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Youle RJ, Strasser A.. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9: 47-59

February 2008
Nature Reviews Molecular Cell Biology 9(1):47-59

February 2008
9(1):47-59

DOI:10.1038/nrm2308

Source
PubMed

Authors:

Andreas Strasser

The Walter and Eliza Hall Institute of Medical Research

BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have been studied intensively for the past decade owing to their importance in the regulation of apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the point of no return for clonogenic cell survival and thereby affect tumorigenesis and host-pathogen interactions and regulate animal development. Recent structural, phylogenetic and biological analyses, however, suggest the need for some reconsideration of the accepted organizational principles of the family and how the family members interact with one another during programmed cell death. Although these insights into interactions among BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the mechanisms they use to activate caspases remains elusive.

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The BCL-2 (B-cell lymphoma-2) gene was discovered

at the t(14;18) chromosome translocation breakpoint

in B-cell follicular lymphomas, where its transcription

becomes excessively driven by the immunoglobulin heavy

chain gene promoter and enhancer on chromosome 14

(REFS 1–3). One key early discovery that introduced a new

paradigm for carcinogenesis was that overexpression of

BCL-2 does not promote cell proliferation as most pre-

viously discovered oncogenes do; rather, overexpression

of BCL-2 inhibits cell death

. Apoptosis has now been

widely accepted as a prominent tumour-suppression

mechanism. Mutations in certain oncogenes that result

in the activation of cell proliferation, such as deregulated

MYC expression, require a second mutation to inhibit

the apoptosis machinery so that tumour promotion can

proceed efficiently

5,6

. Thus, the combined overexpression

of BCL-2 and MYC synergize potently in the develop-

ment of lymphomas and certain other types of cancer

. It

has also become clear that, beyond roles in cancer, BCL-2

and other members of the family are essential for an array

of apoptosis programmes, including developmentally

programmed cell death, tissue turnover and host defence

against pathogens.

In mammals, there are at least 12 core BCL-2 family

proteins, including BCL-2 itself and proteins that have

either three-dimensional (3D) structural similarity or a

predicted secondary structure that is similar to BCL-2

(FIG. 1). These proteins display a range of bioactivities,

from inhibition to promotion of apoptosis. Numerous

so-called BH3-only proteins share homology with each

other and the remainder of the BCL-2 protein family

only through the short BH3 motif

. Other than BID, the

predicted overall structures of the BH3-only proteins

seem to be unrelated and appear to lack a close evolution-

ary relationship to the core members of the BCL-2 family

But, all BH3-only proteins interact with and regulate

the core BCL-2 family proteins to promote apoptosis.

Several of the members of these two classes have been

knocked out in mice to reveal their physiological roles,

redundancy and interactions in vivo (TABLE 1).

This review covers recent insights into the biochemical,

cellular and physiological roles of the BCL-2 family with-

out reiterating the roles of these proteins in cancer and

drug development, which have recently been expertly

reviewed

5,10

. With the recent advances in understanding

BCL-2 family protein interactions, we focus on how such

interactions lead these proteins to change subcellular local-

ization and conformation to regulate their bioactivities.

The latest progress into the differential regulation of

organ development, maintenance and tissue turnover in

mice by BCL-2 family members are also reviewed.

BCL-2 family proteins in apoptosis

All pathways to apoptosis converge on the activation of

caspases, which are cysteinyl aspartate proteases that

coordinate the efficient dismantling and engulfment

of doomed cells (FIG. 2). Two pathways of cell death can

be distinguished by whether they require BCL-2 family

proteins and by which caspases are crucial for their exe-

cution. The intrinsic pathway — also called the BCL-2-

regulated or mitochondrial pathway (in reference to

the role these organelles play) — is activated by various

*Biochemistry Section,

Surgical Neurology Branch,

National Institute of

Neurological Disorders and

Stroke, The National

Institutes of Health,

Bethesda, Maryland 20892,

USA.

‡

The Walter and Eliza Hall

Institute of Medical Research,

Parkville 3050,

Melbourne, Australia.

e-mails:

youler@ninds.nih.gov;

strasser@wehi.edu.au

doi:10.1038/nrm2308

BH3 motif

The amino-acid sequence

LXXXGD, in which X represents

any amino acid. This motif is

conserved between most core

BCL-2 family members and

among BH3-only proteins.

The BCL-2 protein family: opposing

activities that mediate cell death

Richard J. Youle* and Andreas Strasser

‡

Abstract | BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have

been studied intensively for the past decade owing to their importance in the regulation of

apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the

point of no return for clonogenic cell survival and thereby affect tumorigenesis and host–

pathogen interactions and regulate animal development. Recent structural, phylogenetic

and biological analyses, however, suggest the need for some reconsideration of the accepted

organizational principles of the family and how the family members interact with one

another during programmed cell death. Although these insights into interactions among

BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the

mechanisms they use to activate caspases remains elusive.

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BIM/BOD

BAD

BCL-G

BID (2BID)

PUMA/BBC3

BCL-XL (1R2D)

BCL-W (1MK3)

MCL1

BAX (1F16)

BAK (2IMS)

BIK/BLK/NBK

HRK/DP5

WWE

UBA

HECT

MULE

Boo/Diva/BCL-B/BCL2L10

BCL-RAMBO

BOK/MTD

NOXA

BMF

BCL-2 (1G5M)

BCL

-2 homo

ogue

BH3-onl

Anti-apoptotic

Pro-apoptotic

Nature Reviews | Molecular Cell Biology

BH1

BH2

BH3

BH4

TM region

α-helix

UBA

WWE

HECT

TNF receptor family

Cell-surface receptors in the

tumour necrosis factor (TNF)

family.

developmental cues or cytotoxic insults, such as viral

infection, DNA damage and growth-factor deprivation,

and is strictly controlled by the BCL-2 family of proteins.

This pathway predominantly leads to the activation

of caspase-9 (REF. 11) but, at least in certain cell types,

the intrinsic pathway can proceed in the absence of

caspase-9 or its activator, apoptotic protease-activating

factor-1 (APAF1)

The extrinsic or death-receptor pathway is triggered

by ligation of so-called death receptors (members of

the tumour necrosis factor (TNF) receptor family, such as

Fas or TNF receptor-1 (TNFR1)) that contain an intra-

cellular death domain, which can recruit and activate

caspase-8 through the adaptor protein Fas-associated

death domain (FADD; also known as MORT1) at the cell

surface. This recruitment causes subsequent activation of

Figure 1 | Sequence alignment of core BCL-2 family proteins and BH3-only proteins. Green bars depict α-helical

segments from the determined structures (when labelled by Protein Data Bank (PDB) identifier in parentheses) or from

secondary structure prediction (as predicted using PSIPRED). Red lines label regions of predicted transmembrane (TM)

domains (as predicted using TMHMM). Sequence homologies of the BH1 (brown lines), BH2 (grey lines), BH3 (blue lines) and

BH4 (orange lines) regions are shown. The BH1, BH2 and BH3 domains fold to line a hydrophobic pocket that can bind BH3-

only peptides. The BH3 domain, particularly among the BH3-only proteins, mediates interaction between the BH3-only

proteins and core BCL-2 family proteins and thereby promotes apoptosis. The upper five proteins (BCL-2, BCL-XL, BCL-W,

A1 and MCL1) are generally anti-apoptotic. The three proteins in the shaded area are less well studied and cannot be

categorized at this time. The lower 12 proteins are considered to be pro-apoptotic. MULE contains a ubiquitin-associated

domain (UBA), the Trp-Trp-Glu interaction module (WWE) and a HECT ubiquitin ligase domain. BID has a unique role as both

a BCL-2 homologue and a BH3-only protein and links the intrinsic and extrinsic apoptosis pathways (FIG. 2). BIM (also known

as BOD), BAD and BMF are unstructured proteins.

