death executioner Bcl-2 homologue
Bcl-2 and Bcl-xL promote cell survival The Bcl-2 family of proteins regulates apoptosis: some members antagonize cell death while others facilitate it. It has recently been demonstrated that Bcl-2 not only inhibits apoptosis but also restrains cell cycle entry. These two functions can be genetically dissociated. Mutation of a tyrosine
residue within the conserved N-terminal BH4 region has no effect on the ability of Bcl-2 or its closest
homologs to enhance cell survival and does not prevent heterodimerization with death-enhancing family
members Bax, Bak, Bad and Bik. Neither does this mutation override the growth-inhibitory effect of
p53. However, upon stimulation with cytokine or serum, starved quiescent cells expressing the mutant
proteins re-enter the cell cycle much faster than those expressing comparable levels of wild-type
proteins. When wild-type and Y28 mutant Bcl-2 are co-expressed, the mutant is dominant.
Although R-Ras p23 has been reported to bind to Bcl-2, no interaction is detectable in transfected
cells and R-Ras p23 does not interfere with the ability of Bcl-2 to inhibit apoptosis or cell cycle entry.
These observations provide evidence that the anti-apoptotic function of Bcl-2 is mechanistically distinct
from its inhibitory influence on cell cycle entry (Huang, 1997).
Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation; its presence in tumor
cells would be expected to indicate poor prognosis. However, Bcl-2-expressing tumors are often
low-grade and highly responsive to therapy. To investigate this apparent paradox,
the responses of Burkitt lymphoma (BL) cells were examined in vitro to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 promotes BL cell survival following irradiation.
However, a significant proportion of Bcl-2-rescued cells subsequently undergo apoptosis after an
extended period in culture. In different BL lines, Bcl-2 either promotes or
inhibits long-term proliferative activity following gamma-irradiation. This differential regulation of
proliferation correlates both with differential effects of Bcl-2 on the cell cycle and with differences in
p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) have
arrested in G1, whereas those with a mutant allele (wt/mu) are arrested in all phases of the cell
cycle. The proportion of Bcl-2-rescued cells that subsequently undergo apoptosis is reduced by
ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation
reduces both G1-arrest and apoptosis in parallel. These results indicate that while Bcl-2 can delay
apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby
promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may
depend on p53 status and require additional death-repressing or growth-promoting signals (Milner, 1997).
Bcl-2 plays a key role in regulating cell survival in the immune and nervous systems. Mice lacking the
bcl-2 gene have markedly reduced numbers of B and T cells as a result of increased apoptosis,
whereas mice with a transgene causing high levels of Bcl-2 expression in the immune system show
extended survival of B and T cells. Overexpression of Bcl-2 in cultured neurons prevents their
death following neurotrophin deprivation; mice with a bcl-2 transgene under the control of a
neuron-specific enolase promoter have increased numbers of neurons in several regions. Cultured
neurons expressing antisense bcl-2 RNA have an attenuated survival response to neurotrophins,
and neurons of postnatal bcl-2-deficient mice die more rapidly following NGF deprivation in vitro
and are present in reduced numbers in vivo. Bcl-2 also plays a role in
regulating axonal growth rates in embryonic neurons. Sensory neurons from the trigeminal ganglia of
bcl-2-deficient mouse embryos, removed from the embryo on embryonic day 11 or 12, extend axons
more slowly in vitro than do neurons from wild-type embryos of the same age. Serial measurements of
axonal length in the same neurons reveal that there are marked differences in axonal growth rate
between bcl-2-deficient and wild-type neurons, irrespective of whether the neurons are grown with
nerve growth factor, brain-derived neurotrophic factor or neurotrophin-3. Because there is no
significant difference in the numbers of wild-type and bcl-2-deficient neurons surviving with each
neurotrophin at this early stage of development, the effect of Bcl-2 on axonal growth rate is not a
consequence of its well documented role in preventing apoptosis (Hilton, 1997).
Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the
activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL
overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To
address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were
studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or
transfected with both Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibits
apoptosis induced by either anti-Fas or TNF-alpha. Bcl-xL prevents the change in
cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that
recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was
detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In
MCF7/FB cells, caspase-8 is processed normally, while processing of the downstream caspase-7
is markedly attenuated. Apoptosis induced by direct microinjection of recombinant, active
caspase-8 is completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an
anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8
signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step (Srinivasan, 1998).
Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2
family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a
step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase
cascade has remained unclear. Purified active caspase-3 (CPP32/Yama/apopain)
and caspase-1 (ICE) induce apoptosis when microinjected into the cytoplasm of cells; the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are
overexpressed more than sufficiently to prevent Fas-mediated and overexpressed
procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase
cascade (Yasuhara, 1997).
The gene MRIT possesses overall
sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex
of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases.
