1996). Due to the extensive amino acid homology between Bcl-xL and other members of the

KEGULATION OF APOF'TOSIS BY Bcl-2 FAMILY MEMBERS 263

Bcl-2 family, these other family members are also predicted to have a similar tertiary structure.

A THE BH3 DOMAIN OF DEATH AGONISTS INTERACTS WITH A

HYDROPHOBIC CLEFT FORMED BY THE BH1, BH2 A N D BH3 DOMAINS OF BcI-xL

The hydrophobic cleft formed by the BH1, BH2, and BH3 domains of Bcl-x, is the site for interaction with other Bcl-2 family members. Using NMR, it was determined that the BH3 domain of Bak forms an Q helix and binds to the BcI-xL hydrophobic cleft (Sattler et al., 1997). This complex is stabilized through various hydrophobic and electrostatic interactions involving highly conserved residues from the BH1, BH2, and BH3 domains of BcI-xL along with highly conserved amino acids from the BH3 domain of Bak. The nature of this interaction between BcI-xL and various BH3-containing death agonists was confirmed by peptide competition experiments and mutations in either full-length Bcl-x, or full-length BH3- containing death agonists.

Because Bax and Bak are predicted to have the same overall structures as BcI-XL in the absence of heterodimerization, the BH3 domain of the death agonists likely forms intramolecular interactions and participates in the formation of a Bax/Bak hydrophobic cleft. Thus, these intramolecular interactions formed by the BH3 domain of Bax and Bak would need to be disrupted in order for the BH3 domain to form new interactions with the hydrophobic cleft of BcI-xL. Binding of the BH3 domain to Bcl-x, would also require the rotation of the BH3 domain along its helical axis. Details concerning these structural requirements and what might induce these conformational changes are unclear; however, the potential need for Bcl-2 family members to undergo changes is discussed later. Nonetheless, heterodimerization between pro-apoptotic and antiapoptotic Bcl-2 Family members has been suggested to be an important property for Bcl-2 family proteins to regulate cell survival, and these structural studies provide important insight into this property.

B. Bcl-2 FAMILY MEMBERS SHARE STRUCTURAL HOMOLOGY TO

The structure of Bcl-xL bears significant similarity to the pore-forming domain found in various bacterial toxins, particularly diphtheria toxin and

BACTERIAL PORE-FORMING DOMAINS

FIG. 3. The structure of Bcl-xL. (A) Rihbon depiction of the three-dimensional structure of Bcl-xL showing that the protein consists of two central hydrophobic helices (a5 and a6) surrounded by five amphipathic helices. Each alpha helix is labeled, as are the regions corresponding to the BH1, BH2, and BH3 domains. The unstructured loop between amino acids 26 and 76 is not sliown. (B) A space-filled depiction of Bcl-x,~ showing the hydrophobic cleft (light gray) farmed by the BIIl , BII2, and BH3 domains.


the colicins. These pore-forming domains contain two central hydrophobic helices encased by five to eight amphipathic helices (Parker and Pattus, 1993). These domains can be induced to undergo a conformational change from a water-soluble to a membrane-inserted configuration. Bacterial colicins are plasmid-encoded proteins that are secreted by bacteria to kill other bacteria (Cramer et al., 1995). Colicins bind bacterial receptors in the outer membrane to translocate to the inner membrane. Through mechanisms that are not well understood, colicin pore-forming domains are induced to undergo a water-soluble to membrane-bound alteration in structure that results from the insertion of its two central hydrophobic helices. The membrane-bound molecule is then thought to form an ion channel in response to the transmembrane potential of the inner membrane by inserting two other helices to create a four-helix bundle. The creation of an ion channel leads to the depolarization of the bacteria, deenergization, and cell death.