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Death domain

A protein-interaction module

that consists of six α-helices

and that is involved in

apoptosis and other signalling

pathways.

downstream (effector) caspases, such as caspase-3, -6 or

-7, without any involvement of the BCL-2 family. In some

cells, most notably hepatocytes, the extrinsic pathway

can intersect the intrinsic pathway through caspase-8

cleavage-mediated activation of the pro-apoptotic BH3-

only protein BID

13,14

. The C-terminal truncated form of

BID (tBID) translocates to mitochondria and promotes

further caspase activation (caspase-9 and the effector

caspases caspase-3, -6 and -7) through the intrinsic path-

way. In these situations, loss of BID or overexpression

of BCL-XL inhibits cell death

BCL-2 family proteins have opposing apoptotic activities.

BCL-2 family members have classically been grouped

into three classes. One class inhibits apoptosis (BCL-2,

BCL-XL, BCL-W, MCL1, BCL-B (also known as

BCL-2L10) and A1 (also known as BCL-2A1), whereas

a second class promotes apoptosis (BAX, BAK and

BOK (also known as MTD)). A third divergent class

of BH3-only proteins (BAD, BIK (also known as BLK

or NBK), BID, HRK (also known as death protein-5

(DP5)), BIM (also known as BOD), BMF, NOXA and

PUMA (also known as BBC3)) have a conserved BH3

domain that can bind and regulate the anti-apoptotic

BCL-2 proteins to promote apoptosis (FIG. 1). It appears

that the pro-apoptotic family members BAX and BAK

are crucial for inducing permeabilization of the outer

mitochondrial membrane (OMM) and the subsequent

release of apoptogenic molecules (such as cytochrome c

and DIABLO (also known as SMAC)), which leads to

caspase activation. The anti-apoptotic family members,

such as BCL-2 and BCL-XL, inhibit BAX and BAK.

Recent evidence indicates that BH3-only proteins de-

repress BAX and BAK by direct binding and inhibition

of BCL-2 and other anti-apoptotic family members

By contrast, an opposing model postulates direct activ-

ation of BAX and BAK by some BH3-only proteins

(specifically BIM, tBID and PUMA)

(FIG. 2).

Table 1 | Phenotypes of mice that are deficient in BCL-2 family members

BCL-2 family

member

Defects caused by its deletion* Refs

Pro-survival family members

BCL-2 Abnormal death of renal epithelial progenitors, melanocyte progenitors and mature B

and T lymphocytes. Causes fatal polycystic kidney disease (100% mortality by 6 weeks),

premature greying and lymphopoenia (but all of these effects can be rescued by

concomitant loss of the BH3-only protein BIM).

130

BCL-XL Abnormal death of fetal erythroid progenitors and neuronal cells. Causes death around

embryonic day 14 (100% mortality).

129

BCL-W Abnormal death of developing sperm cells. Causes male sterility. 132

A1A Abnormally accelerated death of granulocytes and mast cells in culture. 133

MCL1 Failure in implantation. Conditional knockout causes premature death of immature and

mature B and T lymphoid cells, as well as haemopoietic stem cells.

128

Pro-apoptotic BAX/BAK family members

BAX Mild lymphoid hyperplasia, male sterility due to sperm-cell differentiation defect. 135

BAK No obvious defects detected so far. 136

Pro-apoptotic BH3-only proteins

BIM Lymphoid and myeloid cell hyperplasia, fatal SLE-like autoimmune disease (on mixed

genetic C57BL/6x129SV background), many cell types are abnormally resistant to

cytokine deprivation, deregulated calcium flux and the chemotherapeutic drug taxol;

mild but significant resistance of many cell types to DNA damage and glucocorticoids.

143

BID BID-deficient mice are resistant to Fas-activation-induced hepatocyte killing and fatal

hepatitis; however, some cell types (such as lymphoid cells) are normally sensitive to

Fas-induced apoptosis.

13, 14

PUMA Many cell types are profoundly resistant to DNA damage; many are also resistant to

cytokine deprivation, glucocorticoids and phorbol ester.

150,151

BAD Mild resistance of some cell types to deprivation of epidermal growth factor or insulin

growth factor.

154

HRK Abnormal, although relatively mild, resistance of certain neuronal populations to

deprivation of nerve growth factor.

155,156

BIK No obvious defects detected so far. 158

NOXA

Relatively mild resistance of fibroblasts to γ-irradiation or etoposide, but profound

resistance of these same cells and keratinocytes in the skin to ultraviolet irradiation.

150

*These are phenotypes found in mice. The roles of these proteins may differ in humans. BAD, BCL-2 antagonist of cell death;

BAK, BCL-2-antagonist/killer-1; BAX, BCL-2-associated X protein; BCL-2, B-cell lymphoma-2; A1A, BCL-2-related protein A1A;

BCL-W, BCL-2-like-2; BCL-XL, a BCL-2-like protein; BID, BH3-interacting domain death agonist; BIK, BCL-2-interacting killer;

BIM, BCL-2-like-11; HRK, harakiri (also known as death protein-5); MCL1, myeloid cell leukaemia sequence-1; PUMA, BCL-2

binding component-3; SLE, systemic lupus erythematosus.

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Growth-factor deprivation,

stress, UV, viruses

Inactive

BH3-only

BCL-2

BAX/BAK

Cytochrome c release,

mitochondrial fragmentation

APAF1 assembly

into apoptosome

FAS

Caspase-8

TNFR1

APAF1

Cytochrome c

BID cleavage

Intrinsic pathway Extrinsic pathway

Active

BH3-only

Caspase-3

Apoptosis

Mitochondrial outer

membrane permeabilization

The process by which the outer

membrane of mitochondria

leaks certain soluble

intermembrane space proteins,

such as cytochrome c, into the

cytoplasm.

Apoptosome

The caspase-9 activation

complex that is composed of

APAF1 heptamers and that is

assembled on binding of

APAF1 monomers to

cytochrome c.

