PHYSIOLOGICAL REVIEWS
Vol. 80, No. 2, April 2000
Printed in U.S.A.
Tissue-Specific Bcl-2 Protein Partners in Apoptosis:
An Ovarian Paradigm
SHEAU YU HSU AND AARON J. W. HSUEH
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of
Medicine, Stanford, California
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I. Introduction
II. Ced-9/Bcl-2 Proteins Are Present in the Decision Step of Apoptosis: Lessons Learned
From the Nematode
III. Apoptosis Regulation by Three Subgroups of Mammalian Bcl-2 Proteins
IV. Ovarian Follicle Atresia as a Model of Apoptosis: Construction of an Ovary-Specific Protein-Protein
Interaction Map in Apoptosis Regulation
V. Proapoptotic BAD and BOD Are Ligands for Antiapoptotic Bcl-2 Proteins and Capable
of Transmitting Upstream Signals
VI. Proapoptotic Bok and Bax With a Channel-Forming Domain Regulate Apoptosis by Releasing
Apaf-1 From Suppression by Antiapoptotic Proteins and by Promoting Cytochrome c Release
VII. Cell Death Execution by Caspases: Converging Pathways for Extracellular and Intracellular Signals
VIII. Conclusion
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Hsu, Sheau Yu, and Aaron J. W. Hsueh. Tissue-Specific Bcl-2 Protein Partners in Apoptosis: An Ovarian Paradigm.
Physiol. Rev. 80: 593– 614, 2000.—Apoptosis is an essential physiological process by which multicellular organisms
eliminate superfluous cells. An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of
apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of
apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals. The Bcl-2 proteins can be
divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain. In the mammalian ovary, a high rate of follicular cell apoptosis continues
during reproductive life. With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes
serves as a paradigm to understand apoptosis regulation in a tissue-specific manner. We identified Mcl-1 as the main
ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well
as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands. The activity of the proapoptotic
ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14 –3-3
or hormone-induced P11. In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and,
together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs
and caspases. Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation
of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic
approaches for pathological states.
I. INTRODUCTION
Apoptosis was first discovered by Carl Vogt in 1842
(243). After more than a century of dormancy, Kerr, Wyllie, and Currie reinitiated apoptosis research. They coined
the term apoptosis in 1972 to describe the unique morphology and ladderlike DNA fragmentation found in the
dying adrenal cells after the withdrawal of ACTH support
(119, 255, 257). The morphological hallmarks of apoptosis
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
include cell shrinkage, plasma membrane blebbing, and
apoptotic body formation.
In multicellular organisms, apoptosis or programmed
cell death is an important physiological process to remove
superfluous cells including those that are generated in
excess, have already completed their specific functions,
or are harmful to the whole organism (119, 220, 230).
Apoptosis is particularly important during embryonic development, metamorphosis, tissue renewal, hormone-in593
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SHEAU YU HSU AND AARON J. W. HSUEH
1. Conditions associated with abnormal
apoptosis
TABLE
Autoimmune diseases
Tumorigenesis
Neurodegenerative disorders
Ischemic injury of vascular tissues
Environmental toxins
Viral infection
Radiation
Chemotherapeutic agents
“survival surveillance system” switches on the decision
step of the apoptotic program, thus leading to a subsequent activation of a set of death genes in the execution
phase of apoptosis (186). Because extracellular signals
and cellular receptors that are important for the survival
and apoptosis of different cell types are extremely diverse, redundant but overlapping genes in the apoptotic
pathway have evolved, and different subsets of these
apoptosis regulatory genes are responsible for the decision of cell fate in a tissue-specific manner.
In this review, present understandings on the role of
Bcl-2 proteins in the apoptosis decision step are summarized using ovarian follicle apoptosis regulation as an
experimental paradigm. Several novel Bcl-2 protein partners were isolated from the ovary [Bcl-2-related ovarian
killer (Bok) and Bcl-2-related ovarian death gene (BOD)]
using a yeast two-hybrid protein-protein interaction assay
(85– 87, 90). The function of these genes is discussed to
provide a framework for the understanding of the hormonal regulation of apoptosis in ovarian and other systems. In addition, unique structure-functional attributes
of three subgroups of Bcl-2-related proteins (antiapoptotic channel forming, proapoptotic channel forming, and
proapoptotic ligand) and their role in apoptosis is elucidated.
II. CED-9/BCL-2 PROTEINS ARE PRESENT
IN THE DECISION STEP OF APOPTOSIS:
LESSONS LEARNED FROM THE NEMATODE
The genetic analysis of programmed cell death during Caenorhabditis elegans embryogenesis has contributed significantly to the understanding of molecular
mechanisms underlying apoptosis (73, 83). In combination with biochemical studies using mammalian cells, it is
now clear that programmed cell death in diverse animals
is controlled by evolutionarily conserved cellular machinery. Programmed cell death in C. elegans shares morphological features of apoptosis commonly observed in both
vertebrates and invertebrates. Of the 1,090 somatic cells
formed during development of the nematode, 131 of them
undergo programmed cell death that is under stringent
genetic control (73, 83).
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duced tissue atrophy, and many pathological conditions.
During embryonic development, apoptotic cell death is
widespread and is essential for the sculpturing of body
shape. It is estimated that as much as 85% of embryonic
neurons undergo apoptosis during development of the
central nervous system (8, 9, 174, 187, 230). During metamorphosis in different animals, extensive apoptosis is
necessary for the remodeling of body structure in essentially every part of the organism (104, 117, 220).
In adult tissues, the occurrence of apoptosis is also
common and can be separated into three broad categories. Minimal cell proliferation and a low rate of cell
turnover characterize the first group of tissues, such as
neuron, heart, liver, and kidney. Generally, little apoptosis
can be observed in these tissues (10, 11). The second
group, including hematopoietic tissues, the epithelium
lining intestinal crypts, and male germ cells (spermatogonia) in the testis, exhibits constant cell turnover and has
high rates of stem cell proliferation accompanied by massive apoptosis (18, 255–257). In the third type of tissues,
exemplified by the ovary, a high rate of follicular cell
apoptosis continues during the reproductive life but with
no replenishment of the original stockpile of follicles (91).
The maintenance of a balance between apoptosis and
cell proliferation is crucial for the well being of the multicellular organism. Abnormal apoptosis regulation often
leads to tissue degeneration and/or malformation, thus
leading to pathogenesis found in diseased states (230).
Certain forms of autoimmune disease are due to the suppression of apoptosis in the immune system and the ensuing accumulation of autoreactive lymphoid cells. In
more extreme conditions, the failure of cells to die could
lead to an accumulation of excess cells and, eventually,
tumorigenesis. Similar to the abnormal suppression of
apoptosis, excessive cell death could also lead to pathological states. Increased apoptosis has been associated
with ischemic injury of vascular tissues, graft versus host
diseases, and the development of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease,
Huntington’s disease (230), Charcot-Marie-Tooth type 1A
demyelinating neuropathy (62), spinal muscular atrophies
(200), and toxic epidermal necrolysis (242). Likewise,
excessive apoptosis may be the cause of premature ovarian failure due to an early depletion of ovarian follicle
reserve. Moreover, adverse environmental factors could
also alter apoptosis such as injuries that could be incurred
by environmental toxins, X-irradiation and chemotherapeutic agents, or after a viral infection (Table 1).
With the exception of blastomeres, possibly all differentiated tissues in multicellular organisms are “programmed” to undergo apoptosis through an evolutionarily
conserved “suicide” program (186 –188). To stay alive, the
healthy cells require signals provided by specific survival
factors acting on receptors found intracellularly or on the
cell surface. Once the survival signals are absent, the
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Genetic analyses have demonstrated that about a
dozen genes are essential for the regulation of programmed cell death in C. elegans (79). In addition to
genes responsible for morphological changes and phagocytosis (55, 253, 254), four genes have been shown to be
important in the “decision” and “execution” steps of apoptosis (Fig. 1). Among these four genes, ced-9 encodes a
protein that inhibits the cell death program (78, 79) (Fig.
1). Mutations that abnormally activate ced-9 can prevent
cell death that occurs during normal C. elegans development. Conversely, when ced-9 is inactivated, most of the
cells that would normally live now undergo programmed
cell death, and the animal dies early in development. Of
interest, ced-9 can function properly only if both ced-3
and ced-4 are functionally intact, suggesting that ced-9
functions upstream of the ced-3 and ced-4 genes and
might prevent cell death by keeping the ced-3/ced-4-dependent death program in check in normal cells (78). The
mammalian Bcl-2 family proteins are homologous to the
ced-9 gene, and overexpression of Bcl-2 blocks apoptosis
in nematode, insect, and mammalian cells (6, 79, 240)
(Table 2).