Diphtheria toxin in another molecule that contains a pore-forming domain (London, 1992). This molecule enters cells through receptor-mediated internalization of endosomes. Once in the endosome, the low pH is thought to induce the conformational change that allows the pore-forming domain to create an ion channel. This pore is thought to facilitate the translocation of the diphtheria toxin A fragment into the cytosol where it can ADP-ribosylate protein synthesis elongation factor 2 (EF-2) and inhibit protein synthesis. Unlike the colicins, the formation of a pore by the diphtheria toxin pore-forming domain may not occur through the membrane insertion of four helices from an individual molecule because a peptide that corresponds to the two central hydrophobic helices can mimic the ion channel properties of the entire pore-forming domain (Silverman et al., 1994). This finding, along with studies that analyzed the conductance of the channel in relation to protein concentration, argues that more than one molecule is needed to form an ion channel. However, studies suggesting that the diphtheria toxin ion channel may be monomeric leave this issue unsettled (Huynh et al., 1997).

In summary, structural studies indicate that BcI-xL consists of two central hydrophobic helices that are surrounded by five amphipathic helices. This structure closely resembles the pore-forming domain found in several bacterial toxins and suggests that BcI-xL may also possess the ability to form pores in biological membranes. The ability of Bcl-xL to heterodimerize with BH3-containing death agonists is the result of interactions with a hydrophobic cleft formed by the BH1, BH2, and BH3 domains of BcI-xL. Finally, a large unstructured region is found between BH4 and BH3 domains of Bcl-xL and may serve as a negative regulatory domain.

REGULATION OF APOFTOSIS BY BcI-2 FAMILY MEMBERS 265

C Bc~-xL, Bcl-2, AND Bax CAN FORM ION CHANNELS

Permeability transition, the release of AIF, and the release of cytochrome c are all indications that mitochondria undergo alterations in membrane permeability as a result of apoptotic stimuli. The ability of Bcl-2 to prevent these alterations suggests that it might directly or indirectly function to control membrane permeability. One way that membrane permeability can be controlled is through ion channels. Therefore, the structural similarity between BcI-XL and bacterial pore-forming domains prompted functional studies aimed at determining whether Bcl-xL and other Bcl-2 family members could also form ion channels.

Using planar lipid bilayers and synthetic lipid vesicles, BcI-xL, Bcl-2, and Bax were all found to form ion channels that are voltage and/or pH sensitive (Antonsson et al., 1997; Minn et al., 1997; Schendel et nl., 1997; Schlesinger et nl., 1997). Low pH was shown to promote ion channel formation by these proteins, similar to what is observed with the bacterial pore-forming domains. Low pH is thought to facilitate ion channel formation by inducing the conforinational change that exposes the two central helices to allow for membrane insertion. In addition to insertion, the selectivity of the ion channels formed by Bcl-2, BcI-xL, or Bax is influenced by pH. At more neutral pH values, the ion channels formed by BcI-xL and Bcl-2 are more selective for cations than for anions, whereas Bax is reported to be either mildly cation selective or anion selective. At lower pH values, Bax is reported to have enhanced selectivity, whereas Bcl-2 and Bcl-xL seem to lose the ability to discriminate between cations and anions. These results may be due to the protonation of different amino acid residues that partici- pate in the ion selectivity filter in these molecules. The ion channels formed by the pro-apoptotic and antiapoptotic proteins also show differences with regard to their voltage dependence. The Bcl-2 and B c ~ - x L ion channels behave in an Ohmic fashion, whereby current responds linearly with voltage. In contrast, Bax displays slight rectification in response to positive voltages.

The ion channels formed by Bcl-xL, Bcl-2, and Bax all display multiple conductance states with complex opening kinetics. In general, these proteins form ion channels that can each range in conductance from a few picosieinens to over a nanosiemen. These ion channels can be predominantly open, predominantly closed, or exhibit flickering behavior. Addi- tional studles with BcI-xL indicate that it integrates stably into lipid membranes and allows the passage of large organic cations (A. J. Minn, M. F. Fill, and C. B. Thompson, unpublished data). One explanation for this collective behavior is that these Bcl-2 family members are able to oligomer- ize in the membrane to form larger and smaller ion-conducting units.


