Bcl-2 Family Proteins

Home , FADD, Programmed cell death, TRADD

*John C Reed

The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA

Bcl-2 family proteins serve as critical regulators of evolutionarily conserved step in the programmed cell pathways involved in apoptosis, acting to either inhibit or death machinery ± a hypothesis further supported by promote cell death. Altered expression of these proteins experiments in which human Bcl-2 was found to be occurs commonly in human cancers, contributing to capable of rescuing, at least partially, CED-9 de®cient neoplastic cell expansion by suppressing programmed cell worms (Hengartner and Horvitz, 1994a; Vaux et al., death and extending tumor cell life span. Moreover, 1992). because chemotherapeutic drugs typically exert their These early studies of Bcl-2 and its homologs created cytotoxic actions by inducing apoptosis, the ultimate great interest among apoptosis researchers in under- ecacy of most anticancer drugs can be heavily standing how the Bcl-2 protein performs its cytopro- in¯uenced by the relative levels and state of activation tective functions in cells. Despite over 10 years of of members of the Bcl-2 family. The question of how eort, however, many issues remain unresolved Bcl-2 family proteins function remains debatable, but regarding the biochemical mechanisms of Bcl-2 and new ®ndings are shaping our impressions of these multi- its relatives. functional proteins and revealing the details of how these proteins participate in the regulation of cell life and death. Multiple mechanisms of action

Keywords: Bcl-2; Bax; apoptosis; chemoresistance Bcl-2 appears to defy the dictum: `One gene?One protein?One function.' Rather, Bcl-2 is a multi- functional protein. At least three general functions for Bcl-2 and some of its anti-apoptotic homologs such

Introduction as Bcl-XL have been identi®ed: (a) Dimerization with other Bcl-2 family proteins; (b) binding to non- One of these most prominent families of apoptosis homologous proteins; and (c) formation of ion- regulators is represented by Bcl-2 and its homologs, of channels/pores (reviewed in Green and Reed, 1998; which there are at least 16 in humans (reviewed in Reed, 1997a,b; Schendel et al., 1998; Vaux and Chao and Korsmeyer, 1998; Reed, 1994; Zamzami et Strasser, 1996; Zamzami et al., 1998a). Each of these al., 1998a). The apoptosis-suppressing Bcl-2 gene was functions will be discussed here brie¯y. From the discovered as a proto-oncogene found at the break- outset, any discussion of the functions of Bcl-2 family points of t(14;18) chromosomal translocations in low- proteins must take into consideration their locations in grade B-cell lymphomas. These lymphomas represent cells. Bcl-2 and many of its homologs contain a stretch quintessential examples of human malignancies in of hydrophobic amino-acids at their C-termini (Figure which the neoplastic cell expansion can be attributed 1) that result in post-translational insertion into primarily to failed programmed cell death rather than intracellular membranes, primarily the outer mitochon- rapid cell division. Initial gene transfection studies of drial membrane, nuclear envelope, and endoplasmic Bcl-2 demonstrated that over-production of the protein reticulum (ER) (Krajewski et al., 1993; Lithgow et al., signi®cantly prolongs cell survival in the face of 1994; Yang et al., 1995b; Zha et al., 1996b). classical apoptotic stimuli, including lymphokine deprivation from factor-dependent hematopoietic cells, glucocorticoid treatment of thymocytes and Deadly liaisons: dimerization of Bcl-2 family proteins lymphoid leukemia cells, g-irradiation of thymocytes, and NGF-deprivation from fetal sympathetic neurons A milestone in understanding how Bcl-2 functions (reviewed in Reed, 1994). Conversely, antisense- came when it was discovered that Bcl-2 was capable of mediated suppression of Bcl-2 expression was demon- heterodimerizing with its pro-apoptotic relative Bax strated to induce or accelerate cell death (Reed et al., (Oltvai et al., 1993). The hypothesis thus emerged that 1990). It was thus that Bcl-2 emerged as the ®rst anti-apoptotic proteins such as Bcl-2 and pro-apoptotic example of an intracellular apoptosis-suppresser and proteins such as Bax do battle with each other by the ®rst identi®ed proto-oncogene which contributed to hand-to-hand combat, with the Bcl-2 : Bax ratio neoplasia through eects on cell life span regulation dictating the relative sensitivity or resistance of cells rather than cell division. Subsequent genetic studies of to a wide variety of apoptotic stimuli (Oltvai and C. elegans mutants revealed an anti-apoptotic Bcl-2 Korsmeyer, 1994). However, it was unclear whether homolog, CED-9, implying that Bcl-2 governs an Bcl-2 operated as the eector protein responsible for ensuring survival, with Bax playing the role of trans- dominant inhibitor; or conversely, Bax functioning as an eector of death with Bcl-2 acting as its trans- *Correspondence: JC Reed dominant inhibitor. After multiple attempts to address Bcl-2 family protein JC Reed 3226

Figure 1 The Bcl-2 family. The topologies of several of the Bcl-2 family proteins are depicted, illustrating the BH1, BH2, BH3 and BH4 domains, as well as the transmembrane (TM) domains. Some of these proteins contain BH3-like (BH3-L) domains. The Bcl-XL and Bcl-XS protein arise through alternative mRNA slicing mechanisms from the same gene (Boise et al., 1993). Two xenopus Bcl-2 homologs have also been described (Cruz-Reyes and Tata, 1995). The viral homologs of Bcl-2 are not shown

this issue, probably the most reasonable conclusion is capable of prolonging cell survival even in the absence that both views are correct. of Bax; and conversely, Bax was reported to promote Bcl-2 and Bax appear to have intrinsic independent cell death even without Bcl-2 present. functions as eectors of survival and death, respec- Though Bcl-2 and Bax may be capable of tively. This view of separable, independent functions functioning independently, it is also clear that for Bcl-2 and Bax is supported by both biochemical dimerization among Bcl-2 family members does and genetic arguments. The evidence, for example, provide an important mechanism for controlling their includes investigations of mutants of Bcl-2 and Bax activity. Sequence comparisons of the Bcl-2 family that are incapable of dimerizing but which nevertheless members have revealed up to four conserved domains, display antagonistic activity towards each other and called Bcl-2 Homology (BH) domains: BH1, BH2, BH3 remain capable of repressing or inducing cell death, and BH4 (Figure 1). Mutagenesis studies identi®ed a respectively (Cheng et al., 1996; Simonian et al., conserved domain, BH3, within several of the pro- 1996a,b; 1997; Tao et al., 1997; Wang et al., 1998; apoptotic Bcl-2 family members which was shown to Zha and Reed, 1997). Other support derives from be critical for both dimerization with anti-apoptotic