However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus
sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast
two-hybrid assays, it has been demonstrated that MRIT associates with caspases possessing large and small
prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition,
MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus,
MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family
member (Han, 1997).
Nuclear factor (NF) kappaB is a ubiquitously expressed transcription factor whose function is regulated by the cytoplasmic inhibitor protein, IkappaBalpha. IkappaBalpha activity is diminished in ventricular myocytes expressing Bcl-2. In view of the growing evidence that the conserved N-terminal BH4 domain of Bcl-2 plays a critical
role in suppressing apoptosis, it was ascertained whether this region accounts for the underlying effects of Bcl-2 on IkappaBalpha activity. Transfection of human
embryonic 293 cells with full length Bcl-2 results in a significant 1.9-fold reduction in IkappaBalpha activity with a concomitant increase in DNA
binding and 3.4-fold increase in NFkappaB-dependent gene transcription compared with vector transfected control cells. In contrast, no significant
change in IkappaBalpha activity is detected with either a BH4 domain deletion mutant (residues 10-30) or BH4 domain point substitution mutants, I14G, V15G,
Y18G, K22G, and L23G. However, a small 0.60-fold decrease in IkappaBalpha activity is noted with the BH4 mutant I19G, suggesting
that this residue may not be critical for IkappaBalpha regulation. Furthermore, adenovirus-mediated delivery of an IkappaBalpha mutant to prevent NFkappaB
activation impairs the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha plus cycloheximide in ventricular myocytes. The data provide the first evidence
for the regulation of IkappaBalpha by Bcl-2 through a mechanism that requires the conserved BH4 domain that links Bcl-2 to the NFkappaB signaling pathway for
suppression of apoptosis (de Moissac, 1999).
Apoptosis is triggered when proapoptotic members of the Bcl-2 protein family bearing only the BH3 association domain bind to Bcl-2 or its homologs and block their antiapoptotic activity. To test whether loss of the BH3-only protein Bim could prevent the cellular attrition caused by Bcl-2 deficiency, mice were generated lacking both genes. Mice without Bcl-2 have a fragile lymphoid system, become runted, turn gray, and succumb to polycystic kidney disease. Concomitant absence of Bim prevents all these disorders. Indeed, loss of even one bim allele restores normal kidney development, growth, and health. These results demonstrate that Bim levels set the threshold for initiation of apoptosis in several tissues and suggest that degenerative diseases might be alleviated by blocking BH3-only proteins (Bouillet, 2001).
The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Bcl-2 is shown to interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases (Lin, 2004).
Nur77 (TR3 or NGFI-B), an orphan member of the steroid/thyroid/retinoid nuclear receptor superfamily, plays roles in regulating growth and apoptosis. Nur77 expression is rapidly induced during apoptosis in immature thymocytes and T cell hybridomas, and cancer cells of lung, ovary, colon, and stomach. High levels of Nor1, a Nur77-family member, are associated with favorable responses to several chemotherapeutic agents in patients with diffuse large B-cell lymphoma (Lin, 2004 and references therein).
A paradigm in cellular apoptosis has been discovered, wherein Nur77 translocates from the nucleus to the cytoplasm, targeting to mitochondria and inducing cyt c release. Nur77 mitochondrial-targeting occurs during apoptosis of different types of cancer cells. Sindbis virus-induced apoptosis also involves Nur77 translocation to mitochondria. How Nur77 targets mitochondria and induces apoptosis however has been unclear.
In this study, the mechanism by which Nur77 targets mitochondria and induces apoptosis was been investigated. The results demonstrate that Nur77 interacts with Bcl-2 through its ligand binding domain (LBD) and that the interaction is required for Nur77 mitochondrial targeting and Nur77-dependent apoptosis. Interestingly, Nur77 binds to the Bcl-2 N-terminal loop region, located between its BH4 and BH3 domains, resulting in a conformational change in Bcl-2, which converts it from a protector to a killer protein (Lin, 2004).
The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. A component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL. Deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. These results suggest that the regulation of Bcl-xL deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents (Deverman, 2002).
In addition to its tumor-suppressor activity, Rb is a potent antiapoptotic protein -- loss of Rb in normal fibroblasts confers sensitivity to DNA-damaging agents, and reintroduction of Rb into Rb null tumors confers resistance to these agents. Hence, it was reasoned that Rb must suppress proapoptotic signals. Indeed, Rb suppresses the inactivating deamidation of Bcl-xL, and these findings indicate that the antiapoptotic activity of Rb is dependent upon the ability of Rb to suppress Bcl-xL deamidation. Finally, the data suggest that the inactivation of Rb increases the susceptibility of tumor cells to DNA-damaging agents in part because inactivation of Rb is permissive for Bcl-xL deamidation (Deverman, 2002).