BAX and BAK promote caspase activation by their

effects on mitochondria. Either directly or indirectly,

these two pro-apoptotic BCL-2 family members induce

the release of proteins from the space between the inner

and outer mitochondrial membranes

. This process of

mitochondrial outer membrane permeabilization (MOMP)

results in the release of cytochrome c and other soluble

proteins into the cytosol. Although it is commonly

thought that BAX and BAK form pores in membranes, the

biochemical nature of such pores and how anti-apoptotic

BCL-2 family proteins might regulate them remains a

key and controversial issue in the field of cell death

. At

the same time as cytochrome c release (or immediately

before), BAX and BAK induce mitochondria to fragment

into more numerous and smaller units, which suggests

connections between mitochondrial division processes

and the functions of the BCL-2 family

Once the OMM has been permeabilized, soluble

proteins diffuse from the intermembrane space into the

cytosol, where they promote caspase activation. The best

studied of these proteins is cytochrome c, which binds to

APAF1 and leads to the assembly of a heptameric protein

ring called an apoptosome, which can bind pro-caspase-9

and induce its activation through a conformational

change

20,21

. Cytochrome c–APAF1-dependent activa-

tion of caspase-9 is absolutely required for neuronal and

fibroblast cell-death processes

. However, in addition to

this process, lymphocytes can probably use alternative

APAF1-, caspase-9- and cytochrome c-independent,

but pro-apoptotic BCL-2-family-member-dependent,

pathways for caspase activation and cell killing

12,22

Intriguingly, caspase activation in lymphocytes can be

amplified by APAF1 even when APAF1 has not been

incorporated into the apoptosome

One APAF1-independent pathway of caspase

activation is the relief of caspase inhibition by inhibitor

of apoptosis proteins (IAPs), such as XIAP, which bind

and neutralize certain caspases (such as caspase-9 and

caspase-3). This inhibitory action of IAPs can be antago-

nized by the binding of DIABLO, which is released

from mitochondria after the activation of BAX and/or

BAK. However, DIABLO-deficient mice

, as well as

XIAP-deficient mice

, do not display significant apop-

totic phenotypes, which suggests that novel processes

of caspase activation remain to be discovered. Several

APAF1 related proteins, called NOD-like receptors, regulate

alternative pathways of caspase activation that occur in

non-apoptotic host defence processes that are associated

with innate immunity and serve as examples of pathways

that can also have roles during apoptosis

. One of these

NOD-like receptors, NALP1, can be regulated by BCL-2

and BCL-XL

in manner that is reminiscent of caspase

activation in the worm (BOX 1).

BCL-2 and BCL-XL appear to control cell survival

beyond the APAF1–caspase-9 axis. If caspase activation

is inhibited by loss of APAF1 or caspase-9, or even by the

combined loss of caspase-9 and caspase-2, the rate of acqui-

sition of apoptotic morphology of myeloid progenitors

and mast cells induced by growth-factor withdrawal or

DNA damage can be significantly delayed. However,

although the onset of apoptotic morphology can be

delayed, the cells still lose clonogenic potential and thus

effectively die, unlike cells that overexpress BCL-2 or

BCL-XL

27,28

. Thus, the step of apoptosis regulation that is

controlled by the BCL-2 family appears to be the most gen-

eral final commitment step for the decision between cell

life and death. The disruption of mitochondria by BAX and

BAK may be one cause of eventual clonogenic cell death in

the absence of apoptosome activation. Normally, caspase

activation rapidly and efficiently mediates cell demoli-

tion and removal. When caspases are blocked, certain

features of apoptosis can be lost (or delayed), which causes

the cells to die more slowly by BCL-2-family-mediated

mitochondrial disruption or by novel caspase-activation

pathways that have yet to be characterized.

Figure 2 | Scheme depicting intrinsic and extrinsic pathways of apoptosis. Apoptosis

can be induced by cell surface receptors, such as Fas and tumour necrosis factor

receptor-1 (TNFR1) (extrinsic pathway, right), or by various genotoxic agents, metabolic

insults or transcriptional cues (intrinsic pathway, left). The intrinsic pathway starts with

BH3-only protein induction or post-translational activation, which results in the

inactivation of some BCL-2 family members. This relieves inhibition of BAX and BAK

activation, which in turn promotes apoptosis. Some BH3-only proteins, such as BIM and

PUMA, may also be able to activate BAX and/or BAK (as shown by the dotted line). Once

activated, BAX and BAK promote cytochrome c release and mitochondrial fission, which

leads to the activation of APAF1 into an apoptosome and activates caspase-9 to activate

caspase-3. Caspases in turn cleave a series of substrates, activate DNases and orchestrate

the demolition of the cell. The extrinsic pathway can bypass the mitochondrial step and

activate caspase-8 directly, which leads to caspase-3 activation and cell demolition. The

BCL-2 family regulates the intrinsic pathway and can modulate the extrinsic pathway

when cleavage of BID communicates between the two pathways.

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EGL-1

(BH3-only)

CED-9

(core BCL-2 family)

CED-4

(APAF1-like)

CED-3

(caspase)

Inhibitor of apoptosis

protein

(IAP). One of a family of

proteins that inhibits apoptosis

by binding or degrading

caspases.

NOD-like receptor

A cytosolic receptor that is

homologous to NOD1 and is

involved in innate immunity

pathways.

E3 ligase

One of a family of proteins that

facilitate the transfer of

ubiquitin from a donor protein

to a specific substrate protein

that may signal the target for

proteosomal degradation.

Structure and evolution

The core multi-BH domain BCL-2 family members and,

surprisingly, the BH3-only protein BID (FIG. 1) have con-

served regions of sequence homology and similar pre-

dicted secondary structure. Structures of seven of these

proteins (BCL-XL

, BCL-2 (REF. 30), BCL-W

31,32

, MCL1

(REF. 33), BAX

, BAK

and BID

36,37

) show remarkable

similarity, which is intriguing considering that some are

pro-apoptotic and others are anti-apoptotic. Ks-BCL-2,

a viral homologue of BCL-2 (REF. 38), as well as two viral

proteins without apparent sequence similarity to BCL-2

family proteins, M11L

39,40

and N1L

, display a helical

fold that is similar to that of BCL-XL, and these inhibit

apoptosis, which indicates that several viruses use BCL-2

family members to counteract host defence.

The 3D structures of the seven core BCL-2 family

proteins mentioned above have yet to reveal any distin-

guishing difference between anti-apoptotic members

(such as BCL-XL and MCL1) and pro-apoptotic mem-

bers (such as BAX and BID). All seven proteins are helical

bundles with a hydrophobic helix-turn-helix hairpin that

is flanked on both sides by pairs of amphipathic helices.

Excluding the viral anti-apoptotic BCL-2-like proteins,

BCL-2 homologues appear to have C-terminal membrane-

anchoring domains. In addition, pro-apoptotic BID appears

to be myristoylated to mediate membrane anchorage

In three proteins, BAX, BCL-W and MCL1, the C-terminal

anchor has been included in the structural analysis and

fits into a hydrophobic pocket formed by the BH1, -2

and -3 regions. The same pocket that sequesters the

C-terminal membrane anchor can also bind to peptides

of the BH3-domain sequences of BAK, BAD and BIM

43–45

which suggests that it also functions in dimerization with

BH3-only proteins and/or multi-BH-domain-containing

BCL-2 family members (FIG. 3). An extended BIM BH3

peptide that is 23 amino acids in length binds along the

hydrophobic groove of BCL-XL, although it is inverted in

the C- to N-terminal helical direction relative to the ori-

entation of the BAX and BCL-W C-terminal membrane

anchors

(FIG. 3).