experiments in which Bcl-2 or Bax were expressed in proteins such as Bcl-2 or Bcl-XL and for induction of cellular contexts where either Bcl-2 homologs are apoptosis (reviewed in Kelekar and Thompson, 1998). absent altogether (yeast) or in which either Bcl-2 or Of note, several pro-apoptotic members of the Bcl-2 Bax were selectively eliminated (knock-out mice) family protein contain the BH3 domain as their only (Hanada et al., 1995; Ink et al., 1997; JuÈ rgensmeier et apparent similarity with other members of the family, al., 1997; Kane et al., 1993; Knudson and Korsmeyer, constituting the so-called `BH3-only' branch of the Bcl- 1997; Longo et al., 1997; Sato et al., 1994; Tao et al., 2 family (Figure 1). Most of these proteins, including 1997). Under these conditions, Bcl-2 was found to be Bik/Nbk, Hrk/DP5, Bim/Bad, Blk, and Bad, operate as Bcl-2 family protein JC Reed 3227 trans-dominant inhibitors of anti-apoptotic proteins which share greater amino acid sequence identity with such as Bcl-2 and Bcl-XL, relying exclusively on Bcl-2. Molecular modeling studies suggest that proteins dimerization for their bioactivities. These proteins do such as Bax probably assume the same general not generally dimerize with pro-apoptotic members of structure as Bcl-XL (Schendel et al., 1998). Unlike the Bcl-2 family and cannot homodimerize. An BH3-only proteins, Bax and Bak can homodimerize analogous situation occurs in the nematode Caenorha- with themselves, in addition to heterodimerizing with biditis elegans, an organism in which the genetics of proteins such as Bcl-2, Bcl-XL, or Mcl-1. Currently, the programmed cell death has been extensively studied. In preponderance of data available from structure- the worm, the Bcl-2 homolog CED-9 is inhibited by function analysis of Bax and its close relatives suggest interactions with a BH3-only protein called EGL-1, at that these proteins possess two potentially independent least in certain types of cells (Conradt and Horvitz, mechanisms for promoting cell death. One mechanism 1998). Deletion of the BH3 domains from the BH3- relies upon the BH3 domain as a tool for suppressing only proteins uniformly abolishes their functions as the functions of speci®c anti-apoptotic Bcl-2 family inducers of cell death and prevents their binding to members through dimerization, similar to BH3-only anti-apoptotic proteins such as Bcl-2, Bcl-XL and CED- proteins. The other is an intrinsic, heterodimerization- 9. Thus, dimerization and function are inextricably independent function, probably related to the ability of linked in these proteins. these proteins to insert into membranes as discussed The structural basis for dimerization among Bcl-2 below (reviewed in Kelekar and Thompson, 1998; Reed family proteins has been elucidated using a combina- et al., 1998; Schendel et al., 1998). Analogous to the tion of X-ray crystallography and NMR methods BH3-only proteins, over-expression of fragments of (Muchmore et al., 1996; Sattler et al., 1997). The Bax or Bak which encompass little more than the BH3 structure of the Bcl-XL protein consists of seven domain have been shown to be sucient for binding to amphipathic a-helices joined by ¯exible loops. The Bcl-2 or Bcl-XL and inducing apoptosis in mammalian BH4 and BH3 domains correspond to the ®rst and cells (Chittenden et al., 1995; Simonen et al., 1997) and second amphipathic a-helices in these proteins, as in cell-free systems (Cosulich et al., 1997). In addition, predicted from the 3-dimensional structure of Bcl-XL substituting the BH3 domain of Bcl-2 for that of Bax (Muchmore et al., 1996). The BH1 domain coincides has been reported to convert Bcl-2 from a protector to with a loop located upstream of the ®fth a-helix in Bcl- a killer protein (Hunter and Parslow, 1996). However,

XL and extends partially into this a-helix, whereas the consistent with the idea of an additional BH3- BH2 domain has been assigned to the latter portion of independent mechanism for cell death induction, some the 6th a-helix and loop which follows thereafter. The BH3 domain mutants of Bax that fail to bind Bcl-2 are BH1, BH2 and BH3 domains in combination form the nevertheless still capable of inducing apoptosis and are borders of a hydrophobic pocket located on the surface still suppressible by Bcl-2 or Bcl-XL, despite their of the Bcl-XL protein. Certain mutations that aect apparent inability to dimerize with these proteins residues lining this pocket, such as Gly145Ala in the (Simonian et al., 1996b; Wang et al., 1998; Zha and BH1 domain of human (hu) Bcl-2 protein and Gly159 Reed, 1997). This apparent redundancy in the

Ala in the BH1 domain of hu-Bcl-XL have been shown mechanisms by which Bax kills cells creates difficulties to abrogate their ability to dimerize with Bax (Sedlak in attempting to dissect which of these routes is et al., 1995; Yin et al., 1994). Thus, this surface pocket quantatively more important in various cellular appears to function analogous to a receptor, binding contexts. epitopes located on partner proteins that dimerize with Hints of another BH3-independent mechanism for