However, it cannot be ruled out that individual molecules of the Bcl-2 family are adapting distinct conformations to give rise to the complex properties of the ion channel.

In summary, Bcl-2, BcI-xL, and Bax form ion channels in lipid membranes that have distinct properties, which include ion selectivity, conductance, and voltage dependence, It is presently unclear whether the ability to form ion channels with different characteristics is related to the ability of these proteins to differentially regulate cell survival.

VI. How Do Bcl-2 Family Members Regulate Cell Survival?

The propensity for heterodimerization between pro-apoptotic and antiapoptotic family members has led to the suggestion that heterodimerization is an important mechanism for regulating the cell survival function of Bcl- 2 f'amily members. Mutants of Bcl-2 and Bcl-xL have been described that alter amino acid residues in the BH1 and/or BH2 regions and fail to bind to Bax. When these mutants were assayed for their ability to protect cells from apoptosis, a correlation was found between the inability to heterodimerize with the failure to protect (Sedlak et al., 1995; Yin et al., 1994). Mutants of Bax also have been constructed that fail to hmerize with Bcl-xl, but retain the capacity to antagonize the protective effects of BcI-xL (Simonian et al., 1996). Furthermore, Bax and Bak are cytotoxic to yeast cells, an organisin that contains no known Bcl-2 proteins (Ink et al., 1997; Jurgensmeier et al., 1997; Zha et al., 199613). Cotransformation of Bax or Bak with wild-type Bcl-2 or BcI-xL, but not mutants that fail to bind Bax/Bak, suppresses this yeast toxicity. These data argue that Bax is a direct effector that kills cells and that Bcl-xl/Bcl-2 function to bind and inactivate Bax.

It has been suggested, however, that Bcl-2 can function independently of heterodimerization. Additional mutants of BcI-xL have been described that fail to bind to Bax or Bak but still retain a majority of their protective properties (Cheng et al., 1996). Likewise, KSbcl-2, a viral inhibitor of apoptosis, fails to interact with Bax or Bak (Cheng et al., 1997). Heterodimerization-independent effects were also seen in yeast cells trans- formed with wild-type Bcl-2 (Longo et al., 1997). It was reported that expression of Bcl-2 can protect yeast that normally die when cultured in spent media for several days or when placed under conditions of oxidative stress.

One explanation for these different findings concerning the importance of heterodimerization is provided by structural data on the Bcl-xJBak BH3 complex. Nearly all of the described Bcl-2 and BcI-XL mutants that disrupt heterodimerization were engineered in residues that are not predicted to

REGULATION OF APOPTOSIS BY Bcl-2 FAMILY MEMBEHS 267

be directly involved in interactions with the BH3 domain of death agonists. Thus, unanticipated structural alterations could have been introduced, explaining why some nonbinding mutants are protective while others are not. This concern is supported by findings that show that some of these mutants of Bcl-2 and Bcl-xl, actually do bind to a truncated Bax protein that retains its BH3 domain (Ottilie et al., 1997a). The defect in these mutants may be multifaceted, leading to secondary defects such as the inability to heterodinierize with Bax. With the acquisition of structural information on the heterodiinerization of Bcl-xL with the Bak BH3 domain, new mutants that disrupt critical interacting residues may help to better define the role of heterodherization. In general, however, present data would argue that heterodimerization contributes, at least partly, to the ability of Bcl-2 family members to regulate cell death.