Proapoptotic Bcl-2 family members The NSM cells of the nematode Caenorhabditis elegans differentiate
into serotonergic neurons, while their sisters, the NSM sister cells, undergo
programmed cell death during embryogenesis. The programmed death of the NSM
sister cells is dependent on the cell-death activator EGL-1, a BH3-only
protein required for programmed cell death in C. elegans, and can be
prevented by a gain-of-function (gf) mutation in the cell-death specification
gene ces-1, which encodes a Snail-like DNA-binding protein. The genes hlh-2 and hlh-3, which encode a
Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are
required to kill the NSM sister cells. A heterodimer composed of HLH-2 and
HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory
region of the egl-1 locus in vitro that is required for the death of
the NSM sister cells in vivo. Hence, it is proposed that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus (Thellmann, 2003).
Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been
established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant
member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells
phosphorylated BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerized with BCL-X(L) at
membrane sites to promote cell death. Phosphorylated BAD is sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further
enhances BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating
this checkpoint for apoptosis (Zha, 1996).
Mtd, a novel regulator of apoptosis, has been cloned and characterized. Sequence analysis reveals that Mtd is a member of the Bcl-2 family of proteins
containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA is predominantly detected in
the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA is detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promotes
the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2
family, Mtd does not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd is not inhibited by Bcl-2 or
Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retains its ability to promote apoptosis, further indicating
that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis is not blocked by broad range synthetic caspase
inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of
heterodimerization with survival-promoting Bcl-2 and Bcl-XL (Inohara, 1998).
In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell
fate. From a rat ovarian fusion cDNA library, a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok) has been isolated. Bok has conserved Bcl-2 homology
(BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2
proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic
members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In
addition, negligible interaction is found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induces apoptosis that is
blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok is also suppressed following coexpression with Mcl-1 and BHRF1
but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok is highly
expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that undergoes apoptosis during follicle
atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of
apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover (Hsu, 1997).
The proapoptotic molecule BAX is required for death of sympathetic and motor neurons in the setting
of trophic factor deprivation. Adult Bax-/- mice have more motor neurons than do their wild-type counterparts. These findings raise the possibility that BAX regulates naturally occurring cell death during development in many neuronal populations. To test this idea, apoptosis was assessed in several well-studied neural systems during embryonic and early postnatal development in Bax-/- mice. Remarkably, naturally occurring cell death is virtually eliminated in most peripheral ganglia, in motor pools in the spinal cord, and in the trigeminal brainstem nuclear complex between embryonic day 11.5 (E11.5) and postnatal day 1 (PN1). Reduction, although not elimination, of cell death is found throughout the developing cerebellum, in some layers of the retina, and in the hippocampus. Saving of cells was verified by axon counts of dorsal and ventral roots, as well as facial and optic nerves that reveal a 24%-35% increase in axon numbers. Interestingly, many of the supernumerary axons have very small cross-sectional areas, suggesting that the associated neurons are not normal. It is concluded that BAX is a critical mediator of naturally occurring death of peripheral and CNS neurons during embryonic life. However, rescue from naturally occurring cell death does not imply that the neurons will develop normal functional capabilities (White, 1998).
Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators
of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis.
However, the precise mechanism by which these two families interact to regulate cell death is not yet
known. Overexpression of the Bcl-2 family member Bax induces
apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that
cleave the DEVD tetrapeptide. This apoptotic cell death is suppressed by the viral proteins CrmA
and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and
zVAD-fmk. The Bax-induced apoptosis of COS-7 cells is suppressed by Bcl-xL
and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevent etoposide-induced apoptosis in COS-7
cells. Bcl-xL inhibits the activation of caspase-3-like proteases accompanying
Bax-induced COS-7 cell death, but Bcl-2 does not. These results indicate that the caspase activation is
essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the
Bax-induced caspase activation and apoptosis in COS-7 cells can be differentially regulated. These
results also suggest that Bcl-2 family proteins function upstream of caspase activation and control
apoptosis through the regulation of caspase activity (Kitanaka, 1997).
Expression of the pro-apoptotic molecule BAX has been shown to induce cell death. While BAX forms both homo- and
heterodimers, questions remain concerning its native conformation in vivo and which moiety is functionally active. A physiologic death stimulus, the withdrawal of interleukin-3 (IL-3), is shown to result in the translocation of
monomeric BAX from the cytosol to the mitochondria where it can be cross-linked as a BAX homodimer. In contrast,
cells protected by BCL-2 demonstrate a block in this process: BAX does not redistribute or homodimerize in
response to a death signal. To test the functional consequence of BAX dimerization, a chimeric
FKBP-BAX molecule was expressed. Enforced dimerization of FKBP-BAX by the bivalent ligand FK1012 results in its translocation
to mitochondria and induces apoptosis. Caspases are activated yet caspase inhibitors do not block death; cytochrome c
is not released detectably despite the induction of mitochondrial dysfunction. Moreover, enforced dimerization of
BAX overrides the protection by BCL-XL and IL-3 and kills cells. These data support a model in which a death signal
results in the activation of BAX. This conformational change in BAX manifests in its translocation, mitochondrial
membrane insertion and homodimerization, and a program of mitochondrial dysfunction that results in cell death (Gross, 1998).