Bcl-2 and Bcl-XL. Through a combination of deletional induction of apoptosis by Bcl-2 family members have and mutagenesis studies (Boyd et al., 1995; Chittenden come from structure-function analysis of the Nip3 and et al., 1995; Inohara et al., 1997; Wang et al., 1996b; Nix (BNIP3L) proteins (Chen et al., 1997; 1998a; Zha et al., 1996a,b), peptide competition assays (Diaz Matsushima et al., 1997; Yasuda et al., 1998). Nip3 et al., 1997), and NMR-based structural analyses was ®rst identi®ed by virtue of its ability to bind the (Sattler et al., 1997), it has been determined that the adenovirus Bcl-2 homolog, E1b-19K (Boyd et al., BH3 domain represents the counter-structure on 1993). Nip3 contains a BH3-like domain and induces dimerizing Bcl-2 family proteins which inserts (similar apoptosis when over-expressed in cells, via a pathway to a peptide ligand) into the surface pocket created by which is at least partially suppressible by Bcl-2 (Chen the combination of BH1, BH2, and BH3. Moreover, it et al., 1997; Yasuda et al., 1998). Recently, a close is the hydrophobic face of the BH3 a-helix that is homolog of Nip3 was identi®ed, which is known as required for binding to this pocket (Kelekar et al., Nix or BNIP3L (Chen et al., 1998a; Matsushima et al., 1997; Sattler et al., 1997; Wang et al., 1998). This 1997). Nip3 and Nix contain a BH3-like domain, as ®nding implies that at least some Bcl-2 family proteins well as a C-terminal membrane-anchoring domain that exist in two conformations: one in which the protein targets them to mitochondria, similar to many other creates a receptor-like pocket and the other in which Bcl-2 family members. The membrane-targeting do- the amphipathic a-helix that comprises BH3 rotates main appears to be essential for the proapoptotic outward to expose its hydrophobic surface so that it function of Nip3. In contrast, removal of the BH3 can bury this side of the a-helix into the receptor-like domain destroys binding to Bcl-2 and reduces but does crevice on dimerization partner proteins (Sattler et al., not abolish Nip3's pro-apoptotic activity. Thus, Nip3 1997). and presumably Nix may exert proapoptotic functions The dimerization properties and functional charac- independently of BH3-mediated dimerization with Bcl- teristics of the BH3-only subgroup of pro-apoptotic 2 or other anti-apoptotic Bcl-2 family proteins. Bcl-2 family proteins can be contrasted with pro- One caveat about BH3-independent function of pro- apoptotic proteins such as Bax, Bak, and Bok/Mtd apoptotic Bcl-2 family proteins, however, is that Bcl-2 family protein JC Reed 3228 dimerization is typically assayed in solution, by in vitro for its pro-apoptotic function (Wang et al., 1996b). If binding assays, co-immunoprecipitation from detergent true, then this observation raises the possibility that solubilized cells, or yeast two-hybrid assays using BH3-only proteins may function either as activators of

proteins lacking the C-terminal membrane-anchoring Bax or as inhibitors of Bcl-2/Bcl-XL. The implications domains. It remains possible that some Bcl-2 family for cancer therapy are obvious: Design BH3-mimicking proteins such as Bax and Nip3 do retain the ability to compounds that bind to and suppress anti-apoptotic

interact with anti-apoptotic proteins such as Bcl-2 and proteins such as Bcl-2 and Bcl-XL (Cosulich et al.,

Bcl-XL in membranes, despite deletions or mutations in 1997) or alternatively that associate with and activate their BH3 regions. Indeed, recent chemical crosslinking pro-apoptotic proteins such as Bax and Bak. studies suggest this may be true at least in the case of Bax (Gross et al., 1998). Sex with multiple partners: interactions of Bcl-2 family proteins with other proteins. Running around with a Bad crowd: dynamic movements

of Bcl-2 family proteins To date, Bcl-2 or Bcl-XL has been reported to interact with (or at least co-immunoprecipitate with) a wide Most Bcl-2 family members contain a C-terminal variety of seemingly unrelated proteins, including: (i) hydrophobic stretch of amino acids that anchors them the caspase-activating CED-4 homolog Apaf-1 (Hu et in membranes (Figure 1), predominantly the outer al., 1998; Pan et al., 1998); (ii) the caspase-binding, mitochondrial membrane, endoplasmic reticulum and Death Eector Domain (DED)-containing proteins nuclear envelope. However, at least two pro-apoptotic Bap31 and MRIT (Flip/Flame/Cash/I-Flice/Casper/ BH3-only proteins, Bad and Bid, lack membrane- Clarp) (Han et al., 1997; Ng et al., 1997); (iii) the anchoring domains (Wang et al., 1996b; Yang et al., protein kinase Raf-1 (Wang et al., 1994; 1996a); (iv) 1995a). The locations of these proteins in cells is the GTPase R-Ras (and possibly H-Ras) (Fernandez- dynamically controlled by their association/dissociation Sarbia and Bischo, 1993); (v) the Ca2+-activated with other Bcl-2 family proteins, resulting in a phosphatase Calcineurin (Shibasaki et al., 1997); (vi) regulated translocation between the cytosol and the the Hsp70/Hsc70 molecular chaperone regulator BAG- surface of membranous organelles where other Bcl-2 1 (Takayama et al., 1995); (vii) the p53 binding protein family proteins reside. The Bad protein, for example, 53BP-2 (Naumovski and Cleary, 1996); (ix) a Prion

dimerizes with Bcl-XL and to some extent with Bcl-2, protein (Kurschner and Morgan, 1995); (x) the thereby promoting apoptosis. Phosphorylation of the multiple membrane spanning protein BI-1 (Xu and Bad protein by a variety of kinases, including Akt/Pkb, Reed, 1998); (xi) the spinal muscular atrophy protein and Raf-1 can prevent Bad from interacting with Bcl- SMN (Iwahashi et al., 1997); and (xii) the Adenine

XL, thus ablating its pro-apoptotic function (Blume- Nucleotide Translocator (ANT) of the mitochondrial Jensen et al., 1998; Datta et al., 1997; del Peso et al., inner membrane (Marzo et al., 1998). In most cases 1997; Wang et al., 1996a; Zha et al., 1996c). This where interrogated, thus far, dimerization of Bcl-2 or

regulation of Bad protein activity by phosphorylation Bcl-XL with pro-apoptotic proteins such as Bax, Bad, appears to represent one of the mechanisms by which or Bik disrupts interactions with other proteins, growth factor receptors convey signals for cell survival. implying either that the same hydrophobic pocket Admittedly, however, this is not the only mechanism implicated in dimerization of Bcl-2 family proteins is used by growth factor and neutrophic factor receptors involved, or that dimerization induces conformational

for maintaining cell survival, since Bad is not expressed changes in Bcl-2/Bcl-XL that trigger release of these in all types of cells (Kitada et al., 1998) and since other heterologous proteins. The BH4 domains of Bcl-2 and