Unlike the antiapoptotic role of ced-9, the two downstream genes, ced-3 and ced-4, are necessary for apoptosis. Mutation of these genes prevents cell death that normally occurs during embryogenesis, leading to the
development of worms with excessive cells (60, 77, 262,
265). The ced-3 gene encodes a cysteine protease homologous to proteins of the caspase family in mammals (262).
These cysteine proteases are important for the execution
of apoptosis through proteolytic catalysis of cellular components; unrestricted expression of these proteases leads
to apoptosis in a variety of cells.
The nematode ced-4 gene encodes a hydrophilic
polypeptide containing a P-loop nucleotide (ATP) binding
motif. It presumably functions by forming complexes with
the single polypeptide zymogen form of ced-3, thus promoting its activation by autoprocessing. In living cells, the
ced-4/ced-3 complexes also include the antiapoptotic
ced-9 protein which blocks death by suppressing the
ced-4 from activating ced-3 caspase. In mammalian cells,
the ced-4 homolog apoptosis activating factor (Apaf)-1
(265), is essential for caspase activation and forms complexes with specific initiator caspases upstream of the
apoptosis caspase cascade (93, 178, 217). Because ced-4/
Apaf-1 physically forms complexes with both ced-9/Bcl-2
proteins and ced-3/caspases in mammalian cells, it has
been hypothesized that ced-4/Apaf-1 can bridge between
the ced-9/Bcl-2 family members in the apoptosis decision
step and caspases in the execution phase. Although mammalian Apaf-1 shares significant sequence homology with
ced-4, it contains additional functional motifs including
the NH2-terminal CARD caspase recruitment domain
(CARD) domain and the COOH-terminal WD-40 repeats,
2. The expanding Bcl-2 family
of apoptosis-regulating proteins
TABLE
Antiapoptotic channel members
Ced-9, Bcl-2, Bcl-x, Bcl-w, Mcl-1, Bfl-1/A1, NR-13
BHRF1 (Epstein-Barr virus), LMW5-HL (African swine fever virus),
ORF16 (herpes virus saimiri), KSbcl-2 (Kaposi sarcoma-associated
herpes virus)
Proapoptotic channel members
BAX, Bak, Bok/Mtd, Diva/Boo
Proapoptotic ligands with BH3 domain only
EGL-1, BAD, Bik/Nbk/Blk, BOD/Bim, BID, Harakiri/DP5, NIP3, NIP3L/
NIX
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FIG. 1. Conserved regulation of apoptosis from nematodes to human. On the basis of genetic analyses, four genes
have been shown to function at different points of the apoptotic pathway in Caenorhabditis elegans during embryogenesis. These genes are indispensable for the normal progression of apoptosis during early development, and they can
be categorized into 3 different groups encoding apoptosis decision factors (antiapoptotic ced-9 and proapoptotic egl-1),
a bridging factor (ced-4), and apoptosis execution proteolytic enzyme (ced-3). The ced-9 gene has evolved into a large
group of anti- and proapoptotic channel-forming proteins (represented by Bcl-2 here) in the mammals, whereas the
proapoptotic EGL-1 is related to the mammalian proapoptotic ligands with BH3 domain only (represented by BAD). The
mammalian ced-4 gene counterpart is Apaf-1, whereas the ced-3 gene has evolved into a large group of cysteine proteases
known as caspases that are essential for the degradation of cellular structures. The antagonisitc relationship between
Ced9/Bcl-2 and EGL-1/BAD proteins are reflected by broken arrows.
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SHEAU YU HSU AND AARON J. W. HSUEH
mologs of ced-3, ced-4 and ced-9 as well as other uncharacterized factors (69, 109, 146, 152, 190, 191, 204, 222).
III. APOPTOSIS REGULATION BY THREE
SUBGROUPS OF MAMMALIAN
BCL-2 PROTEINS
The nematode ced-9 gene and its mammalian homolog bcl-2 protect cells from apoptosis. The bcl-2 (B-cell
leukemia/lymphoma 2) gene was first isolated as a protooncogene at the breakpoint of a t(14,18) chromosomal
translocation associated with follicular B-cell lymphoma
(40, 234). This translocation leads to overexpression of
Bcl-2 in lymphoid cells due to an aberrant regulation of
the Bcl-2 gene under the control of the immunoglobulin
heavy chain gene promoter/enhancer. Because of the
gained survival advantage provided by Bcl-2, lymphoid
cells become susceptible to the accumulation of additional genetic alterations (e.g., c-Myc mutation), thus
leading to tumorigenesis (42, 221).
The Bcl-2 protein is localized to the mitochondria, perinuclear membrane, and smooth endoplasmic reticulum
(29, 80). When overexpressed, the Bcl-2 protein suppresses
apoptosis induced by a variety of agents both in vitro and in
vivo (5, 7, 169, 170). In transgenic mice, overexpression of
Bcl-2 in the lymphoid system results in increased immunoglobulin-secreting cells, elevated serum immunoglobulins,
exaggerated antibody response to immunization, development of spontaneous systemic autoimmune disease, and an
increased incidence of malignant B-cell lymphoma in aging
transgenic animals (42, 155, 213, 221).
However, the apoptosis-suppressing action of Bcl-2 is
not universal, and this protein cannot prevent apoptosis
in many occasions (45, 237). For example, cell death
incurred by withdrawal of trophic factors such as interleukin-2, interleukin-6 (170), or ciliary neurotrophic factor
from dependent cells (5) is not altered by Bcl-2 overexpression. In transgenic mice overexpressing Bcl-2 in the
lymphoid system, apoptosis induced by superantigens in
immature thymocytes is also not suppressed (114, 209,
239). Likewise, mice deficient in Bcl-2 do not show an
overt abnormality during development, and only cells in
hair follicles, kidney, and lymphoid systems show defects
in apoptosis (216, 241). Thus the suppression of apoptosis
by Bcl-2 is restricted to selected apoptotic stimuli in
restricted tissues.
With the use of homologous gene isolation, proteinprotein interaction screen, subtraction cloning, and viral
gene analysis, an expanding family of Bcl-2-related proteins has been identified in mammals. On the basis of
structural and functional attributes, Bcl-2 proteins can be
divided into three subgroups (Fig. 2): 1) the antiapoptotic
channel-forming Bcl-2 proteins with four BH domains
(BH1 to -4) and a transmembrane anchor sequence, 2) the
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suggesting that the mammalian Apaf-1 could regulate
apoptosis using newly evolved mechanisms. Recent studies suggested that the CARD domain and WD-40 repeats
are important for interactions with specific caspases and the
Apaf-1 cofactor cytochrome c, respectively (93, 178, 217).
Another nematode gene egl-1 encodes a proapoptotic
protein sharing a conserved BH3 (Bcl-2 homology) domain originally characterized in mammalian Bcl-2 family
proteins but without other common functional motifs (41,
48). EGL-1 activates apoptosis by binding to and directly
suppressing the activity of ced-9, and loss-of-function mutations of egl-1 in worms prevent cell death. It is likely
that the binding of EGL-1 to ced-9 releases the cell death
activator ced-4 from the constraint imposed by ced-9, thus
allowing ced-4 to activate ced-3 and apoptosis. A subgroup of mammalian Bcl-2-related proapoptotic proteins,
including BAD, Bim/BOD, Bik/Nbk/Nlk, BID, Nip3, Nip3L/
Nix, and Harakiri/DP5, is homologous to the nematode
EGL-1 gene in having only the BH3 domain (25, 26, 76, 90,
97, 98, 115, 171, 245, 263) (Table 2). These proteins are
likely to have the same evolutionary origin and functional
characteristics as the nematode egl-1 gene. Structural and
functional analyses have revealed that these proapoptotic
proteins are ligands to antiapoptotic Bcl-2 proteins and
require the conserved BH3 domain for apoptosis induction.
In addition to the decision and execution steps of
apoptosis, removal of dying cells is also an important
process to maintain tissue homeostasis. The corpses of
dead cells in developing embryos of the nematode are
quickly engulfed by neighboring cells and degraded. A
group of genes, which includes ced-1, -2, -5, -6, -7, -8, and
-10, has been found to be important for the efficient
removal of corpses of dying cells (54, 55, 60). Although
the identity of these genes has not been fully characterized, ced-5 and its mammalian homolog DOCK180 are
essential for cell surface extension of engulfing cells
(254), whereas ced-7 and its homolog ABC transporters
probably translocate molecules that mediate homotypic
cell surface adhesion between the dying and engulfing
cells (253).