If heterodiinerization is a method to regulate the activity of Bcl-2 family members, what is the biochemical nature of this activity? The finding that Bcl-2 family members can form ion channels suggests that this biochemical property may be important in the ability of Bcl-2 family members to control apoptosis. Bcl-2 and Bcl-x12, which localize to the outer mitochondrial membranes, can prevent swelling and other alterations in mitochondrial properties both in isolated mitochondria, cell-free systems, and in viuo. One possibility is that many of these apoptosis-associated alterations of the mitochondria result in swelling due to perturbations in the mechanism that mitochondria utilize to maintain volume homeostasis. The permeability transition pore, or components thereof, has been proposed to regulate mitochondrial volume. Opening of this pore leads to large amplitude swelling of isolated mitochondria (Zoratti and Szabo, 1995). Because this pore is thought to be composed of both outer and inner initochondrial membrane proteins, one possibility is that Bcl-2 family members act in conjunction with these proteins to prevent opening of the pore. This hypothesis is supported by electron microscopy and cell fractionation studies that suggest that Bcl-2 may be localized preferentially to initochondrial contact sites, areas where the inner and outer mitochondrial membrane meet (de Jong et al., 1994; Hockenberry et al., 1990). Alternatively, Bcl-2 family members may be able to act as stand-alone channels at contact sites and/or the outer mitochondrial membrane to influence mitochondrial homeostasis. Although it is thought that the outer mitochondrial membrane is generally permeable to large molecules, evidence suggests that the permeability of this membrane is regulated by resident channels (Mannella, 1992). The formation of another permeability pathway by Bcl-2 family members on the outer mitochondrial membrane may be important under certain conditions, such as those that occur during apoptosis. Consistent with this idea, evidence suggests that expression of Bcl-x,~ in cells that have received an

268 ANDY J . MINN et 01.

apoptotic stimulus may prevent the accumulation of protons in the mitochondrial intermembrane space (Vander Heiden et al., 1997).

Assuming the ion channel properties of Bcl-2 family members do control apoptosis, there are several possible mechanisms that may account for how anti- and pro-apoptotic family members are able to have opposite effects on cell survival. For example, the Bax ion channel may promote mitochondrial dysfunction, whereas the Bcl-2 ion channel may retard this process. Bax was shown to cause the release of cytochrome c when expressed in yeast (Manon et al., 1997), suggesting that Bax may directly promote mitochondrial changes that, in a mammalian cell, would lead to apoptosis. The role of heterodimerization may be to regulate the formation of ion channels by controlling insertion into membranes. Alternatively, since it is possible that BcI-XL regulates apoptosis independently of heterodimerization, the formation of separate ion channeIs by Bax and Bcl-xL may give rise to different channels that are able to neutralize each other’s effects. Another possibility is that the formation of hybrid or nonfunctional channels through intermembrane interactions between opposing Bcl-2 family members, which are distinct from those that occur in solution, may prevent apoptosis. Consistent with some of these possibilities, Bcl-2 was shown to inhibit Bax ion channel activity (Antonsson et al., 1997).

If Bcl-2 family members are able to form ion channels that regulate mitochondrial homeostasis, they could influence the propensity for cytochrome c and AIF to redistribute to the cytosol during apoptosis. In turn, caspase activation may be either prevented or promoted. Although some of the data discussed so far would support such a model, another model to regulate caspase activity has also been suggested based on the finding that Bcl-xL, as well as CED-9, interacts with CED-4 (Chinnaiyan et al., 199713; Wu et al., 1997b). Both CED-9 and Bcl-xL mutants that were previously found to be defective in inhibiting apoptosis also failed to bind to CED-4. Therefore, because CED-9 can inhibit the ability of CED-4 to promote the activation and processing of CED-3, Bcl-2 and BcI-XL may, in a similar fashion, prevent caspase activation by binding to a mammalian homolog of CED-4, such as Apaf-1, to form an inactive complex. Apaf-1 is, in fact, involved in the cytochrome-c dependent activation of caspase 3, as previously discussed. Heterodimerization between Bcl-2 family members may serve to act as competitive dimerization substrates for Apaf-1 binding. For example, Bax can act to cause cell death by preventing Bcl- 2 and BcI-XL from binding to proteins Iike Apaf-1. This is supported by experiments demonstrating that Bax and a Bak BH3 peptide are able to inhibit Bcl-xL from interacting with CED-4 (Chinnaiyan et al., 1997b; Ottilie et al., 1997b).