potentially relevant substrates, including caspase-family Bcl-XL appear to be required for several of these cell death proteases, have been identi®ed for some of protein interactions, perhaps explaining why the these kinases such as Akt (Cardone et al., 1998). proteins listed above generally do not bind to Bax or The Bid protein also exhibits dynamic movements Bak (Figure 1) and suggesting that there may be more from cytosol to membranes. The post-translational to the story than the BH3-binding pocket. It is modication that controls Bid translocation however is tempting to speculate that these interactions of Bcl-2

proteolysis rather than phosphorylation. Bid can be or Bcl-XL with other proteins may account for their cleaved by caspase-8, the pinnical protease in the Fas/ cytoprotective eects in the settings of Ca2+ overload TNF-pathway for apoptosis (for reviews see Salvesen (Calcineurin), p53 induction (53BP-2); heat shock and Dixit, 1997a; Wallach et al., 1997; Yuan, 1997). (BAG-1), and exposure to mitochondrial poisons Upon removal of its N-terminus, Bid then translocates (ANT). Though formal proof of this is lacking, Bcl-2 to the surface of mitochondria, apparently dimerizing nevertheless does exhibit some interesting eects that with Bcl-2 family proteins via its BH3 domain (Li et can potentially be dissociated from cell death control al., 1998; Luo et al., 1998). Exactly how cleavage and that may be linked directly to its interactions with activates Bid remains unclear. Possibly it exposes the these unrelated proteins, including preventing Calci- hydrophobic face of the BH3 a-helix so that it is neurin-mediated dephosphorylation and activation of available for dimerization, though alternative mechan- the transcription factor NF-AT (Shibasaki et al., 1997), isms have not been excluded. Moreover, caspase- blocking transcriptional repression by p53 (Sabbatini et independent mechanisms for Bid translocation to al., 1995; Shen and Shenk, 1994) and targeting Raf-1 membranes and dimerization with Bcl-2 family as well as BAG-1/Hsc70 complexes to mitochondria members may also exist. Interestingly, mutagenesis (Takayama et al., 1998; Wang et al., 1996a). Bcl-2, Bcl-

studies of the BH3 domain of Bid suggest that its XL, and Bcl-W also have eects on cell proliferation

binding to Bax rather than Bcl-2 or Bcl-XL is critical that can be dissociated from their anti-apoptotic Bcl-2 family protein JC Reed 3229 functions, a topic which will not be discussed here downstream of Apaf-1 activation in some cellular (Borner, 1996; Huang et al., 1997). Taken together, contexts (reviewed in Hengartner, 1998). therefore, these observations provide further support Additional evidence that Bcl-2 family proteins can for the concept of Bcl-2 family proteins as multi- regulate Apaf-1 come from the discovery of a new functional molecules. family member, Diva (Inohara et al., 1998). The Diva Arguably, of all the proteins with which Bcl-2 and protein contains BH1, BH2, BH4 and a BH3-like

Bcl-XL can potentially interact, those directly linked to domain (Figure 1). Diva is a pro-apoptotic protein that caspase activation seem likely to be among the most reportedly forms complexes with Apaf-1, displacing important. Strong genetic evidence from C. elegans Bcl-XL. The BH3 domain of Diva is not required for implies a critical role for CED-9 interactions with Apaf-1 binding or apoptosis; and Diva does not

CED-4 (Hengartner and Horvitz, 1994b), a caspase- heterodimerize with Bcl-2, Bcl-XL or several cellular activating protein which contains a CARD (Caspase anti-apoptotic Bcl-2 family proteins. However, the Bcl- Recruitment Domain) domain and putative ATP- 2 homolog, Kbcl-2, of the Kaposi's sarcoma-associated binding P-loop (Chinnaiyan et al., 1997; Irmler et al., herpesvirus does bind and inhibits Diva-induced 1997). Moreover, physical interactions between CED-9 apoptosis (Inohara et al., 1998). The implication is and CED-4 have been demonstrated, and correlated that Bcl-XL and Diva compete for binding to Apaf-1, with CED-9's ability to suppress CED-4 mediated with Bcl-XL presumably inhibiting and Diva enabling activation of caspases (Chinnaiyan et al., 1997; Irmler Apaf-1-induced activation of caspases. The adenovirus et al., 1997; Seshagiri and Miller, 1997; Spector et al., Bcl-2 homolog E1b-19K also may bind and interfere 1997; Wu et al., 1997a,b). In addition, dimerization of with Apaf-1 (Han et al., 1998), providing another the BH3-only protein EGL-1 with CED-9 reportedly example in which a Bcl-2 family member apparently displaces CED-4, freeing it to activate caspases (del modulates this caspase-activating protein. Unless Peso et al., 1998). cytochrome c-independent pathways for Apaf-1 activa- The only CED-4 homolog thus far recognized in tion are uncovered, presumably these observations are vertebrate animals is Apaf-1 (Zou et al., 1997), though limited to post-mitochondrial steps in apoptotic path- recent results from Apaf-1 knock-out mice imply the ways where death signals have already triggered release possible existence of others (Cecconi et al., 1998; of this cofactor required for Apaf-1 activation. The Yoshida et al., 1998). Apaf-1 contains a CED-4-like possibility exists however that other CED-4/Apaf-1 domain but unlike CED-4 is not constitutively active. homologs will eventually be discovered that do not Rather, Apaf-1 lies in a dormant state until supplied require cytochrome c for their activation. with dATP (or ATP) and another critical protein, cytochrome c, which is released from mitochondria during apoptosis (reviewed in Green and Reed, 1998; Shot full of holes: Bcl-2 family proteins as pore-forming Reed, 1997a). Once activated by cytochrome c and proteins dATP/ATP, Apaf-1 forms complexes with pro-caspase- 9 (Li et al., 1997b), inducing caspase-9 activation. The ®rst suspicion that some Bcl-2 family proteins Caspase-9 then cleaves and activates downstream might be capable of forming pores or ion-channels in caspases, resulting in apoptosis. membranes came with the determination of the 3