It is clear that a number of evolutionarily conserved
genes could regulate a common cell death pathway in
different organisms. Although genetic studies in nematode provided valuable insight into the basic mechanism
of apoptosis conserved in diverse species, recent biochemical studies indicated that the increasing complexity
in cellular differentiation of higher organisms has led to
the development of additional regulatory mechanisms in
the apoptosis program. It appears that Bcl-2 proteins, in
addition to forming dimers with each other and ced-4/
Apaf-1, may also form channels in the mitochondria outer
membrane to regulate the release of cytochrome c, an
important Apaf-1 cofactor for the activation of specific
caspases in the “apoptosome” which comprises of ho-
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BCL-2 PROTEINS IN APOPTOSIS
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proapoptotic channel-forming proteins with all but the
BH4 domain, and 3) the proapoptotic ligands containing
only the BH3 domain (2, 69, 191). The first two subgroups
of proteins are believed to be anchored on the mitochondria membrane, whereas the third subgroup of proteins
acts as ligands that dimerize with the membrane anchored, channel-forming Bcl-2 “receptors” (116, 158). The
BH3 domains in the third subgroup are essential for the
binding activity of these ligands.
The mitochondria-anchored antiapoptotic Bcl-2 proteins, represented by ced-9 and Bcl-2, likely form ion
channels capable of maintaining membrane homeostasis
and preventing cytochrome c release, thus enhancing cell
survival. These proteins also interact with ced-4/Apaf-1 to
prevent their activation of associated caspases, thus leading to the suppression of the caspase cascade and apoptosis. In addition to ced-9 and Bcl-2, several other Bcl-2
proteins, including Bcl-xL, Bcl-w, Mcl-1, and Bfl-1, have
similar antiapoptotic activity and functional motif arrangement but with a unique and overlapping tissue distribution pattern. Bcl-xL and Bcl-w were identified by
homologous screens (19, 68), whereas Mcl-1 (myeloid cell
lymphoma-1) and Bfl-1/A1 were identified by subtraction
cloning (35, 36, 56, 138, 194). Viral infection can trigger
host cell apoptosis to limit viral propagation, and some
viruses have developed mechanisms to prevent host cell
demise. Of interest, several viral genes, the adenovirus
E1B 19K (251), the Epstein-Barr virus BHRF-1 (106, 120,
225), the African swine fever virus LMW5-HL (166),
ORF16 (165), and Ksbcl-2 (28), encode proteins that are
structurally and functionally homologous to the mammalian antiapoptotic Bcl-2 proteins. Suppression of apoptosis by these viral proteins extends the life span of host
cells and increases the efficiency of viral replication.
Unlike the channel-forming antiapoptotic Bcl-2
members, Bcl-2 family proteins in the second subgroup
(Bax, Bak, and Bok) not only antagonize the survival
action of antiapoptotic Bcl-2 proteins but also actively
trigger apoptosis in transfected cells (33, 34, 63, 86, 121,
173). This subgroup of Bcl-2 proteins has BH1, -2, and -3
domains and the membrane-anchoring region but is
missing the NH2-terminal BH4 domain important for
apoptosis inhibition (94). They can dimerize with antiapoptotic Bcl-2 proteins, thus relieving the ced-4/Apaf-1
from the suppression exerted by antiapoptotic Bcl-2
proteins and promoting caspase activation (178, 217).
These mitochondria-anchored proapoptotic Bcl-2 proteins could also promote apoptosis by altering mitochondria membrane homeostasis and increasing cytochrome c release (70, 109, 137, 217, 236).
The third subgroup of Bcl-2 proteins, homologous
to the recently identified nematode EGL-1 protein, consists of proapoptotic ligands with only the BH3 domain.
These proteins are capable of interacting with selective
channel-forming Bcl-2 proteins to promote cell death
and may serve as adaptor proteins linking upstream
signaling pathways to the decision step of the apoptosis
program. Several Bcl-2 proteins in this subgroup have
been identified based on protein-protein interaction
screens using the yeast two-hybrid system or bacterial
expression cloning. Like the nematode EGL-1, several
of the proapoptotic ligands (BAD, BOD/Bim, and BID)
only have the BH3 domain (90, 245, 261). In addition,
some proteins of this subgroup (Bik/Nbk, Blk, Harakiri/
DP5, NIP3L/Nix, and NIP3) have an additional COOHterminal transmembrane region for membrane anchoring (25, 26, 75, 76, 97, 98, 151).
It is now becoming clear that Bcl-2 and related proteins are multifunctional proteins, and protein-protein interactions play an important role in their regulation of
apoptosis. One of the mechanisms by which the Bcl-2
proteins regulate apoptosis is through homodimerization
and heterodimerization with proteins in the same family.
The BH3 domain in the proapoptotic Bcl-2 proteins serves
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FIG. 2. Three subgroups of Bcl-2 proteins
and their Bcl-2 homology (BH) domain arrangement. Bcl-2 proteins can be divided into 3 subgroups based on their structural and functional
properties. The first subgroup consists of channel-forming antiapoptotic proteins with four BH
domains and a transmembrane region for anchoring to the mitochondria. The second subgroup of proapoptotic Bcl-2 proteins have BH1,
-2, and -3 domains but without the BH4 domain.
These proteins also have a transmembrane region important for mitochondria anchoring. The
third subgroup is the proapoptotic ligands possessing only the BH3 domain with or without
the transmembrane region. They serve as ligands for the channel-forming Bcl-2 proteins by
forming functional heterodimers to regulate apoptosis.
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SHEAU YU HSU AND AARON J. W. HSUEH
Volume 80
as the ligand for binding to the receptor domain (encompassing the BH3, BH1, and BH2 domains) in antiapoptotic
members (162, 203). As shown in Figure 3, the prototypic
antiapoptotic Bcl-2 protein contains a unique BH4 domain, believed to be important for interaction with Apaf-1,
thus preventing caspase activation. In addition to the
COOH-terminal transmembrane region essential for anchoring to the mitochondria, endoplasmic reticulum, or
nuclear membrane, the ␣-helices 5 and 6 encompassing
the BH1 and BH2 regions are important for channel formation in the regulation of cytochrome c release by mitochondria (109, 152, 203, 204). Although no specific molecules have been identified to interact with these channel
domains of Bcl-2 proteins, recent studies have shown that
channel-forming Bcl-2 family proteins could interact with
multiple mitochondrial proteins to regulate cytochrome c
release through permeability transition pores of mitochondria (146, 147, 164, 210).
The multiple Bcl-2-related proteins found in vertebrates interact in a partner-specific manner and play tissue- and pathway-specific roles in apoptosis regulation
(86, 99, 100, 208, 214). The expression pattern of Bcl-2
homologs in different tissues appears to be distinct but
overlapping; some are widely distributed, whereas others
are more restricted. Despite the wide distribution of Bcl-2
proteins in a variety of tissues, studies on mutant mice
deficient in different members of the bcl-2 gene family
reinforce the notion that Bcl-2 homologs regulate apoptosis in a tissue-specific manner. In Bcl-2-deficient mice,
most tissues develop normally, and only cells in hair
follicles, kidney, small intestine, neurons, and lymphoid
system show defects in apoptosis (112, 216, 227, 241). In
contrast, targeted disruption of Bcl-X in mutant mice
leads to lethality around embryonic day 13 (161). Histological studies revealed excessive cell deaths in develop-
ing neurons and lymphoid cells of mutant embryos, suggesting that Bcl-X supports the viability of cells in the
nervous and hematopoietic systems during fetal life. Likewise, mice deficient in Bax appear healthy, and only cells
of lymphoid and gonadal lineage show aberrations in cell
death. In these mice, hyperplasia in T and B cells results
in enlarged thymus and spleen, whereas the accumulation
of premeiotic germ cells in seminiferous tubules leads to
male infertility (125). Furthermore, mice that lack the
widely distributed Bcl-w gene are viable and healthy except that the males are infertile (182, 198). The testis
develop normally, and the initial prepubertal wave of
spermatogenesis is largely unaffected; however, the seminiferous tubules are disorganized because of enhanced
apoptosis during adulthood. Likewise, mice lacking Bfl-1
are normal and indistinguishable from their wild-type littermates. However, hair loss on the head and face are
obvious by 8 –12 wk of age in these Bfl-1-deficient mice
(74). Although the tissue-specific phenotypes detected in
these transgenic mice could be due to differences in the
apoptosis regulatory mechanisms in diverse cells, the
findings clearly show that the ced-9 homologs in mammalian cells not only have diverged in their functional characteristics but also have adopted unique roles in specific
cell lineage.