Classically, BH3-only proteins have been defined as

having homology to the core BCL-2 family members in

only the BH3 domain. Recent sequence analyses indicate

that, except for BID, the BH3-only proteins have pre-

dicted secondary structures or determined 3D structures

that are unrelated to the core BCL-2 family members,

and, except for BID, they probably acquired BH3 motifs

by convergent evolution

. One particular example of

a BH3-motif-containing protein that is not otherwise

related to the core BCL-2 family is MULE, an E3 ligase

that reportedly targets MCL1 for ubiquitylation and pro-

teasomal degradation. MULE has a BH3 domain and can

loosely be considered to belong to the class of BH3-only

proteins, which interact with and regulate other mem-

bers of the BCL-2 family

46,47

(FIG. 4). Even the autophagy

regulatory protein beclin-1 reportedly binds to BCL-2

through a BH3 domain, although further biochemical

and genetic experiments are needed to establish a func-

tional connection

. BAD, BMF and BIM are intrinsically

unstructured

and, along with PUMA, these proteins

are not likely to be core BCL-2 family homologues on the

basis of secondary structure predictions (FIG. 1).

So, BH3-only proteins include various proteins that

share a single motif that allows them to bind and regu-

late the core BCL-2 family members. BID, by contrast, is

the one BH3-only protein with a determined structure

that places it squarely in the core BCL-2 family mem-

bers, which perhaps explains why BID shares certain

properties with multidomain BCL-2 family members,

such as the ability to oligomerize

and to permeabilize

membranes

. Thorough phylogenetic analyses of the

BCL-2 family have generated important insights into

the origins of the core BCL-2 family members (BOX 2)

and the BH3-only proteins, and suggest that many of

these proteins might have biological activities beyond

regulation of cell death

9,52

BCL-2 family protein activation

BH3-only proteins are pro-apoptotic and function as

initial sensors of apoptotic signals that emanate from

various cellular processes. BH3-only protein expression

can be induced by transcription factors. For example,

NOXA and PUMA are induced by the tumour sup-

pressor p53 in response to DNA damage

53–55

, and BIM

is induced by the class O forkhead box transcription

factor-3A (FOXO3A) in response to growth-factor

deprivation

and by the transcription factors CEBPα

Box 1 | The mechanism of CED-9, the C. elegans orthologue of BCL-2

Genetic analyses of the apoptosis pathway in Caenorhabditis elegans and recent

biochemical insights are consistent with the model shown in the figure above. EGL‑1, a

BH3‑only protein, is transcriptionally induced by developmental cues for programmed

cell death. EGL‑1 binds to the BCL‑2 homologue CED‑9, thereby freeing CED‑4, an

AAA+ ATPase that is related to apoptotic protease‑activating factor‑1 (APAF1), which is

normally sequestered by CED‑9. The released CED‑4 assembles into a tetrameric

apoptosome and activates the protease activity of the caspase CED‑3. This model differs

from the cytochrome c release model of mammalian cells (FIG. 2). It is not anticipated

that homologous proteins regulate the same process by different mechanisms, so some

underlying common process among BCL‑2 family members of C. elegans and mammals

may await discovery.

Two studies indicate that CED‑9 may do more than prevent CED‑4 activation. In

worms with mild ced‑3 loss‑of‑function mutations in which some excess cells survive,

weak loss‑of‑function ced‑9 alleles actually increase cell survival, which suggests that

CED‑9 also has pro‑apoptotic activity

163

. This might indicate that, depending on its

conformation, CED‑9 can have BCL‑2‑like (that is, anti‑apoptotic) or BAX‑like (that is,

pro‑apoptotic) activity

. In addition, loss of CED‑9 activity inhibits cell death due to

overexpression of drp‑1 (REF. 122), which further suggests that the sole core BCL‑2

family protein in C. elegans can function in both pro‑ and anti‑apoptotic modes.

Certain mammalian BCL‑2 family members have also been reported to be convertible

between anti‑ and pro‑apoptotic forms

164,165

. CED‑9 resides on mitochondria, as many

mammalian BCL‑2 family proteins do, but how this localization relates to its

biochemical action remains unclear. Similar to programmed cell death in mammals and

flies, mitochondria become fragmented during apoptosis in the worm

122

upstream of

caspase activation, showing that there is one common denominator involving

mitochondria in all three systems.

Future studies should explore how BCL‑2 family members function in sponges,

echinoderms and insects. One recent study in D. melanogaster came to the surprising

conclusion that although the two BCL‑2 family members are required for certain stress‑

induced apoptosis pathways, they are not required for developmentally programmed

cell death

166

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ER stress

The accumulation of unfolded

or incompletely glycosylated

proteins in the endoplasmic

reticulum (ER) results in stress

that may lead to apoptosis.

Dynein motor complex

A molecular machine that

transports cargo along

microtubules.

JAK–STAT pathway

The Janus kinase (JAK)–signal

transducer and activator of

transcription (STAT) pathway is

a signalling pathway that is

activated by growth factors and

cytokines.

(CCAAT-enhancer binding protein-α) or CHOP (CEBP

homologous protein) in response to endoplasmic reticu-

lum (ER) stress

. BH3-only proteins can also be activated

post-translationally; for example, BAD is activated by

loss of phosphorylation in response to growth-factor

deprivation

; BID is activated by caspase-8-mediated

proteolysis

59,60

; BIM is activated by release from the

dynein motor complex

or by loss of extracellular signal-

regulated kinase (ERK)-mediated phosphorylation

(which targets it for ubiquitylation and proteasomal

degradation in healthy cells)

62,63

; BMF is activated by

release from actin–myosin motor complexes

; and BIK

is activated by an unknown mechanism in response to

inhibition of protein synthesis

Regulation of the expression levels of anti-apoptotic

BCL-2 family proteins is another way in which cells

can regulate apoptosis. For example, BCL-XL can be

transcriptionally induced by growth factors through the

Janus kinase–signal transducer and activator of trans-

cription (JAK–STAT) pathway to promote cell survival

MCL1 is rapidly degraded by the ubiquitin–proteasome

pathway in response to cytokine deprivation or other

death stimuli (such as ultraviolet (UV) radiation) and

can be upregulated post-transcriptionally to prevent

apoptosis by inhibiting the rate of degradation

46,67

Regulation of the expression levels of the pro-apoptotic

proteins BAX and BAK is less apparent and the pro-

teins appear to be constitutively expressed at more

or less constant levels. BAX and BAK are primarily

post-translationally regulated by other members of the

BCL-2 family.