Bcl-XL has been reported to bind Apaf-1, interacting dimensional structure of Bcl-XL (Muchmore et al., with the CED-4-like region in this protein (Hu et al., 1996), revealing striking similarity to the pore forming 1997; 1998; Pan et al., 1998). How this binding of Bcl- domains of some types of bacterial toxins, e.g.,

XL to Apaf-1 aects apoptosis, however, remains diphtheria toxin (DT) and the colicins. The structures obscure. In some experimental systems, Bcl-XL of DT, colicins, and Bcl-XL contain a pair of core apparently causes displacement of pro-caspase-9 from hydrophobic helices that are long enough to penetrate Apaf-1. In others, a terinary complex containing Bcl- the lipid bilayer and which are shielded from the

XL, Apaf-1, and pro-caspase-9 reportedly can be aqueous environment by the other ®ve to seven detected. Formal proof that Bcl-XL is capable of amphipathic helices that orient their hydrophobic inhibiting Apaf-1-mediated processing and activation surfaces towards the central core helices and their of caspase-9 is lacking, though correlative evidence hydrophilic surfaces outward. Studies of the bacterial from cell transfection studies does exist in which Apaf- toxins suggest that under conditions of a voltage 1 mutants were employed that lack a C-terminal gradient and low pH, the central hydrophobic helices negative regulatory domain (Hu et al., 1998). How- insert through the lipid bilayer with the surrounding ever, addition of large amounts of recombinant Bcl-XL amphipathic helices folding up analogous to the or Bcl-2 protein to cytosolic extracts fails to interfere opening of an umbrella (Cramer et al., 1992; Duche with Apaf-1 under conditions in which addition of et al., 1996; Merrill et al., 1990; Parker et al., 1992; cytochrome c can be used to trigger Apaf-1 activation Shin et al., 1993). In the case of colicins, additional a- and processing of pro-caspase-9 (Deveraux et al., helices subsequently also insert into membranes. 1998). Moreover, Bcl-2 either fails to bind Apaf-1 or Molecular modeling studies suggest that anti-apopto- does so very weakly, implying that Apaf-1 binding is tic proteins such as Bcl-2 and some types of pro- not essential for Bcl-2's cytoprotective function. apoptotic proteins such as Bax share the same fold

Interestingly, however, over-expression of Bcl-2 or with Bcl-XL (Schendel et al., 1998).

Bcl-XL has been reported to delay apoptosis induced As predicted by their structures, Bcl-2, Bcl-XL, and by microinjection or electroporation of cytochrome c in Bax have been shown to form ion-channels at least in some types of cells (Zhivotovsky et al., 1998), but not vitro when added to synthetic membranes under others (Garland and Rudin, 1998; Li et al., 1997a), conditions previously shown to be conducive for suggesting that Bcl-2 block apoptosis at the level of or channel formation by DT and the bacterial colicins Bcl-2 family protein JC Reed 3230 (Antonsson et al., 1997; Minn et al., 1997; Schendel et al., extracts, Bax fails to insert into mitochondrial 1997; Schlesinger et al., 1997). The relative conductances, membranes but does so when provided with extracts ion-selectivities and dynamic characteristics of the chan- from apoptotic cells. Deletion of an N-terminal domain

nels formed by Bcl-2, Bcl-XL, and Bax appear to dier. from Bax results in constitutive targeting to mitochon- Though far more work needs to be done, the gen-eral dria, implying that this domain plays a regulatory suggestion is that the cytoprotective proteins Bcl-2 and function (Goping et al., 1998). Forced dimerization of

Bcl-XL tend to form small channels that assume a mostly Bax can also induce its translocation from cytosol to closed conformation, preferring cations, whereas the mitochondrial membranes (Gross et al., 1998). Inter- pro-apoptotic protein Bax tends to form larger channels, estingly, unlike Bid, translocation of Bax is not which assume a mostly open conformation and prefer caspase-dependent, though it is somehow prevented

anions (reviewed in Schendel et al., 1998). The structural by over-expression of Bcl-2 or Bcl-XL. basis for these dierences can presumably be attributed Addition of Bax to isolated mitochondria induces at least in part to dierences in the charges of the amino cytochrome c release through a mechanism that is acid residues predicted to line the aqueous lumen of caspase-independent but suppressible by co-addition of

channels created by the putative pore-forming ®fth and Bcl-XL (JuÈ rgensmeier et al., 1998). The observation sixth a-helices, but many issues remain unresolved, in- that translocation of Bax to mitochondria, membrane cluding the questions of precisely which a-helices insert insertion, and release of cytochrome c can also take into membranes and the stoichiometry of membrane- place in the presence of broad-spectrum irreversible inserted molecules which hypothetically might oligomer- caspase inhibitors such as zVAD-fmk, implies that Bcl- ize to form large channels. Also unclear is how channel 2 family proteins possess an Apaf-1-independent formation relates to the mutual anta-gonism displayed mechanism for controlling cell life and death. Bax by anti-apoptotic proteins such as Bcl-2 and pro- has also been reported to associate with mitochondria apoptotic proteins such as Bax. In this regard, evidence and induce cytochrome c release when expressed in has been obtained that under some in vitro conditions, yeast (Manon et al., 1997) which have no caspases. Bcl-2 can prevent channel formation by Bax (Antonsson When ectopically expressed in both budding and ®ssion et al., 1997), but this observation has not been trans- yeast, Bax and Bak confer a lethal phenotype that ferable to other assays (Schendel et al., 1998). Indeed, the depends on the presence of the putative pore-forming intrinsic channel activities of Bcl-2 family proteins may a5 and a6 helices and which is suppressible by Bcl-2 or

have little relevance to their mechanism of action. Bcl-XL but not by broad-spectrum caspase inhibitors Instead, inactions with other proteins in mem-branes (Ink et al., 1997; JuÈ rgensmeier et al., 1997; Matsuyama may be the key to understanding how they function. et al., 1998). Though no direct evidence thus far been presented that Bcl-2 family proteins can form channels in vivo, evidence is amassing to suggest that proteins such as More than one way to skin a mitochondrion: Bcl-2 fa- Bax do insert into membranes and exist in two mily proteins and the mitochondrial permeability transi- conformational states that may represent active and tion pore latent versions of the protein. For example, in at least some types of cells, the Bax protein resides largely in The issue of how cytochrome c escapes from the cytosol, despite the presence of a typical mitochondria during apoptosis remains controversial membrane-anchoring sequence near its C-terminus and more than one mechanism may be possible, (Hsu et al., 1997; Wolter et al., 1997). Following depending on the particular stimulus and the type of exposure of cells to various apoptotic stimuli, Bax cell involved (Green and Reed, 1998; Reed, 1997a).