IV. OVARIAN FOLLICLE ATRESIA AS A MODEL
OF APOPTOSIS: CONSTRUCTION OF AN
OVARY-SPECIFIC PROTEIN-PROTEIN
INTERACTION MAP IN APOPTOSIS
REGULATION
Although it appears that the intracellular regulators
of cell death are conserved during evolution, the death of
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FIG. 3. Different functional domains have been identified in Bcl-2 proteins. As shown in the prototypic Bcl-2 protein,
the BH4 domain is important for binding to downstream Apaf-1 and its homologs, whereas the COOH-terminal
transmembrane region is important for mitochondria anchoring. For homo- or heterodimerization among Bcl-2 members,
the BH3 region serves as the ligand domain, whereas the region spanning the BH3, -1, and -2 regions serves as the
receptor domain. In addition, the ␣5- and ␣6-regions are important for channel formation in the mitochondria. To amplify
the apoptotic cascade, caspases could cleave a consensus site found between BH4 and BH3 domains to allow the
conversion of antiapoptotic Bcl-2 proteins to proapoptotic ones.
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BCL-2 PROTEINS IN APOPTOSIS
cell apoptosis by activating the cAMP-dependent pathway
and increasing the production of paracrine and autocrine
factors such as estrogens, interleukin-1, nitric oxide, and
insulin-like growth factor I (IGF-I) (Fig. 4). These factors
in turn promote cell survival through the activation of
nuclear estrogen receptor, the cGMP-dependent pathway,
and protein tyrosine phosphorylation, respectively (91).
Because the progression of apoptosis in ovarian follicles
is dependent on the cooperative regulation of different
paracrine and autocrine factors, it is likely that none of
these factors is singularly obligatory in the control of
follicle growth or demise. Instead, a balance of these
different survival and apoptotic factors may decide
whether a follicle will continue development or undergo
apoptosis. How the various extracellular hormones and
their intracellular signal transduction mediators are
linked to the intracellular decision step of apoptosis in
ovarian follicles remains, however, largely undefined.
To elucidate the hormonal regulation of ovarian follicle atresia, we first tested whether the Bcl-2 system is
important in the regulation of ovarian cell apoptosis in
vivo. Transgenic mice overexpressing Bcl-2 in the ovary
were generated by using the mouse inhibin-␣ gene promoter/enhancer to drive the Bcl-2 transgene (88, 89). In
these mice, a suppression of follicular cell apoptosis,
enhancement of folliculogenesis, and an increased incidence of benign ovarian teratoma development were
found, indicating that Bcl-2 associated regulatory system
is operating in the ovary. However, endogenous expression of Bcl-2 in follicle cells was not evident based on in
situ and immunostaining approaches, suggesting Bcl-2
family proteins other than Bcl-2 are involved in the regulation of ovarian cell apoptosis.
Taking advantage of the heterodimerization property
of Bcl-2-related proteins, we constructed an ovarian fusion cDNA library and used Bcl-2 as bait to search for
endogenous Bcl-2-interacting preys in the yeast two-hybrid protein-protein interaction screen. After each Bcl-2-
FIG. 4. Stage-dependent regulation of follicle
survival. On the basis of studies on rat ovarian
follicles, gonadotropins are the major survival factors that suppress ovarian cell apoptosis through
the activation of cAMP-dependent pathways. In addition, the apoptosis-suppressing action of gonadotropins is augmented by multiple local factors, including interleukin-1, estrogens, and insulin-like
growth factor I (IGF-I), which in turn promote cell
survival by activating the cGMP-dependent pathway, nuclear estrogen receptor, and tyrosine phosphorylation, respectively.
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a cell is usually under the control of multiple extracellular
factors, and a balance of survival and apoptotic signals
determines a cell’s fate. The multifactorial mode of apoptosis regulation is exemplified by the hormonal control
of follicle demise in the mammalian ovary. In the ovary,
apoptosis occurs mainly at the early antral follicle stage,
and ⬎99% of ovarian follicles undergo atresia during reproductive life (84, 91, 156). Morphological and biochemical studies have demonstrated that the death of both
somatic and germ cells in the ovary is mediated by apoptosis (16, 91, 96, 179, 232). Ovarian follicle cell apoptosis
is regulated by multiple hormones including pituitary glycoprotein hormones as well as local growth factors, cytokines, a diffusible gas (nitric oxide), and steroids (16,
91). Of interest, many of these factors are important both
for promoting cell proliferation and for maintaining cell
viability. In contrast, Fas ligand, tumor necrosis factor
(TNF)-␣, interleukin-6, and gonadotropin-releasing hormone-like peptides act as intraovarian apoptotic factors
through specific apoptotic pathways (17, 110, 184, 185,
201, 259). Furthermore, in vivo studies have implicated
estrogens as antiapoptotic hormones in ovarian follicles,
whereas androgens are proapoptotic (15, 16). It has been
clearly demonstrated that the majority of follicles undergo apoptosis when they reach the early antral (in
rodents) or antral (in human) stages unless rescued by
gonadotropins (mainly follicle-stimulating hormone) (15,
37–39). However, the exact mechanisms underlying this
programmed follicle demise and role of granulosa and
thecal cells are not clear. Future studies on the potential
role of various intraovarian apoptotic factors during follicle development are of interest.
The diverse hormones function through endocrine,
paracrine, or autocrine mechanisms. Pituitary gonadotropins appear to be the most important survival factors for
ovarian follicle cells because gonadotropin treatment increases the expression of local survival factors in ovarian
follicles. Specifically, the gonadotropins suppress ovarian
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allowed one to construct a network map of Bcl-2-interacting proteins in the ovary to understand the intracellular mechanisms of ovarian follicle cell apoptosis and its
regulation by upstream survival and/or apoptotic factors.
Although the exact link between ovarian Bcl-2 proteins
and the ovarian ced-4/Apaf-1 proteins remains to be elucidated, recent reports indicated that Apaf-1 is expressed
in human and mouse granulosa cells (105, 197), and treatment with gonadotropins suppresses Apaf-1 expression.
Furthermore, Diva/Boo, a recently discovered, ovary-specific Bcl-2 protein (100, 214), is capable of binding to
Apaf-1. Future studies on the role of Diva/Boo in the ovary
could allow the identification of additional ovarian Apaf-1
homolog and the exact ovarian Bcl-2 protein partner for
Apaf-1 and Diva/Boo.
It is becoming clear that a given tissue has a set of
Bcl-2 proteins, and apoptosis is regulated through selective dimerization of channel-forming antiapoptotic Bcl-2
proteins, channel-forming proapoptotic Bcl-2 proteins,
and the BH3-only apoptotic ligands (69, 191). The present
approach could serve as an experimental paradigm for the
construction of Bcl-2 network maps in diverse tissues.
Several useful points have been gained from these studies: 1) the protein-protein interaction network map constructed is tissue specific, 2) novel Bcl-2 and interacting
genes in the apoptosis pathway can be identified, and 3)
posttranslational modification of proteins such as the
phosphorylation of BAD plays an important role in protein dimerization.
Although the two-hybrid system is a powerful tool, it
has limitations: 1) Gal-4 fusion proteins used in the yeast
cell screen may behave differently from native proteins,
2) observed interactions in yeast may not take place
inside mammalian cells due to differential cellular localization or cell type-specific expression, and 3) some fu-
FIG. 5. Three stages of ovarian cell apoptosis are shown. Taking advantage of the dimerization property of
Bcl-2-related proteins, Bcl-2 was used as bait to search for endogenous Bcl-2-interacting preys in the yeast 2-hybrid
system. After characterization of each prey, new bait was chosen and, in turn, used for a subsequent round of screens.
Repeating this procedure multiple times allowed the identification of BAD as an important Bcl-2 member in the ovary
that bridges the upstream signaling proteins, 14 –3-3 and P11, to the Bcl-2 family of proteins. In addition, the antiapoptotic
Mcl-1 was found to be the likely mammalian ced-9 homolog in the ovary and Mcl-1 dimerized with Bax and two novel
ovarian proapoptotic Bcl-2 members, Bok and BOD, in the decision step of apoptosis. The antiapoptotic protein, Mcl-1
or the recently discovered DIVA/Boo, may interact with downstream ovarian ced-4/Apaf-1 homolog which, in turn,
activates caspases in the execution step of apoptosis.