When BH3-only proteins are induced or activated,

they interact with core BCL-2 family proteins to pro-

mote apoptosis. The binding of BH3-only proteins or

BH3 peptides to specific anti- and pro-apoptotic BCL-2

family members has been determined by using yeast

two-hybrid analysis, plasmon resonance binding assays

and by cell-free mitochondria and liposome permea-

bilization studies

68–72,73

. Together, these assays indicate

that some BH3-only proteins, such as BIM and PUMA,

bind all anti-apoptotic BCL-2 family members, whereas

others, such as BAD and NOXA, bind only certain anti-

apoptotic BCL-2 family members (FIG. 4). In addition to

interaction with anti-apoptotic BCL-2 family members,

several reports show synergy of BID or BIM with BAX

in cell-free membrane permeabilization assays, which

suggests that some BH3-only proteins may directly

bind and activate BAX and BAK

70–72

. However, it is dif-

ficult to detect binding of full-length BID, tBID or BIM

to BAX or BAK

, although a modified BH3 peptide can

bind BAX and/or BAK

. Other models in which BH3-

only proteins directly activate BAX and BAK are called

into question owing to results from Bim/Bid double

knockout mice and their cell lines, which show that

these putative direct activators of BAX and BAK are not

required for many apoptotic pathways

. Thus, known

BH3-only proteins appear to induce apoptosis primarily

by inhibiting anti-apoptotic BCL-2 family members,

thereby liberating BAX and BAK to cause MOMP and

activation of the caspase cascade (FIG. 2). The precise

biochemical mechanisms that lead to the activation

of BAX and BAK remain a mystery and constitute

the ‘holy grail’ of apoptosis research.

Dynamics of subcellular localization

The anti-apoptotic BCL-2 protein is embedded in the ER,

the nuclear envelope and the OMM by a hydrophobic

C-terminal membrane-spanning domain, with most of

its amino acids in the cytosol

75,76

. Although BCL-2 in any

of these subcellular locations can block apoptosis, the

functions of BCL-2 at the ER and the nuclear envelope

are less clear than those on mitochondria and have

recently been reviewed

77,78

In contrast to BCL-2, BAX is mostly cytosolic and

sequesters its hydrophobic C-terminal membrane anchor

in its BH3-binding pocket (FIG. 3), with a minor fraction

lightly bound to the OMM

. BAX appears to exist as a

monomer in the cytosol of cells rather than being bound

to any anti-apoptotic BCL-2 family members

. During

apoptosis induction, BAX translocates specifically to mito-

chondria (see Supplementary information S1 (movie)),

Figure 3 | Space-filling models of the structures of BAX, BCL-W and BCL-XL

bound to a BIM BH3-region peptide. Comparing the structures of full-length BCL-2

family members with those bound to BH3 peptides from other BCL-2 family members

suggests how subcellular localization might be linked to protein–protein interactions

among family members. (The structures in the lower panels are rotated 90 degrees from

those in the top panels.) Full-length BAX (a,b) and BCL-W (c,d) fold with the C-terminal

helix (grey) sequestered in a hydrophobic pocket. This C-terminal domain is

experimentally deleted in BCL-XL (e,f), which binds to an extended BH3 domain peptide

of BIM (yellow) in the homologous pocket that is occupied by the membrane anchor in

BAX, BCL-W and MCL1. The BIM peptide orientates in the pocket in the opposite

direction to the endogenous regions of the C terminus (that is, in the C- to N-terminal

direction). Because the C-terminal helix is involved in membrane binding and is thought

to penetrate deeply into membranes (FIG. 5), this helix would become displaced from the

hydrophobic pocket on mitochondrial translocation (see Supplementary information S1

(movie)). Emptying this pocket of the C-terminal helix would enable it to bind BH3

domains (yellow) from other BCL-2 family members, allowing hetero- or homodimer

formation. Alternatively, binding of BH3-only proteins to BAX or BCL-W in the cytosol

could displace the C-terminal helix from the pocket and trigger mitochondrial

translocation. Non-structured amino acids in BAX (1–12) and BCL-W (1–8 and 170–178)

have been excluded from the models.

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BCL-2

BCL-XL

MCL1

BCL-W

PUMA

tBID

BIM

NOXA

BAD

where it inserts into the OMM as an integral membrane

protein

using its C-terminal membrane anchor

perhaps with organelle targeting specified by defined

regions in the N terminus

. This translocation step of

BAX, although reversible in certain situations, usually

correlates closely with the irreversible commitment

of cells to die and to the cytochrome c release step

discussed below.

BOK also translocates from the cytosol to mito-

chondria during apoptosis

, whereas BAK already

resides on the OMM (see subcellular localization of

BCL-2 family members in Supplementary information

S2 (table)) in healthy cells, where it has been reported

to be bound to MCL1 (REF. 67) and to BCL-XL

Notably, BAK–MCL1 and BAK–BCL-XL interaction

experiments rely on detergent extraction of membrane

proteins that, in some cases, can cause artefactual

interactions among BCL-2 family proteins

. Although

the OMM channel protein VDAC2 (voltage-dependent

anion channel-2) also reportedly binds to BAK

and

is important for BAK import into the OMM

, Vdac1/

Vdac2/Vdac3 triple knockout mice display normal

apoptosis

, indicating little or no role for VDACs in

BCL-2 family protein regulation. The mitochondrial

localization of BAK may be a consequence of MCL1

binding into the BH3 pocket of BAK and displacing

the C-terminal membrane anchor, allowing it to

interact with membranes. Although the BH3 domain

is the obvious candidate domain of MCL1 for this inter-

action, it is not exposed in the soluble MCL1 struc-

ture

. The C-terminal membrane anchor of BCL-XL

has been proposed to mediate binding to BAX by

fitting into its BH3-binding pocket in trans

, which

suggests another way by which BCL-XL and MCL1 can

interact with BAK. On apoptosis induction, MCL1 is

degraded (at least in certain cell types in response to

certain cytotoxic stimuli)

67,90

and/or the MCL1–BAK

and BCL-XL–BAK interactions are disrupted by BH3-

only proteins, such as NOXA, BIM

or BIK

, which

frees BAK to promote apoptosis.

Because the translocation of BAX to the mitochon-

dria correlates with pro-apoptotic activity, it is curi-

ous that BCL-2 is constitutively membrane bound but

BCL-XL

79,91

, BCL-W

and MCL1 (REF. 90) exist partly

in the cytosol and translocate from the cytosol to mito-

chondria during apoptosis. Their binding to BH3-only

proteins appears to be the trigger, perhaps by displacing

the C-terminal membrane anchors of BCL-XL

and

BCL-W

by occupancy of the hydrophobic pockets.

The intracellular translocation probably correlates

with conformational changes and deep insertion of

BCL-XL and BCL-W into the OMM. Whether this

translocation of anti-apoptotic BCL-2 family members

represents a mechanism to inhibit apoptosis, inactiva-

tion by BH3-only proteins or even conversion into

pro-apoptotic effectors remains unclear

92,93

. It has, for

example, been postulated that binding of a BH3-only

protein changes the conformation of the pro-survival

BCL-2-like protein so that it can then initiate formation

of BAX and/or BAK oligomers in the mitochondrial

and other intracellular membranes to cause initiator

caspase activation

Figure 4 | BH3-only protein binding specificity for BCL-2 homologues. BIM and PUMA bind to all five anti-apoptotic

BCL-2 family members tested. By contrast, NOXA only binds to MCL1 and A1, and BAD binds selectively to BCL-W, BCL-2

and BCL-XL. tBID binds avidly to BCL-XL, BCL-W, MCL1 and A1, but only weakly to BCL-2. BH3-only proteins do not

appear to bind strongly to BAX or BAK. These binding specificities recapitulate the ability of these proteins to activate

apoptosis. For example, BIM, BID or PUMA alone can induce apoptosis, whereas a combination of NOXA and BAD is

required. This probably enables the fine specificity of apoptosis regulation in different tissues and during changes in

cellular developmental stages. The BH3 domain is shown in red and the five anti-apoptotic BCL-2 family proteins are

shown in the middle of the figure.