translocates from the cytosol to the membranes of The closest structural homolog of Bcl-XL, diphtheria mitochondria, where it inserts as de®ned by resistance toxin (DT) creates pores that allow the transport of a to extraction by alkaline or high salt. This insertion protein (the ADP-ribosylating A-subunit of the toxin) process is also accompanied by the exposure of an N- across lysosomal membranes (Donovan et al., 1982). terminal epitope and increased protease sensitivity of Thus, the notion has arisen that Bax might create a the N-terminus of Bax, consistent with the `umbrella' channel in the outer membrane which allows model of insertion mentioned above (Gross et al., cytochrome c leakage into the cytosol, thus departing 1998). Chemical crosslinking studies further suggest from its normal location in the space between the inner that once integrated into membranes, Bax can form and outer mitochondrial membranes (IMS). dimers and larger oligomers. In addition, one report An alternative mechanism for cytochrome c release has suggested that Bax may also undergo a secondary envisions a marked change in the permeability of the translocation from the outer to the inner membrane inner membrane of mitochondria, resulting in osmotic (Marzo et al., 1998). If true, this could potentially swelling of the organelle and rupture of the outer provide an explanation for some of the controversy membrane. During apoptosis induced by myriad insults over whether Bcl-2 family proteins (including Bcl-2 and and stimuli, the electrochemical gradient (DC) across

Bcl-XL) reside in the inner versus outer membrane the inner mitochondrial membrane becomes dissipated (Hockenbery et al., 1990; Kharbanda et al., 1997); it (reviewed in Petit et al., 1996). This loss of DC has would also create a scenario reminiscent of the been attributed to the opening of a large conductance bacterial colicins which similarly translocate to the inner membrane channel commonly referred to as the inner membranes of bacteria, resulting in membrane mitochondrial PT pore. Though the structure and depolarization and cell death (Cramer et al., 1995). biochemical composition of the PT pore remain poorly It is presently unclear what controls the transloca- de®ned, its constituents are thought to include both tion of Bax from cytosol to mitochondria. When added inner membrane proteins such as the adenine nucleo- to isolated mitochondria in the presence of cytosolic tide translocator (ANT) and outer membranes proteins Bcl-2 family protein JC Reed 3231 such as the Voltage-Dependent Anion Channel which fail to suppress apoptosis in intact cells (Hirsch (VDAC) which operate in concert, presumably at et al., 1997; Marzo et al., 1998; Zamzami et al., 1998b). inner and outer membrane contact sites, to create an Finally, direct interactions of Bax and the ANT have inner membrane channel with *1.5 kDa diameter been reported (Marzo et al., 1998). Moreover, the (reviewed in Bernardi et al., 1994). Interestingly, ANT agonist atracyloside appears to be incapable of immuno-electron microscopic studies suggest that Bcl- inducing ANT channel opening in the absence of Bax 2 is concentrated at these contact sites in the outer in vitro when reconstituted into liposomes and in membrane of mitochondria (de Jong et al., 1994). The isolated mitochondria derived from Bax knock-out ANT is the channel protein within the inner membrane mice (Marzo et al., 1998). thought to be chie¯y responsible for PT (reviewed in Though accumulating evidence suggests a close Bernardi et al., 1994). PT can be measured directly in collaborative interaction between Bcl-2 family proteins isolated mitochondria based on an increase in their and the PT-pore, it remains controversial whether light-absorbance caused by mitochondria swelling in cytochrome c release occurs as a result of mitochon- isotonic medium or indirectly by the failure of such drial PT or is the cause of it. For example, mitochondria to take up cationic ¯uorescent dyes comparisons of the time-courses of cytochrome c which rely upon an intact electrochemical gradient release and mitochondrial DC loss in cells undergoing (DC) for entry into mitochondria. The opening of this apoptosis caused by growth factor deprivation or non-selective channel in the inner membrane allows for cytotoxic anticancer drugs have provided evidence an equilibration of ions within the matrix and IMS of that cytochrome c release can precede DC loss in at mitochondria, thus dissipating the electrochemical least some types of cells (Bossy-Wetzel et al., 1998; gradient and uncoupling the respiratory chain Kluck et al., 1997; Yang et al., 1997). In addition, (reviewed in Petit et al., 1996). Secondary conse- evidence has been obtained suggesting that in some quences of PT pore opening include generation of circumstances where cytochrome c release appears to reactive oxygen species (ROS) and cessation of precede PT pore opening, caspase activation may be mitochondrial ATP production. In addition, the required for the loss of DC but not for the release of volume dysregulation that occurs upon PT pore cytochrome c (Bossy-Wetzel et al., 1998). Thus, PT opening reportedly leads to entry of water into the pore opening may be a relatively late event in such protein-rich matrix of mitochondria, causing the matrix cases, occurring as a secondary consequence of Apaf-1- space to expand. Since the inner membrane with its mediated caspase activation. Moreover, apoptosis and folded cristae possesses a larger surface area than the cytochrome c release can occur in the absence of any outer membrane, this matrix volume expansion can evidence of the mitochondrial swelling predicted