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interacting protein obtained from the ovarian library was
characterized, new baits were chosen and, in turn, used
for second round screens for additional interacting molecules. Repeating this procedure multiple times allowed
the identification of several known and novel Bcl-2 family
proteins as well as their interacting regulators in the
ovary. First, a Bcl-2-interacting protein, BAD, was found
as an important proapoptotic ligand in the ovary that
bridges the upstream signaling proteins, 14 –3-3 and P11,
to the channel-forming antiapoptotic Bcl-2 family proteins
(87) (Fig. 5). Of interest, 14 –3-3 proteins preferentially
bind phosphorylated BAD, whereas the use of an underphosphorylated BAD mutant as bait allowed the isolation
of P11 and Mcl-1 as BAD-interacting proteins (130a). It
appears that the antiapoptotic Mcl-1 is a mammalian ced-9
homolog in the ovary which, in turn, dimerizes with two
novel ovarian proapoptotic Bcl-2 members, Bok and BOD,
as well as the known proapoptotic Bax in the decision
step of apoptosis (86, 90). The antiapoptotic protein Mcl-1
may prevent apoptosis by interacting with unknown
downstream ovarian ced-4/Apaf-1 homolog capable of activating caspases in the execution step of apoptosis. The
antiapoptotic activity of Mcl-1 could also be modulated by
the proapoptotic family proteins including Bok, BOD,
BAD, and Bax. Studies on ovarian Mcl-1 mRNA levels
indicated that the expression of this antiapoptotic protein
is induced by survival hormones in the ovary (130a).
Because the phosphorylation status of BAD plays an
essential role on its proapoptotic activity, gonadotropins
and other upstream survival factors likely enhance the
phosphorylation of BAD to allow the binding of 14 –3-3
proteins, leading to the dampening of BAD-induced cell
killing (49, 65, 87, 264). Likewise, some survival factors
could induce the synthesis of P11 that also binds to BAD
and decreases its proapoptotic action. Thus these findings
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BCL-2 PROTEINS IN APOPTOSIS
V. PROAPOPTOTIC BAD AND BOD ARE
LIGANDS FOR ANTIAPOPTOTIC BCL-2
PROTEINS AND CAPABLE OF
TRANSMITTING UPSTREAM SIGNALS
BAD and BOD were isolated from the ovarian fusion cDNA library based on their binding to antiapoptotic Bcl-2 proteins (87, 90, 111). Both BAD and BOD
belong to the subgroup of proapoptotic ligands with
only the BH3 domain. After overexpression in transfected cells, these ligands induce apoptosis. Because
both BAD and BOD lack the transmembrane region
important for anchoring to intracellular membranes,
they function by serving as cytoplasmic proapoptotic
ligands for membrane-bound channel-forming Bcl-2
proteins through heterodimerization. Indeed, coexpression with specific antiapoptotic Bcl-2 proteins abolishes the cell killing activity of BAD or BOD.
BOD, also known as Bim (171), is expressed in
multiple tissues. Analysis of protein-protein interactions in the yeast two-hybrid system demonstrated that
BOD interacts with diverse antiapoptotic Bcl-2 proteins
including Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and the
Epstein-Barr viral-derived BHRF-1 (90). Thus the widely
distributed BOD could serve as an apoptosis mediator in
diverse cell lineages through dimerization with a variety
of antiapoptotic proteins. There are several splicing variants of BOD that vary in their NH2-terminal sequences
(BOD-L, BOD-M, and BOD-S) in ovarian tissues; all three
forms contain the BH3 domain and promote cell death in
transfected cells. Similar to several BH3-only proapoptotic ligands, mutation analysis demonstrated that the
BH3 domain of BOD is essential for its heterodimerization
with antiapoptotic Bcl-2 members and for its cell-killing
ability (76, 98, 115).
Similar to BOD, BAD has been shown to heterodimerize with the antiapoptotic Bcl-2 proteins through
its BH3 domain (115, 263). In addition to its role as a
ligand for antiapoptotic Bcl-2 proteins, BAD has been
found to interact with upstream signaling molecules capable of regulating its proapoptotic activity. With the use
of the yeast two-hybrid system to search for BAD-binding
proteins in the ovary, multiple isoforms of 14 –3-3 were
identified. The highly conserved ubiquitous 14 –3-3 proteins are expressed in perhaps all eukaryotic organisms
including yeast, plants, insects, and mammals with at
least seven different isoforms found in mammalian cells
(139, 247, 258). Proteins of the 14 –3-3 family bind diverse
enzymes and signaling molecules including Raf-1 kinase,
B-Raf, phosphatidylinositol 3-kinase, CDC25 phosphatases, Bcr, Cbl, and polyoma middle-T antigen (4, 144,
193). They are important adaptors in intracellular signaling, cell cycle control, oncogenesis, and neurotransmitter
biosynthesis. Crystallographic studies further indicated
that different isoforms of 14 –3-3 dimerize to generate a
complex with two ligand-binding sites (139, 258), thus
allowing the assembly or anchoring of different signaling
proteins and cytoskeletal elements.
In transfected cells, overexpression of 14 –3-3 suppresses apoptosis induced by BAD, indicating that interactions between them may allow coordination of cell
death and other 14 –3-3-regulated intracellular signaling
pathways (87, 264). The 14 –3-3 proteins have been shown
to bind to their interacting proteins through a consensus
serine phosphorylation sites, and serine phosphorylation
of BAD is important for 14 –3-3 binding and cell survival.
Point mutation of BAD in one (S137A), but not the other
(S113A), putative binding site found in diverse 14 –3-3
interacting proteins abolished the interaction between
BAD and 14 –3-3 without affecting interactions between
BAD and Bcl-2. In mammalian cells, cotransfection with
14 –3-3 suppressed apoptosis induced by wild-type BAD
or the S113A BAD mutant, but not by the S137A mutant
that was incapable of binding 14 –3-3 (87). The 14 –3-3
proteins could suppress BAD activity by sequestering the
phosphorylated BAD in an inactive state or by bridging
the phosphorylated BAD with a putative inhibitor of BAD
through the consensus 14 –3-3 phosphorylation site.
Because IGF-I and insulin activate the Akt/PKB kinase to phosphorylate BAD, BAD phosphorylation has
been suggested to be an important mechanism by which
upstream survival factors suppress apoptosis (47, 49,
128). Thus gonadotropin and other survival factors could
increase the serine phosphorylation of BAD in ovarian
cells to enhance the binding of BAD to the constitutively
expressed 14 –3-3, thus maintaining BAD in an inactive
state. The proapoptotic BAD is functional only after withdrawal of the survival signals followed by the dephosphorylation of BAD (Fig. 5).
Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD incapable of
binding 14 –3-3 proteins, this mutant was used to screen
for additional BAD-interacting proteins in the ovary
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sion proteins encoded by apoptosis-inducing genes may
be lethal to yeast cells, thus preventing the identification
of such genes. To avoid these potential pitfalls, it is important to perform the following steps to further characterize each candidate gene: 1) confirmation of direct protein-protein interactions by coprecipitation using
endogenous or recombinant proteins, 2) expression of
protein pairs in mammalian cells to identify their putative
roles in apoptosis, 3) examination of temporal and spatial
expression pattern of candidate genes in the tissues of
interest, 4) mutations of putative phosphorylation sites to
control the degree of protein phosphorylation in yeast
cells, and 5) the use of selective domains of genes as bait
to avoid a lethal phenotype or potential stereochemical
hindrance.
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VI. PROAPOPTOTIC BOK AND BAX WITH A
CHANNEL-FORMING DOMAIN REGULATE
APOPTOSIS BY RELEASING APAF-1 FROM
SUPPRESSION BY ANTIAPOPTOTIC
PROTEINS AND BY PROMOTING
CYTOCHROME C RELEASE
The Bok gene was first identified as a Mcl-1 binding
protein in the ovary using the yeast two-hybrid system
(86). Unlike other channel-forming proapoptotic Bcl-2
proteins including Bax and Bak, Bok preferentially dimerizes with antiapoptotic proteins Mcl-1, Bfl-1, and BHRF1
but does not interact with Bcl-2, Bcl-w, and Bcl-xL. Although low levels of mRNA for Bok, also named as Mtd,
are present in diverse tissues (99), it is expressed at high
levels in several reproductive organs, including ovary,
testis, and uterus (86). The distinct heterodimerization
property and tissue distribution pattern of Bok suggested
that it is a tissue-specific mediator of cell death and could
promote apoptosis by interacting with selective antiapoptotic protein. This notion is consistent with the hypothesis
that apoptosis is regulated through the dimerization of
Bcl-2 family proteins in a tissue-specific manner.