Box 2 | Phylogenomics of BCL-2 family proteins

BCL‑2 gene orthologues have been identified in all metozoan animals examined so far

The earliest metazoan that has been analysed, the sponge

, contains two BCL‑2 related

genes that most closely resemble mammalian BOK

, which has so far received little

attention in mouse and human systems. Interestingly, Monosiga brevicollis, a single cell

choanoflagellate that is closely antecedent to sponges, has no identifiable BCL‑2 family

member in its recently sequenced genome (C. Wang, personal communication), which

suggests that BCL‑2 family genes evolved with multicellular life forms. However, some

recent viral gene products have been found with the signature helical fold of BCL‑2, and

these function in apoptosis regulation

39–41

, which suggests that additional structural

orthologues of BCL‑2 might exist in eukaryotes without discernible primary sequence

homology. The number of apparent BCL‑2 family member genes in different orders

varies widely. The sea urchin (Strongylocentrotus purpuratus) has ten homologous genes,

significantly more than insects (Drosophila melanogaster; two core BCL‑2 family

members) and round worms (Caenorhabditis elegans; one core BCL‑2 family member)

(BOX 1). C. elegans also has one prominent BH3‑only gene, egl‑1, which appears to be

crucial for most (and perhaps all) developmentally programmed somatic cell death

160

and another BH3‑only gene, ced‑13, which might have a role in stress‑induced cell

killing

161

. Zebrafish contain genes for nine versions of core BCL‑2 family members and

homologues of eight of the best‑studied BH3‑only genes

162

. Humans and mice have a

similar set of 12–13 structural homologues of BCL‑2 family proteins, which indicates

that family member organization is stable among mammals.

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N terminus

C terminus

BAX

Soluble, cytosolic

conformation

in healthy cells

Model of membrane-inserted

apoptotic state of BAX

Conformational changes during apoptosis

Both pro- and anti-apoptotic BCL-2 family members

undergo dramatic conformational changes during apop-

tosis to unfold and insert deeply into the lipid bilayer (FIG. 5).

For example, BAX changes conformation to reveal a hidden

epitope in its N terminus

80,95,96

, shows increased proteolytic

sensitivity

and forms oligomeric complexes

97–99

around

the time it translocates to mitochondria.

Two steps in the activation process of BAX can be

discerned: an initial translocation to mitochondria, and

then the N-terminal conformational change that is likely

to be coupled to membrane insertion and oligomeriza-

tion. The translocational step appears to be reversible

under certain conditions

100

. Amino acids 14–23 in the

N terminus of BAX are hidden when the protein is

in its ‘healthy cell’ conformation, but become exposed in

the early stages of apoptosis. The monoclonal antibody

6A7 binds to these amino acids and can be used to iden-

tify the rearrangement of this specific region of BAX

This BAX epitope has been crystallized bound to the

6A7 monoclonal antibody, revealing the large extent of

remodelling of the BAX N terminus that occurs dur-

ing apoptosis

101

. BAK also changes conformation

102

and

oligomerizes

during apoptosis. How many units of

BAX or BAK form these oligomers is still unclear, and it

appears that these oligomers are distributed over a wide

range of molecular weights.

BCL-2 also changes conformation during apoptosis

owing to binding of BH3-only proteins. Although

a model of this membrane-inserted form of BCL-2

(REF. 103) resembles that of BAX

104

, the anti-apoptotic

BCL-2 family proteins do not proceed to form oligomers,

unlike the pro-apoptotic family members. The conforma-

tion of anti-apoptotic BCL-2 family members in solution

(FIG. 3) probably represents the conformation found in

healthy cells, which does not bind to pro-apoptotic BAX.

However, BCL-2 and BCL-XL may bind BAX and BAK

after they insert into membranes, possibly when they

adopt conformations that resemble those induced by

detergents

, and may cap the BAX or BAK oligomers

and inhibit chain elongation

105

. One report concluded

that only the form of BCL-2 found in the early stages of

apoptosis could bind BAX and BAK and further inhibit

BAX or BAK oligomerization to promote cell survival

However, this would yield the counter-intuitive situation

in which BH3-only proteins could inhibit apoptosis by

promoting a change in the conformation of BCL-2 so

that it more actively inhibits BAX and BAK. Other work

found that the anti-apoptotic activity of BCL-W was

inactivated on insertion of BCL-W into the membrane

during apoptosis

Altering mitochondria

BCL-2 family members interact with mitochondria

either constitutively or on induction of apoptosis and,

although they might have activities in other cellular

compartments, it is clear that they regulate apoptosis by

their impact on the OMM.

Pro-apoptotic BCL-2 proteins induce cytochrome c

release. The OMM becomes permeable to soluble inter-

membrane space proteins at around the same time as

BAX is translocated and BAK undergoes conformational

change. Cytochrome c, DIABLO, adenylate kinase, the

Ser protease OMI, apoptosis-inducing factor (AIF),

deafness dystonia protein (DDP), endonuclease G and

a cleaved form of OPA1 (a mitochondrial dynamin-like

GTPase) have all been reported to be released from the

mitochondrial intermembrane space into the cytosol of

cells undergoing apoptosis

17,106–108

. Cytochrome c and

DIABLO release have been consistently shown to be

important for caspase activation.

Figure 5 | Conformational changes in BCL-2 family members during apoptosis. BAX undergoes extensive

conformational changes during the mitochondrial translocation process (see Supplementary information S1 (movie)). The

protein changes from a soluble cytoplasmic protein in healthy cells to one that appears to have at least three helices

inserted into the mitochondrial membrane in apoptotic cells. The C-terminal helix (blue) and the amphipathic helices (red)

in the soluble, cytoplasmic form of BAX (left) are thought to insert deeply into the mitochondrial membrane (right) during

early stages of apoptosis. In addition to changes in membrane topology, the N-terminal amino acids 13–27 (green) change

from a conformation that is buried in the protein folds and inaccessible to the monoclonal antibody 6A7 to a conformation

that is fully accessible to antibody binding during apoptosis. Immunostaining of cells with antibody 6A7 is a good marker

of the conformational change

. However, certain steps of this conformational change, such as the N terminus exposure,

may be reversible when apoptosis triggers are aborted. Other members of the BCL-2 family undergo similar changes in

conformation during apoptosis, including BAK, in which an N-terminal epitope is exposed. Interestingly, even anti-

apoptotic members of the BCL-2 family change conformation, and models of BCL-2 topology in the membrane that are

similar to those of BAX have been proposed

103

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Erythropoiesis

The production of red blood

cells.