to eventually cause the outer membrane to rupture, result from PT-pore opening (reviewed in Green and releasing proteins located within the intermembrane Reed, 1998; Reed, 1997a). Nevertheless, it is also clear space (IMS) into the cytosol. Recent reports suggest that Bcl-2 can protect against oxidants and other that not only cytochrome c but also other proteins of stimuli that directly aect constituents of the PT-pore, potential relevance to apoptosis escape from the IMS thus preventing swelling and rupture of mitochondria after treatment of mitochondria with PT-pore inducers, (reviewed in Kroemer, 1997; Zamzami et al., 1998a). including (a) AIF, a protein that induces apoptosis-like Furthermore, indirect evidence has been obtained destruction of nuclei (Susin et al., 1996); and (b) through use of pharmacological inhibitors of the caspases-2, -3 and -9, which evidently are sequestered mitochondrial cyclophilin that regulates the PT pore, in these organelles under at least some circumstances supporting the idea of a functional interaction between (Mancini et al., 1998; Susin et al., 1998). the PT-pore complex (PTPC) and Bax ± even in settings Bcl-2 family proteins have been reported to associate where Bax induces cytochrome c release from isolated with PT-pore constituents and regulate the function of mitochondria without swelling (JuÈ rgensmeier et al., this entity in both isolated mitochondria and intact 1998). cells. Over-expression of Bcl-2 or Bcl-XL, for example, How does one reconcile these dierences? One reportedly confers protection upon mitochondria, possibility is that both views are correct. Bax, for making it more dicult for many stimuli to induce instance, may be capable of forming large pores in the PT pore opening, including agents that directly act on outer membrane, assisted by interactions with the ANT these organelles such as cyanide, oligomycin, and or other PTPC proteins. Conversely, sustained opening atracyloside (Kluck et al., 1997; Marchetti et al., of the ANT, which induces depolarization of the inner 1996; Shimizu et al., 1996; Susin et al., 1996; Yang et membrane, may be aided by Bax ± either through al., 1997). Conversely, over-expression of the pro- interactions at contact sites where inner and outer apoptotic protein Bax in cells or addition of Bax to membranes abutt or by translocation of Bax to the isolated mitochondria has been demonstrated to induce inner membrane. Bcl-2 and Bcl-XL theoretically could loss of DC through a caspase-independent mechanism protect mitochondrial membranes by (a) preventing (Finucane et al., 1998; Marzo et al., 1998; Xiang et al., Bax-induced pores in the outer membrane (presumably 1996). Bax also reportedly co-puri®es with the multi- by blocking Bax's oligomerization with itself or with protein PT pore complex from mitochondria and PTPC proteins); (b) by interfering with Bax's regulates its activity when reconstituted in synthetic translocation to the inner membrane; or (c) by liposomes in vitro (Hirsch et al., 1997; Marzo et al., translocating themselves to the inner membrane and 1998). Moreover, these preparations of partially somehow preventing osmotic swelling in their putative puri®ed PT pore complexes retain many of the capacity as ion-channel proteins. Because these expected functional characteristics when reconstituted possibilities are not mutually exclusive, the relative in liposomes, including suppression of pore opening by contributions of swelling-dependent and swelling- recombinant Bcl-2 protein but not by mutants of Bcl-2 independent release of cytochrome c and other Bcl-2 family protein JC Reed 3232 proteins from the IMS may be dictated by a variety of This delayed non-apoptotic cell death is preceded by tissue-speci®c dierences in the relative levels of and indeed probably caused by mitochondrial damage various proteins within the outer and inner mem- which results in release of cytochrome c from these branes of mitochondria. Quite likely, the speci®c death organelles. The loss of cytochrome c from mitochon- stimulus applied will also feature heavily in determin- dria not only provides a mechanism for Apaf-1- ing which mechanism is employed. mediated activation of caspases in the cytosol, but it also interrupts electron transport in the inner membrane, since cytochrome c is responsible for Near-death experiences: what de®nes commitment to cell transfer of electrons from complex III to IV in the death? respiratory chain. Interrupting electron chain-transport has several potential consequences, including causing Recent studies have demonstrated that blocking generation of free-radicals, producing loss of the apoptosis induced by growth factor deprivation, electrochemical gradient across the inner membrane, staurosporine, or anticancer drugs is not necessarily and causing ATP depletion (reviewed in Green and synonymous with blocking cell death commitment. Reed, 1998; Reed, 1997a). Thus, the escape of cyto- Caspase-family cell death proteases represent the chrome c from mitochondria can induce cell death by ultimate eectors of apoptosis. Despite suppressing two independent routes ± apoptosis (Apaf-1/caspases) these proteases with irreversible caspase inhibitors such and necrosis (electron chain-transport) (Figure 2). as zVAD-fmk or similar compounds, and despite Interestingly, though caspase inhibitors often fail to preventing all manifestation of apoptosis, cells which prevent mitochondrial damage and commitment to cell have been deprived of growth factors or damaged with death induced by growth factor deprivation and chemotherapeutic drugs nevertheless may die through anticancer drugs, clonigenic survival studies indicate