Functional analysis indicated that Bok is a proapoptotic protein, and its cell killing ability can be dampened
following coexpression with its dimerization partners
Mcl-1 and BHRF-1 (86). Bok, together with Bax and Bak,
belong to the subgroup of proapoptotic channel-forming
Bcl-2 proteins containing BH1, BH2, BH3, and a COOHterminal transmembrane region (2). During the analysis of
Bok mRNA and gene structure, we found a Bok splicing
variant in which the region encoded by exon 3 is absent,
suggesting the existence of a truncated short form
(Bok-S) of the full-length Bok protein (Bok-L) (85). The
skipping of exon three encoding 43 amino acids maintained the original reading frame and retained the BH2
and the COOH-terminal membrane anchoring domains;
however, parts of the BH3 and BH1 domains were deleted. Of interest, functional analysis indicated that Bok-S
is still capable of inducing apoptosis while the truncated
Bok has lost its ability to heterodimerize with Mcl-1,
BHRF-1, and Bfl-1, suggesting that an intact BH3 domain
of Bok is not essential for apoptosis regulation and the
short splicing variant induces apoptosis through a heterodimerization-independent mechanism (85).
Although the BH3 domain was originally shown to be
essential for the heterodimerization property of different
proapoptotic Bcl-2 proteins and for their cell killing property, recent studies indicated that the BH3 domain and
associated heterodimerization property are dispensable
for apoptosis induction by the channel-forming proapoptotic protein Bax and Bak (108, 191). Likewise, mutation
analysis indicated that the BH3 domain in Bok is not
essential for apoptosis induction and substitution of conserved residues in the BH3 domain of Bok also abates its
ability to dimerize with antiapoptotic proteins. Similar to
Bok-S, mutants of related proapoptotic proteins Bax and
Bak with their homologous BH3-BH1 region deleted also
retained proapoptotic activity, thus suggesting a common
mechanism of action is shared by this subgroup of channel-forming proapoptotic proteins.
Although proapoptotic Bcl-2 proteins could function
by antagonizing the activity of antiapoptotic proteins mediated by intact BH3 domains, structure-functional characterization indicated that the ␣5- and ␣6-helices spanning
BH1 and BH2 domains are important for channel formation in the mitochondria and for the regulation of cell
death by these proteins. Deletion of the channel-forming
domain of Bax completely abolished its proapoptotic activity in yeast while substitution of the Bcl-2 channelforming domain with Bax sequences converts it into a
proapoptotic protein (152). Of interest, truncation of the
region between BH3 and BH1 from Bok-L does not affect
the homologous ␣5- and ␣6-regions proposed to be impor-
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using the yeast two-hybrid system. P11, a nerve growth
factor (NGF)-induced neurite extension factor, was
found to be a BAD interacting protein. P11 is also known
as 42C or calpactin I light chain and belongs to the S100
family of calcium binding proteins (64, 149, 150). It was
originally identified as an early response gene following
NGF stimulation of a rat pheochromocytoma (PC12) cell
line. In addition, P11 was also increased after transformation induced by viral oncogenes in diverse cell lines (43,
175). Of interest, overexpression of P11 induces neurite
outgrowth and enhances PC12 cell survival in the absence
of NGF (150).
P11 interacted preferably with the mutant BAD but
less effectively with the wild-type protein (87). In transfected cells, coexpression of P11 attenuated the proapoptotic effect of both wild-type BAD and S137A BAD mutant. In addition, direct P11 binding to BAD was also
demonstrated in vitro. Like 14 –3-3, P11 may also function
as a BAD binding protein to dampen the apoptotic activity
of BAD. Because the 14 –3-3 family of proteins could
interact with key signaling proteins, whereas P11 is an
early response gene induced by survival factors, extracellular hormonal signals could modulate BAD-induced
apoptosis through induction of this BAD-binding protein
in addition to regulating the interaction between BAD and
14 –3-3 via BAD phosphorylation (87, 264). BAD appears
to play an important role in mediating communication
from different upstream signal transduction pathways to
the Bcl-2-regulated apoptotic decision step. Although how
BOD activity is regulated by upstream signaling molecules has not been reported, BOD and other BH3-only
proapoptotic ligands could also function as ligands that
bridge upstream signals to membrane-bound channelforming Bcl-2 receptor proteins.
Volume 80
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BCL-2 PROTEINS IN APOPTOSIS
ant does not dimerize with antiapoptotic proteins but
probably forms mitochondrial channels to regulate apoptosis. Although multiple reports suggested a role for
several Bcl-2 proteins (e.g., Bax, Bak, Bok) in the formation of mitochondria channels, it is important to note that
no direct in vivo evidence for such channel is presently
available, and additional studies are needed to confirm
this hypothesis.
VII. CELL DEATH EXECUTION BY CASPASES:
CONVERGING PATHWAYS FOR
EXTRACELLULAR AND INTRACELLULAR
SIGNALS
It is becoming apparent that mitochondrial changes
play a central role in apoptosis regulation in a three-step
model of apoptosis: 1) a premitochondrial decision step
during which upstream signal transduction pathways are
activated to regulate Bcl-2 proteins; 2) a mitochondrial
phase, during which mitochondrial homeostasis is lost
resulting in the release of proteins that activate caspases
as well as the disruption of mitochondria electron transport, oxidative phosphorylation, and ATP production; and
3) a postmitochondrial phase, during which proteins released from mitochondria cause the activation of Apaf-1
and initiator caspases followed by sequential activation of
a cascade of effector caspase zymogens.
In the mitochondria phase, the channel-forming Bcl-2
family proteins could alter mitochondrial inner transmembrane potential through the opening of a large-conductance channel known as the mitochondrial permeability transition (PT) pore (146, 147). Pharmacological
FIG. 6. Model of apoptosis regulation by
proapoptotic Bcl-2 family proteins. At least
three mechanisms could be involved in the action of proapoptotic Bcl-2 proteins: 1) the subgroup of proapoptotic Bcl-2 proteins with only
the BH3 domain, represented by the soluble
BAD and BOD proteins, heterodimerizes with
membrane-bound antiapoptotic proteins to regulate apoptosis; 2) the subgroup of membranebound proapoptotic Bcl-2 channel-forming proteins, represented by Bok-L, heterodimerizes
with antiapoptotic proteins (Mcl-1 or Bfl-1) or
functions as mitochondrial channels to regulate
apoptosis; and 3) the unique Bok-S does not
dimerize with antiapoptotic proteins but probably forms mitochondrial channels to regulate
apoptosis. Regulation of Bok-S levels by upstream signals could rapidly induce apoptosis
without direct interference by antiapoptotic
proteins present in the same cell.
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tant for channel formation of Bax (191). In addition, the
hydropathicity property between the 5⬘-end of the BH1
region and the COOH-terminal transmembrane domain is
not altered by the truncation found in the 5⬘-end of the
channel formation region of Bok-S (85).
It is likely that the ␣5- and ␣6-regions found in Bok-S
comprise a module sufficient for channel formation to
promote apoptosis, thus mediating apoptosis through a
heterodimerization-independent pathway (210). Findings
that Bok-L and Bok-S could also promote apoptosis independent of heterodimerization further suggest that this
subgroup of proapoptotic proteins could induce apoptosis through channel formation in addition to their role as
“ligands” for antiapoptotic Bcl-2 proteins. Thus Bok-S
represents a new form of proapoptotic protein consisting
of only minimal functional modules and manifests its
proapoptotic action without direct interactions with antiapoptotic proteins. Bok-S expression in the ovary and
uterus could provide a short circuit to promote cell demise in hormone-dependent cell populations that express
abundant antiapoptotic proteins (such as Mcl-1), thus
allowing swift removal of superfluous cells during reproductive cycles.