The initial finding that the structure of BCL-XL resem-

bles that of the translocation domain of diphtheria toxin

led to the proposal that BCL-XL might form pores in

membranes

. In trying to understand how pore-formation

activity is related to biological activity, a confusing factor

is that both pro- and anti-apoptotic BCL-2 family

members appear to be able to form membrane channels

in vitro

29,109,110

. Incubation of BAX with isolated mitochon-

dria induces cytochrome c release

111

, and incubating BAX

with liposomes allows the release of large (up to 10

Da)

dextran molecules

112

, which is consistent with the lip-

idic pores that were identified in lipid bilayer studies

113

BCL-XL inhibits BAX-induced cytochrome c release

from isolated mitochondria

111

and dextran release from

liposomes

. Furthermore, the BH3-only protein BID can

synergize with BAX to cause cytochrome c release in cell-

free assays, either by activating BAX or by preventing anti-

apoptotic BCL-2 family members from inhibiting BAX

and BAK. Thus, one model for BAX and BAK activation

is that they form large pores in the OMM that allow the

release of proteins into the cytosol, inducing caspase activ-

ation. However, the biochemical nature of this putative

BAX–BAK pore, such as the number of molecules of BAX

that comprise the pore, remains unknown.

Curiously, certain cell types (such as some neuro-

nal populations and cardiomyocytes) can survive the

cytochrome c release step, at least for a limited amount

of time

114,115

. In such cells, caspases might be stringently

regulated by caspase-inhibiting IAPs. In these cases,

apoptosis requires the release of DIABLO from mito-

chondria to relieve the IAP inhibition and thereby allow

caspase activation. This might be a specialized adapta-

tion of normally long-lived post-mitotic cells (which are

essential for animal survival) to supply these cells with

additional protection from cell-death activation and to

prevent their accidental death.

Roles in mitochondrial fragmentation and morphology.

Confocal and electron microscopy analyses of BAX

translocation to mitochondria reveal that the earliest

detectable form of ‘activated’ BAX does not localize to

the entire OMM, but is found concentrated at small

focal regions on the mitochondrial surface

116

. BAK also

moves into these ‘BAX foci’ during apoptosis induction.

This focal cluster form of BAX observed by microscopy

has an altered N-terminal conformation and probably

reflects the in situ state of BAX and BAK as oligomers.

These sites of BAX coalescence often develop into

mitochondrial division sites

117

, linking BAX to the pro-

motion of the mitochondrial fragmentation processes

that occur almost simultaneously with the release of

cytochrome c

118

(FIG. 2).

Inhibition of mitochondrial fission in vitro by

downregulation of the mitochondrial dynamin family

member DRP1 delays cytochrome c release and can

decrease caspase activation, which suggests that the

organelle division machinery somehow participates in

the regulation of apoptosis

119

. Deletion or mutation of

Drp1 in D. melanogaster

120,121

and drp-1 in C. elegans

122

also inhibits apoptosis in vivo (BOX 1). Conversely, inhi-

bition of mitochondrial fusion by loss of a different

mitochondrial dynamin family member, OPA1, induces

spontaneous apoptosis

123

Unexpectedly, healthy cells that lack both BAX and

BAK have altered mitochondrial morphology and slower

mitochondrial fusion rates, which indicates that BAX

and BAK affect mitochondrial morphogenesis machin-

eries even in the absence of apoptotic stimuli

124

. Recent

work showing that OPA1 controls mitochondrial cristae

formation and that tight cristae junctions can inhibit

cytochrome c release during apoptosis suggests how

mediators of mitochondrial fission and fusion might

have a role in cytochrome c release and apoptosis

125

. In

contrast to cytochrome c and OPA1, the release of other

mitochondrial intermembrane space proteins (such

as DIABLO) is not inhibited by DRP1 knockdown

126

which underscores the suggestion that the role of the

mitochondrial fission machinery in apoptosis might be

indirectly linked to the cytochrome c release step. Ectopic

expression of human BCL-XL and C. elegans ced-9 in

mammalian cells has been found to affect mitochondrial

morphogenesis, which shows that it is possible to sepa-

rate the process of organelle fusion regulation by BCL-2

family members from the regulation of apoptosis

127

Physiological roles of BCL-2 proteins

BCL-2 family members have essential roles in the mouse

from early embryogenesis through to adult tissue homeo-

stasis. The nervous system, haematopoietic tissues and

spermatogenesis are particularly dependent on BCL-2

family protein regulation (TABLE 1).

Anti-apoptotic BCL-2 proteins. MCL1 and BCL-XL

are both essential for normal embryogenesis. Mcl1

–/–

embryos die before implantation at the blastocyst stage

128

and Bcl-x

–/–

mice (in which the entire Bcl-x locus (incor-

porating Bcl-xl and Bcl-xs) was knocked out) survive only

until fetal day 13.5, displaying severe defects in erythropoi-

esis and neuronal development

129

. By contrast, although

BCL-2-deficient mice survive to birth, they have defects

in the immune system, hair follicles and renal epithe-

lial cells, and all succumb to polycystic kidney disease

by ~4–8 weeks of age (the age of death is influenced by

genetic background)

130,131

. Bcl-w-knockout male mice are

sterile owing to defective spermatogenesis, but otherwise

both females and males are developmentally normal

132

Analysis of the essential functions of A1 by gene targeting

is complicated by the fact that, in contrast to humans,

mice have four a1 genes. Mice that lack A1A are essen-

tially normal, but their granulocytes and mast cells

undergo apoptosis abnormally rapidly in culture

133,134

Pro-apoptotic BCL-2 proteins. Bax-knockout mice are

viable and females are fertile, but both males and females

have mild overgrowth of neurons and mild lymphoid

hyperplasia, and males have a severe defect in sperm-

cell differentiation, which results in sterility

135

. The

Bak

–/–

phenotype in mice is even less pronounced than

that of the Bax

–/–

mice: their fertility and most of their tis-

sues are normal

136

, although they do exhibit mild platelet

hypertrophy owing to a requirement for BAK to mediate

the turnover of these anuclear cellular fragments

137

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SLE-like autoimmune

disease

A rodent pathology that

resembles human systemic

lupus erythematosus, which is

commonly known as lupus.

Remarkably, however, Bax/Bak double knockout

mice display various severe defects, indicating extensive

redundancy in their activities

136

. A large fraction of Bax/

Bak double knockout mice die during embryogenesis

(particularly on an inbred C57BL/6 background; D.C.S.

Huang, unpublished observations) or perinatally (on a

mixed genetic background). The neonates display vari-

ous developmental deficits — such as webs between their

digits, imperforate vaginas and abnormally increased

numbers of lymphoid and myeloid cells — that are caused

by the persistent survival of cells that normally undergo

developmentally programmed death

136,138

. In vitro experi-

ments with cells from Bax/Bak double knockout mice

have shown that BAX and BAK are required for most

forms of stress-induced apoptosis

139

and that these cells

are even resistant to enforced expression of BH3-only

proteins

140,141

. These results demonstrate that BAX and

BAK are essential for apoptosis induction downstream

of the BH3-only proteins.

Importantly, the heart, liver, lungs and many other

organs develop normally in Bax/Bak double knockout

mice

136

. This might indicate that apoptosis, or at least

BAX/BAK-dependent apoptosis, is not crucial for normal

morphogenesis and normal cell turnover in these organs.

However, it remains possible that the closely related (but

only relatively poorly studied) protein BOK has a crucial

role in these tissues. It will therefore be informative to

generate Bok

–/–

, Bax/Bok and Bak/Bok double knockout

mice and, perhaps most importantly, Bax/Bak/Bok triple

knockout mice.