what appears to be delayed necrosis (Amarante- that Bcl-2 and Bcl-XL can prevent or reduce cell death Mendes et al., 1998; Bossy-Wetzel et al., 1998; Brunet under the same circumstances (reviewed in Green and et al., 1998; Hirsch et al., 1997; McCarthy and Dixit, Reed, 1998). Thus, at least where examined so far, Bcl- 1998; Ohta et al., 1997; Vander Heiden et al., 1997). 2 family proteins appear to govern a cell death

Figure 2 Mechanisms of Bcl-2 Action. Bcl-2 may be able to interfere at two points in mitochondria-dependent cell death pathway: (1) preventing cytochrome c release from mitochondria and (2) interfering with Apaf-1. Loss of cytochrome c from mitochondria can result in apoptosis via caspase activation and necrosis due to cessation of electron chain-transport. A feedback loop entails caspase-mediated induction of mitochondrial PT pore opening and recruitment of additional mitochrondria into the process Bcl-2 family protein JC Reed 3233 commitment step upstream of caspase activation caspases via interactions with speci®c adaptor proteins (Figure 2). This does not however exclude the (Fadd; Tradd) that bind the death domains of these possibility that Bcl-2 also exerts a protective eect receptors (Wallach et al., 1997; Yuan, 1997). Activa- downstream of cytochrome c release, as suggested by tion of the caspases which interact with TNF-family some recent reports (RosseÂ et al., 1998; Zhivotovsky et receptor complexes (e.g., caspases-8 and 10) can trigger al., 1998). It does, however, provide further evidence a cascade of proteolysis, involving processing and that Bcl-2 has caspase- and Apaf-1-independent activation of the zymogen forms of other downstream functions, thus deviating from the CED-9/CED-4/ caspases (e.g., caspases-3, 6 and 7) which serve as the CED-3 model derived from genetic and biochemical ®nal eectors of apoptosis (reviewed in Salvesen and studies of programmed cell death in C. elegans. Dixit, 1997b). In many types of cells, this `death Caspases, however, can operate both upstream and receptor' pathway for apoptosis runs via a Bcl-2- downstream of mitochondria, further complicating the independent pathway, circumventing the participation issue of de®ning the point or points at which Bcl-2 of mitochondria or other organelles where Bcl-2 and blocks the cell death pathways. Several studies have many of its homologs reside as integral membrane provided evidence that active caspases can induce proteins. mitochondrial PT pore opening, at least as de®ned by However, in some cells, Bcl-2 is capable of loss of DC (Hirsch et al., 1997; Susin et al., 1997). The suppressing Fas- or TNF-induced apoptosis. The basis mechanisms responsible may entail proteolytic activa- for this dierence in Bcl-2 sensitivity appears to reside tion of pro-apoptotic proteins such as Bid (Li et al., in whether caspase-8 does or does not require a 1998; Luo et al., 1998) or proteolytic removal of the N- mitochondria-dependent ampli®cation step to achieve terminal BH4 domains from Bcl-2 and Bcl-XL, sucient activation of downstream eector caspases converting them to killer proteins (Cheng et al., for apoptosis. For example, addition of recombinant 1997). These observations imply the existence of a caspase-8 at relatively high concentrations to cytosolic feed-forward mechanism by which cytochrome c extracts results in activation of downstream caspases as release from a few mitochondria can induce caspase well as apoptosis-like destruction of exogenously added activation, thereby recruiting more mitochondria into nuclei. In contrast, if lower concentrations of active the process (Figure 2). caspase-8 are added, then downstream caspase activa- Can cells be rescued once cytochrome c release has tion and nuclear destruction are minimal unless been triggered? In most instances tested thus far, mitochondria are added to the cytosolic extracts inhibition of caspases has proven insucient to rescue (Kuwana et al., 1998). The in vivo correlate of these such cells. However, at least one report suggests that experiments involving addition of caspase-8 to cell some cells can tolerate mitochondrial depletion of extracts is found in comparisons of cell lines where Bcl- cytochrome c, provided caspases are inhibited (Chen et 2 is either eective or ineective as a suppresser of Fas- al., 1998b). This observation implies that release of induced apoptosis (Scadi et al., 1998). In Bcl-2- cytochrome c does not irreversibly commit all cells to suppressible cell lines, only small amounts of caspase-8 death, implying that whatever the mechanism by which activation are induced by Fas. In contrast, in Bcl-2- cytochrome c escaped from mitochondria, it did not independent cell lines, abundant amounts of caspase-8 involve irreparable damage to these organelles. In this processing and activation are observed. The molecular regard, it warrants noting that the mitochondria of basis for the dierences in the eciencies of Fas- some types of cells are relatively quiescent, with most induced caspase-8 activation remain unde®ned. ATP production coming from anerobic glycolysis. Comparisons of the eects of various Bcl-2 family Thus, these cells may tolerate mitochondrial depletion proteins on TNF- or Fas-induced apoptosis also of cytochrome c, at least on a temporary basis, provide an illustration of the molecular diversity of provided caspases are kept under control ± either these apoptosis-suppressers. For example, the adeno- arti®cially by pharmacological caspase inhibitors or virus Bcl-2 homolog E1b-19K, which is anchored in the naturally by IAP-family caspase-inhibitory proteins or nuclear envelope via associations with nuclear lamins, phosphorylation-mediated inactivation of caspases that has been reported to bind Fadd (MORT-1), the operate within the cytochrome c pathway (Cardone et adaptor protein required for caspase recruitment to al., 1998; Deveraux et al., 1997). Fas- and TNFR1 (Han et al., 1998). E1b-19K- mediated sequestration of Fadd therefore presumably prevents it from interacting with Fas and TNFR1 Bcl-2-independent pathways for apoptosis complexes at the plasma membrane. Bcl-2 evidently does not have this capability. Nevertheless, like E1b- While Bcl-2 clearly can govern a cell death commit- 19K, the Bcl-2 protein has been reported to have ment step upstream of caspase activation, it has been mitochondria-independent functions, including a reten- shown that Bcl-2-independent pathways for caspase tion of at least a subset of its apoptosis suppressing activation and apoptosis induction also exist (reviewed activity even when target to the ER, and interactions in Vaux and Strasser, 1996). For example, in some (but with a caspase-binding ER protein Bap-31 (Ng et al., not all) types of cells, apoptosis triggered by Fas 1997; Zhu et al., 1996). (CD95) and certain members of the Tumor Necrosis Factor (TNF) family of `death receptors' is not blocked by over-expression of Bcl-2 or other anti-apoptotic Conclusions members of the Bcl-2 family. Fas, and similar TNF- family receptors that contain `death domains' within Bcl-2 family proteins can potentially modulate cell their cytosolic tails directly induce caspase activation death pathways through multiple mechanisms, includ- through ligand-induced recruitment of cytosolic pro- ing caspase-independent eects on mitochondria and Bcl-2 family protein JC Reed 3234 regulation of Apaf-1/CED-4-family proteins. The gene expression, protein turnover, alterations of quantitatively most important of these various protein conformations, protein phosphorylation, pro- mechanisms for controlling cell death most likely teolytic processing, and protein translocation. Contin- varies, depending on the particular type of cell and ued attempts to understand the molecular basis of Bcl- the cell death stimulus involved. The diversity of anti- 2 family protein function and the details of their

death mechanisms attributed to Bcl-2, Bcl-XL,and regulation will reveal strategies for exploiting tissue- some of their homologs suggests that evolutionary speci®c and context-dependent dierences, thus leading pressure has built several potential functions into these to new therapeutic opportunities for the treatment of proteins, permitting coordinated regulation of cell life cancer and other diseases. and death decisions. Diverse mechanisms for regulating the levels and functions of Bcl-2 family proteins probably also exist for the purpose of achieving precise control over cell life span regulation in various Acknowledgements cellular contexts. Those revealed thus far include I thank T Brown and E Smith for manuscript and ®gure transcriptional and post-transcriptional control of preparation.

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