At least three mechanisms could be proposed for the
action of proapoptotic Bcl-2 proteins (Fig. 6): 1) the subgroup of proapoptotic Bcl-2 proteins with only the BH3
domain (e.g., the BAD and BOD proteins) heterodimerizes
with membrane-bound antiapoptotic proteins to regulate
apoptosis; 2) the subgroup of channel-forming proapoptotic Bcl-2 proteins with BH1, BH2, BH3, and TM domains,
represented by Bok-L, heterodimerizes with antiapoptotic
proteins (Mcl-1/Bfl-1) or functions as mitochondrial channels to regulate apoptosis; and 3) the unique Bok-S vari-
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SHEAU YU HSU AND AARON J. W. HSUEH
cysteine proteases require an Asp residue at the P1 site
for cleavage (69, 231). In addition, the pentameric peptide
signature, QAC(R/Q/G)G, surrounding the putative active
site cysteine, is highly conserved in all caspase family
proteins. Unlike most other posttranslational modifications, the caspase cascade is irreversible. Once the
caspases in the execution step are activated, the apoptotic process normally proceeds to completion. Most
caspases are synthesized as precursors, and their activities are tightly regulated at the activation step and/or
through interaction with specific inhibitors. Although the
role of cytochrome c in the activation of caspases is
gaining support, the potential role of different ions (such
as K⫹ and Na⫹) in this process should also be considered
(20, 95).
Active caspases are formed by cleavage of a proprotein precursor at internal aspartate residues (231). These
residues divide the proprotein into NH2-terminal, middle,
and COOH-terminal domains. After proprotein processing, the middle and COOH-terminal domains associate to
become an active protease. The NH2-terminal domain,
which is highly variable in sequence and length, is involved in the regulation of proprotein activation. After the
initial activation, two units of the middle and COOHterminal domains associate to form an active tetramer
complex.
Based on their positions in the apoptosis protease
cascade, caspases can be divided into two subgroups. The
“initiator” caspases, such as caspase-6, -8, and -9, initiate
the disassembly process directed by upstream apoptotic
signals. The downstream “effector” caspases, such as
caspase-3 and -7, are activated by initiators and function
in the subsequent degradation of cellular components,
thereby precipitating morphological and biochemical
FIG. 7. Formation of the active complex of apoptosome to initiate the
caspase cascade. The execution step of
apoptosis depends on the formation of an
active apoptosome complex consisting of
Apaf-1, cytochrome c released by mitochondria, and procaspase-9. Once the
procaspase-9 is cleaved in the active apoptosome complex, the active enzyme, in
turn, activates downstream caspases
such as caspase-3, leading to the degradation of multiple substrates important
for the integrity of the cellular organelles.
When Apaf-1 is bound by antiapoptotic
Bcl-2 proteins, they are present as an
inactive complex, and Apaf-1 is not capable of activating caspases. The suppressing action of antiapoptotic Bcl-2 “receptors” on caspase activation can be
relieved after binding of the proapoptotic
ligands (BAD and BOD). In addition, the
levels of cytochrome c in the apoptosome
also determine the activation of caspase.
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studies indicated that antiapoptotic Bcl-2 proteins prevent PT pore opening whereas proapoptotic ones induce
it, perhaps through direct interaction with inner membrane proteins such as the adenine nucleotide translocator. In the postmitochondria execution phase of apoptosis, the array of activated caspases amplifies the apoptotic
process through proteolytic degradation of vital cellular
components as well as the activation of additional catabolic enzymes such as the nucleases (DFF40/CAD) involved
in internucleosomal DNA fragmentation (140, 141).
The regulation of mitochondrial homeostasis by
channel-forming anti- and proapoptotic Bcl-2 proteins
controls the release of cytochrome c (and other uncharacterized mitochondrial apoptosis-inducing factors) from
the outer surface of the inner mitochondrial membrane to
the cytoplasm. The liberated cytochrome c becomes associated with Apaf-1 and procaspase-9 to form the active
apoptosome complex (93, 137, 178, 217) (Fig. 7).
Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CARD domain in the presence of cytochrome c and dATP, an event that leads to the conversion
of the latent procaspase-9 to its active form (81). Activated caspase-9, in turn, cleaves and activates downstream caspases (e.g., caspase-3). Caspases and mitochondria can engage in a circular self-amplification loop
to accelerate the apoptotic process. An increase in mitochondrial membrane permeability would cause the release of caspase activators, and caspases, once activated,
could in turn increase the mitochondrial membrane permeability through conversion of antiapoptotic Bcl-2 family proteins to proapoptotic ones (27, 260) and the activation of proapoptotic ligands (71, 133, 142).
At least 14 mammalian homologs of the ced-3
caspase gene have been isolated (3, 92, 168, 231). These
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BCL-2 PROTEINS IN APOPTOSIS
(44, 235), actin (148, 196), nuclear lamins (189, 223), spectrin, keratin-18 (181), keratin-19 (181), and gelsolin (66,
172)], 2) signal transduction [the catalytic subunit of the
DNA-dependent protein kinase (215), PITSLRE kinase (14),
protein kinase C-␦ (61), focal adhesion kinase (67, 132, 250),
MEKK-1 (252), PAK65 (130), PKN (224), p21-activated kinase 2 (244), and 90-kDa heat shock protein-␤ (181)], 3)
transcription, translation, and cell replication [U1 70-kDa
ribonucleoprotein (24), heteronuclear ribonucleoproteins
C1 and C2 (248), DNA topoisomerase I (202), large subunit
of replication factor C (14, 195), translation initiator factor
4G (145), transcription factors SREBP-1 and SREBP-2 (26),
Rb protein (19, 20), MDM2 (180), and STAT1, (122)], 4)
triplet nucleotide expansion (Huntingtin disease protein, atrophin-1, ataxin-3, and the androgen receptor) (59, 249), and
5) DNA fragmentation [the inhibitor of caspase-activated
DNase (DFF45/ICAD) (101)]. Cleavage of these and additional caspase targets results in the abrogation of the DNA
repair mechanisms, detachment of the apoptotic cells from
surrounding cells, disruption of the cytoskeleton structure,
blockage of vital signal transduction, and initiation of DNA
fragmentation. The resulting biochemical and morphological changes eventually lead to the formation of apoptotic
bodies and their engulfment by macrophages or other scavenger cells (118, 257).
In addition to cleaving proteins essential for cell
integrity, caspases also act on upstream Bcl-2 proteins to
further amplify the progression of apoptosis. Of interest,
the NH2-terminal BH4 domain of the antiapoptotic Bcl-xL
proteins can be cleaved by caspase-3, and the liberated
COOH-terminal fragment of Bcl-2 lacking the BH4 domain
potently induces apoptosis (27, 260). Thus cleavage of
Bcl-xL during the execution phase of cell death converts
Bcl-xL from a protective to a lethal protein. Likewise, the
cleavage of proapoptotic ligand BID by caspase-8 allows
the translocation of BID from cytoplasm to the mitochondria and further amplification of the apoptotic signals
through destruction of mitochondria homeostasis (71,
FIG. 8. Converging pathways of apoptosis
are shown. Apoptosis can be initiated by diverse
mechanisms. The withdrawal of tissue-specific
survival factors could regulate anti- and proapoptotic Bcl-2 proteins and trigger caspase activation by dissociating Apaf-1 from antiapoptotic Bcl-2 proteins and by increasing the
release of cytochrome c from the mitochondria.
As the result of genome instability, intracellular
death signals could increase the levels of p53,
which, in turn, enhance apoptosis by increasing
the expression of proapoptotic Bcl-2 proteins
(such as Bax), leading to caspase activation. In
addition, extracellular death ligands could bind
to death receptors and activate death effector
proteins through death domain adapters, also
leading to caspase activation and apoptosis.
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changes characteristic of apoptosis (69). Although effector caspases are perhaps activated in every apoptotic cell,
only selective initiator caspases are activated by specific
signaling pathways in a given dying cell after binding to
pathway-specific adaptor proteins.
It appears that death caspases are present in all cells,
ready to be activated by upstream apoptotic signals. The
interaction between initiator caspases and upstream signaling molecules is mediated through their NH2-terminal
regulatory sequences such as death effector domain
(DED) and CARD domains (13, 81, 131, 211, 219). The
NH2-terminal CARD domain of procaspase-9 is important
for its association with Apaf-1 in the apoptosome complex
and for its subsequent conversion to the active enzyme
(Fig. 8). Similarly, the NH2-terminal DED domain of the
caspase-8 proprotein binds to DED domain-containing
adaptor proteins, which, in turn, interact with upstream
death domain-containing receptors including Fas antigen,
TNF receptor I, DR3, DR4, DR5, and DR6 (32, 163, 176,
207, 212). Activation of these receptors after ligand occupancy leads to the formation of a death-inducing signaling
complex (DISC) containing oligomers of receptors, receptor-associated DED domain-containing adapters (such as
TRADD and FADD), and procaspase-8, followed by the
autoactivation of caspase-8 (123, 157). These diverse
structure-functional characteristics of procaspases suggest that caspases play tissue-, signal-, and pathway-specific roles.