BH3-only proteins. Gene-targeting experiments have

also helped to define the essential functions of BH3-

only proteins (reviewed in REF. 142). Loss of BIM causes

abnormal accumulation of lymphoid and myeloid cells

and, on a mixed (C57BL/6x129SV) genetic background,

fatal SLE-like autoimmune disease

143

. BIM is crucial for

the deletion of autoreactive T and B cells

144,145

during

their development and for the termination of immune

responses

146

. In vitro experiments demonstrated that BIM

is essential for apoptosis that is induced by growth-factor

deprivation of a surprisingly broad range of cell types,

including lymphocytes

143

, osteoclasts

, mast cells

147

epithelial cells, endothelial cells

and neurons

148,149

BIM-deficient lymphocytes are also less vulnerable to

deregulated calcium flux and have only minor resistance

to γ-irradiation or treatment with glucocorticoids

143

Loss of BID has little effect on developmental apop-

tosis and, although it renders mice resistant to Fas-

induced hepatocyte apoptosis and fatal hepatitis

13,14

lymphoid cells from Bid

–/–

mice are normally sensitive

to Fas ligand

. PUMA, by contrast, is crucial for DNA-

damage-induced apoptosis, which is mediated by p53

(REFS 150–152). Curiously, although γ-radiation and UV

radiation both trigger apoptosis in a p53-dependent

manner, PUMA is essential for γ-radiation-induced

apoptosis and NOXA is essential for UV-radiation-

induced apoptosis within the same cell type (trans-

formed fibroblasts)

153

. This suggests that, depending on

the type of DNA damage and the nature of the molecular

mechanism of damage detection, p53 might be activated

in subtly different ways, thereby determining which of

its two pro-apoptotic BH3-only target genes is activated

preferentially. Alternatively, different forms of DNA

damage might activate distinct pathways that act in par-

allel with p53 signalling to determine whether PUMA,

NOXA or both are induced. PUMA is also crucial for

cell death that is induced by certain p53-independent

apoptotic stimuli, including cytokine deprivation or

treatment with glucocorticoids or phorbol ester

150,151

Mice that lack the BH3-only proteins that can only

bind some pro-survival proteins (BAD, BIK, HRK, BMF

or NOXA) have mild phenotypic abnormalities. This

is consistent with the hypothesis that these BH3-only

proteins are relatively weak killers compared to BIM,

PUMA or BID, which bind to anti-apoptotic BCL-2

family members more promiscuously. Mice that lack

BAD, BIK, HRK, BMF or NOXA are essentially normal

in appearance and are normally fertile. In BAD-deficient

mice, some cell types have subtle resistance to epidermal

growth factor or insulin growth factor deprivation

154

;

however, although these Bad

–/–

mice were reportedly

abnormally prone to lymphoma development, this could

not be reproduced in a subsequent study (P.N. Kelly

and A.S., unpublished observations). Neuronal popu-

lations from Hrk

–/–

mice exhibited some resistance to

nerve-growth-factor deprivation

155,156

, but this was less

pronounced than the protection afforded by loss of

BAX

157

, which indicates that other BH3-only proteins

are probably also involved.

Because many cells express more than one BH3-only

protein and several apoptotic stimuli can activate more

than one BH3-only protein, functional overlap appears

to be likely, and this has indeed been confirmed in early

studies on double knockout mice that lack two BH3-only

proteins. Although Bim

–/–

and Bik

–/–

male mice both have

normal spermatogenesis, severe defects that cause male

sterility became apparent in Bim/Bik double knockout

mice

158

. As in Bax

–/–

males

135

, the failure to produce mature

sperm cells in Bim/Bik double knockout mice was a result

of the abnormal accumulation and persistence of imma-

ture progenitors, which prevent differentiating cells from

getting access to specialized niches on stromal cells.

Analysis of mice that lack both BIM and PUMA has

shown that these two BH3-only proteins are the most

crucial for apoptosis initiation in response to many death

stimuli in a broad range of cell types, particularly those

of haemopoietic origin

159

. For example, although loss of

either BIM or PUMA alone renders lymphoid and mye-

loid cells resistant to cytokine deprivation or treatment

with glucocorticoids, only the combined loss of both pro-

teins provided as much protection as the overexpression

of BCL-2 or the combined loss of BAX and BAK

159

BH3-only versus core BCL-2 proteins. The breeding of

mice that lack both a BH3-only protein and a BCL-2

pro-survival family member has helped to clarify func-

tional relationships between these proteins. Remarkably,

loss of a single allele of Bim prevents the fatal polycystic

kidney disease and lymphopoenia caused by loss of

BCL-2 (Bim

+/–

Bcl-2

–/–

mice), and loss of both Bim

alleles even prevents the abnormal death of melanocyte

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progenitors and premature greying

131

. These results indi-

cate that when BCL-2 is absent in renal epithelial stem

cells, lymphoid cells and melanocyte progenitors, the

physiological levels of BIM are not sufficiently opposed

and cause abnormal apoptosis, presumably by neutralizing

the activity of other pro-survival BCL-2 family members,

such as BCL-XL or MCL1.

Concluding remarks

Our understanding of the regulation of BCL-2 family

members and their roles in tissue dynamics of mam-

mals has greatly expanded in recent years. BH3-only

proteins sense signals to induce apoptosis and relay this

information to core BCL-2 family members to initiate

cell death. BAX and BAK are induced to change confor-

mation and permeabilize the OMM. How BAX and BAK

function in this process remains unclear despite inten-

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BIM, is essential for developmentally programmed

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Shutdown of an acute T cell immune response to viral

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only protein PUMA is essential for p53-mediated

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independent stimuli, such as cytokine deprivation

or treatment with glucocorticoids.

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induced apoptosis of lymphoid cells in vivo. Blood

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153. Naik, E., Michalak, E. M., Villunger, A., Adams, J. M.

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BH3-only protein Noxa. J. Cell Biol. 176, 415–424

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154. Ranger, A. M. et al. Bad-deficient mice develop diffuse

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homologue of mammalian BCL-2, indicating that

the control of apoptosis is evolutionarily conserved.

164. Cheng, E. H. et al. Conversion of Bcl-2 to a Bax-like

death effector by caspases. Science 278, 1966–1968

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166. Sevrioukov, E. A. et al. Drosophila Bcl-2 proteins

participate in stress-induced apoptosis, but are not

required for normal development. Genesis 45,

184–193 (2007).

DATABASES

Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=gene

BCL-2

UniProtKB: http://beta.uniprot.org/uniprot

APAF1 | BAD | BAK | BAX | A1 | BCL-B | BCL-W | BCL-XL | BID |

BIK | BIM | BMF | BOK | caspase-8 | caspase-9 | DIABLO | HRK |

MCL1 | PUMA | XIAP

FURTHER INFORMATION

Richard J. Youle’s homepage:

http://neuroscience.nih.gov/Lab.asp?Org_ID=81

Andreas Strasser’s homepage:

http://www.wehi.edu.au/facweb/indexresearch.php?id=24

SUPPLEMENTARY INFORMATION

See online article: S1 (movie) | S2 (table)

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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