This model, whereby Bcl-2 family members regulate cell death by binding to CED-4-like proteins, is not incompatible with a model in which Bcl-2 family inembers regulate mitochondrial function through ion channel formation. Bcl-2 family members may act at multiple steps to regulate apoptosis (Fig. 4). On one level, these proteins may influence the redistribution of cytochrome c and indirectly regulate caspase activation by forming ion channels in mitochondrial membranes. On another level, Bcl-2 family members may be capable of reinforcing their influence on cell fate by interacting with CED-4-like proteins. Because a cell has numerous mitochondria, even if a minority release cytochrome c, Bcl-xL and Bcl-2 can still prevent apoptosis by binding to Apaf-1 and preventing the cytochrome c-dependent activation of caspases. In contrast, Bax can function not only to promote the release of cytochrome c from mitochondria, but also prevent BcI-xL from inhibiting Apaf-1.

VIi. Conclusion

The biological significance of apoptosis is now widely appreciated, although the mechanism that controls apoptosis is still poorly understood. As seems to be true with many fundamental biological processes, disruptions in pathways that control these processes likely contribute to many human diseases, making the elucidation and understanding of these pathways of potential importance. Although apoptosis is a complex process, much data suggest that Bcl-2 family members regulate a focal point’where a motley of upstream apoptotic signals first converge. From this point on, however, the processes that execute the cell death program seem to diverge. This phenomenon seems to result from the ability of Bcl-2 family members to control the activation of caspases. Data suggest that Bcl-2 family members may regulate caspase activation by preventing alterations in mitochondrial homeostasis that would otherwise lead to the release of mitochondrial proteins that are capable of activating downstream caspases. The formation of ion channels by Bcl-2 family members may be one mechanism by which these proteins control mitochondrial processes. A direct link between Bcl- 2 family members and downstream caspases has also been discovered by studies that demonstrate that both the former and the latter proteins are able to interact with CED-4. The control of CED-Pmediated processing of caspases through interactions with Bcl-2 family members may be yet another way to regulate apoptosis. In addition to the multitude of proteins that associated with Bcl-2 family members mentioned in this review, many other interesting proteins can also associate and, in some cases, influence cell survival. Some additions to this list include Raf-1 (Wang et al., 1996a), calcineurin (Shibasaki et al., 1997), p28 Bap31 (Ng et al., 1997), and

FIG. 4. Bcl-2 family members may regulate apoptosis at multiple steps in the cell death pathway. One shared feature that results from multiple apoptotic stimuli is an early alteration in mitochondrial function that manifests as organelle swelling, rupture of the outer mitochondrial membrane, eventual loss of transmembrane potential, and the release of mitochondrial proteins, such as cytochrome c and AIF. Antiapoptotic Bcl-2 family members, such as Bcl- xL, are able to prevent these characteristic mitochondrial changes, whereas proapoptotic Bcl-2 family members, such as Bax, are able to promote these changes. Bcl-x,, and Bax may independently regulate mitochondrial physiology through the formation of ion channels and/or through the regulation of each others function by heterodimerization. The release of mitochondrial proteins, such as cytochrome c, along with other factors, such as dATP, results in the activation of Apaf-1. Apaf-1 binds to downstream caspases and processes them into proteolytically active forms. Bcl-xL may bind to Apaf-1 and prevent this conversion, whereas Bax may complex with BcI-xL to prevent it from binding to Apaf-1.

REC:ULATION OF APOPTOSIS BY Bcl-2 FAMILY MEMBERS 271

cytochroine c (Kliarbanda et al., 1997). Future studies will need to test the importance of the biochemical activities identified for members of the Bcl-2 family, determine the relative importance of the interactions between Bcl-2 family members and the proteins they interact with, and how these protein interactions regulate function.

ACKNOWLEDGMENT The authors thank Aineeta Kelekar tor her tliouglitful discussions and critique of this

manuscript.

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This article was accepted for publication on December 15, 1997.

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