The effector caspases cleave cellular proteins to disable
critical homeostatic and repair processes essential for cell
survival; they also disassemble key structural components
including cytoskeleton and nuclear DNA (69). The DNA
repair enzyme poly(ADP)-ribose polymerase (PARP) is one
of the first cellular substrates shown to be cleaved by
caspase-3 during apoptosis (46, 72, 129, 167). Subsequently,
a variety of caspase substrates important for diverse cellular
functions were identified. These include proteins that are
essential for 1) maintenance of cellular structures [␣-fodrin
605
606
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death, and the caspase-8 null embryos exhibit defects
in heart muscle development (238).
In contrast, mice deficient in caspase-1, caspase-2, or
caspase-11 develop normally and show limited functional
impairments in specific organs (12, 21, 135, 136, 246). In
caspase-2 null mice, the major apoptosis defect was found
in the developing motor neurons (12). In caspase-1 and
caspase-11 null mice, the production of interleukin-1␣ and
-1␤ was impaired; however, no aberrant apoptosis could
be observed (21, 135, 136, 246). These findings are consistent with the hypothesis that apoptosis in most tissues
is regulated by a coordinated array of tissue-specific factors at the decision as well as execution phases of apoptosis. Although the important role of the caspase cascade in the execution phase of apoptosis is clearly
documented, it is important to note that there are multiple
examples of apoptosis induction without the apparent
involvement of known caspases (1, 30, 183).
After an explosion of literature on apoptosis regulation in recent years, converging pathways of apoptosis
regulation have become apparent (Fig. 8). In addition to
the regulation of apoptosis by extracellular survival signals mediated through anti- and proapoptotic Bcl-2 proteins, intracellular death signals including genomic instability and the excessive accumulation of free oxygen
radicals could also trigger apoptosis. The tumor suppressor protein p53 is a “gatekeeper” of genome integrity (124,
153, 190) and increases in p53 levels as the result of cell
cycle imbalance or DNA damage induce the synthesis of
Bax, leading to apoptosis (160, 192). In addition, mitochondrial dysfunction as the result of the collapse of the
mitochondrial inner transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions and outflow of matrix calcium and glutathione could also lead to cytochrome c release and
activation of the apoptosis cascade (190, 192). These direct connections between genomic instability, cellular aberration, and different steps of apoptosis could have
evolved to ensure the proper removal of cells that are
damaged or harmful to the whole organism.
In addition to extracellular survival factors, apoptosis in many cells is also regulated by extracellular death
factors. These death factors are members of the TNF
family of cytokines and include Fas ligand (also known as
Apo-1/CD95 ligand), TNF-␣, TRAIL/Apo-2 ligand, and
TWEAK. These membrane-bound or soluble proteins induce apoptosis in target cells by binding to specific members of the TNF receptor superfamily called death receptors (31, 53, 82, 143, 177, 205, 206, 212). The stimulation of
death receptors by their respective ligands induces the
formation of a death-inducing signaling complex (DISC)
(123, 212). The intracellular domain of the death receptor
binds to the adaptor molecules FADD, which in turn
recruits catalytically inactive procaspase-8 to allow their
intermolecular autocatalytic cleavage, thus releasing ac-
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133, 142). Furthermore, caspase activity has been directly
linked to the formation of ladderlike DNA fragments in
apoptotic cells. During apoptosis, the cleavage of an inhibitor of nuclease (DFF45/ICAD) relieved the inhibition
of nuclease DFF40/caspase-activated deoxyribonuclease
(CAD), thus allowing DFF40/CAD to induce nuclear DNA
fragmentation (101, 140, 141, 159, 228).
Two additional families of proteins important for the
regulation of caspase activity have been identified. One
group, including NIAP, cIAP1, cIAP2, x-IAP, and survivin,
acts as substrate inhibitors of caspases to block the activation of different effector caspases (caspase-3 and
caspase-7) (50 –52, 57, 58). Overexpression of these inhibitor proteins leads to the suppression of apoptosis triggered by diverse proapoptotic signals in both mammalian
and insect cells. The other group of caspase regulators
includes FLICE inhibitory protein (FLIP) and death effector protein in testis (DEFT) (13, 102, 103, 113, 131, 218,
219, 229, 233). These proteins regulate apoptosis through
direct interaction with initiator caspases such as procaspase-8 and procaspase-10 through their death effector
domains (103, 168, 212). Caspase-9 has also been shown
to be regulated by mitogen-regulated PKB/Akt kinase
through phosphorylation (22). Because the phosphorylated form of caspase-9 is catalytically inactive, these
findings suggest that upstream survival factors could promote cell survival through direct phosphorylation and
inactivation of caspases. It is now becoming clear that the
intricate mechanisms involved in caspase regulation are
not only important to ensure against accidental activation
of these death enzymes but also for coordinating signals
triggered by diverse extracellular survival and apoptotic
factors. Although the role of these proteins in gonads has
not been specifically investigated, DEFT, FLIP, caspase-9,
and inhibitor of apoptosis proteins (IAP) are expressed at
high levels in gonads (50, 52, 107, 131, 199, 226, 229, 233),
suggesting that these caspase regulators could play important roles in the regulation ovarian cell death as well.
Of interest, recent studies showed that apoptosis of granulosa cells from preantral and early antral follicles is
associated with reduced protein levels of IAP2 and xIAP
while gonadotropin treatment increases IAP2 and xIAP
content (134).
Mutant mice with deletion of caspase genes have
been generated. Mutation of caspase-9 results in embryonic lethality and defective brain development
(126). In addition, fibroblasts isolated from caspase-9
null mice showed defects in cytochrome c-mediated
cleavage of procaspase-3. Likewise, mutation of
caspase-3 causes perinatal death and all mutant mice
die at 1–3 wk of age (127). The major morphological
changes were found in the brain and include a variety
of hyperplasia and disorganized cell deployment. Targeted disruption of caspase-8 also leads to embryonic
Volume 80
April 2000
BCL-2 PROTEINS IN APOPTOSIS
VIII. CONCLUSION
It is clear that active cell suicide plays an important
role in the elimination of superfluous, damaged, or malignant cells to allow the general well-being of the whole
organism. In a normal organism, cells are alive because
they are constantly stimulated by diverse survival factors.
If survival signals are removed, apoptosis is initiated to
ensure that superfluous cells would die. Alternatively,
death signals could also actively target unwanted cells to
undergo apoptosis.
The signaling mechanism for apoptosis is modulated
by a diverse array of intracellular regulators. Studies using the yeast two-hybrid system provide an experimental
paradigm to identify tissue-specific Bcl-2 proteins and
their binding proteins in the decision step of apoptosis. In
the ovary, the Mcl-1 and other antiapoptotic proteins are
believed to heterodimerize with proapoptotic Bcl-2 proteins (BAD, BOD, Bok, and Bax), and the ratio of these
protein pairs could effect downstream events including
the release of cytochrome c and binding to ovarian Apaf-1
in the regulation of caspase activation. The proapoptotic
ligands, BAD and BOD, likely interact with upstream binding proteins to mediate the action of survival factors. In
addition, mechanisms both dependent and independent of
heterodimerization mediated by Bok variants may be important for cell death regulation for reproductive tissues
that exhibit constant or cyclic cell turnover during the
reproductive life. Further characterization of these Bcl-2
proteins and the recently discovered DIVA/Boo would
allow a better understanding of the intracellular decision
step of apoptosis, particularly for hormone-regulated cell
death.
In addition to the maintenance of cell survival by
diverse survival factors mediated through the Bcl-2 decision step of apoptosis, death ligands also enhance apoptosis by acting through death receptors. In addition,
corpses for apoptotic cells are removed by phagocytosis
mediated by conserved cell surface markers. Because
aberrant apoptosis regulation is the basis of tumorigenesis and various degenerative diseases, these pathological
conditions can be corrected after pharmacological manipulation of apoptosis. Tumor cells can be induced to undergo apoptosis, whereas inhibitors of caspases could
reduce myocardial reperfusion injury in vivo by attenuating cardiomyocyte apoptosis within the ischemic area at
risk. Future studies on specific Bcl-2 proteins and their
binding proteins in individual tissues could allow manipulation of cell fate in a tissue-specific manner. Elucidation
of the regulatory mechanisms regarding follicular cell
death may provide new therapeutic modalities for ovarian
diseases including premature ovarian failure and polycystic ovarian syndrome.
This work was supported by National Institute of Child
Health and Human Development Grant HD-31566.
Address for reprint requests and other correspondence: A.
J. W. Hsueh, Div. of Reproductive Biology, Dept. of Gynecology
and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305-5317.
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