Following exposure of cells to stimuli that trigger programmed cell death (apoptosis), cytochrome c is
rapidly released from mitochondria into the cytoplasm, where it activates proteolytic molecules known
as caspases that specifically cleave the amino-acid sequence DEVD and are crucial for the execution
of apoptosis. The protein Bcl-2 interferes with this activation of caspases by preventing the release of
cytochrome c. These molecular interactions have been studied during apoptosis induced by the protein Bax, a pro-apoptotic homolog of Bcl-2. In cells transiently transfected with bax, Bax
localizes to mitochondria and induces the release of cytochrome c, activation of caspase-3, membrane blebbing, nuclear fragmentation, and cell death. Caspase inhibitors do not affect Bax-induced
cytochrome c release but block caspase-3 activation and nuclear fragmentation. Unexpectedly, Bcl-2
also fails to prevent Bax-induced cytochrome c release, although it co-localizes with Bax to
mitochondria. Cells overexpressing both Bcl-2 and Bax show no signs of caspase activation and
survive with significant amounts of cytochrome c in the cytoplasm. These findings indicate that Bcl-2
can interfere with Bax killing, both downstream and independent of cytochrome c release. This activity mediated by Bcl-2 may be due to an interaction of Bcl-2 with the cytochrome c receptor Apaf-1/CED-4 (Drosophila homolog: Apaf-1-related-killer), a recently identified mediator of caspase-3 activation by cytochrome c (Rosse, 1998).
'BH3 domain only' members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction.
Tumor necrosis factor alpha (TNFalpha) treatment induces a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15
and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targets normal mitochondria and
releases cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of
hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also
required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induces BID cleavage and p15 BID becomes an integral
mitochondrial membrane protein. However, BCL-XL/BCL-2 prevents the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpires
and cells die nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not
be required for cell death (Gross, 1999b).
DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells
were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated
with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. DP5 is induced during neuronal
apoptosis in cultured sympathetic neurons. DP5 gene expression and the specific interaction of DP5 with Bcl-xl was analyzed during neuronal death induced by
amyloid-beta protein (A beta). DP5 mRNA is induced 6 h after treatment with A beta in cultured rat cortical neurons. The protein encoded by DP5 mRNA
shows a specific interaction with Bcl-xl. Induction of DP5 gene expression is blocked by nifedipine, an inhibitor of L-type voltage-dependent calcium channels,
and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the
increase in cytosolic calcium concentration caused by A beta. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A
beta, and its binding can impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play
significant roles in neuronal degeneration following exposure to A beta (Imaizumi, 1999).
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an
apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which
Bax, a proapoptotic Bcl-2 family member, has been deleted, do not undergo apoptosis in 5 mM
potassium, yet do undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM
NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells
decrease glucose uptake to <20% of control. Protein synthesis also decreases rapidly in both
wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM
potassium. Both wild-type and Bax -/- neurons increase mRNA levels of c-jun, and caspase 3
(CPP32) and increase phosphorylation of the transactivation domain of c-Jun after K+ deprivation.
Wild-type granule cells in 5 mM K+ increase cleavage of DEVD-aminomethylcoumarin
(DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule
cells do not cleave DEVD-AMC. These results place BAX downstream of metabolic changes,
changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of
caspases; they indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type
cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, block cleavage of DEVD-AMC and block an increase in DNA degradation.
However, these inhibitors have only a marginal effect on the prevention of cell death, suggesting a
caspase-independent death pathway downstream of BAX in cerebellar granule cells (Miller, 1997).
BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD
prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors
of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase,
was activated by IL-3 in a PI 3-kinase-dependent manner. Only active forms of Akt are
found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in
response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt
pathway (del Peso, 1997).
Growth factor deprivation is a physiological mechanism to regulate cell death. An interleukin-2 (IL-2)-dependent murine T-cell line was used to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, it was found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with the appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras (Ayllon, 2000).
BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. The function of BAK has been evaluated in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability (Fannjiang, 2003).
Bax (Bcl2-associated X protein) is an apoptosis-inducing protein
that during normal development participates in cell death and also participates in
various diseases. Bax resides in an inactive state in the cytosol of
many cells. In response to death stimuli, Bax protein undergoes
conformational changes that expose membrane-targeting
domains, resulting in its translocation to mitochondrial membranes,
where Bax inserts and causes release of cytochrome c and
other apoptogenic proteins. It is unknown what controls conversion
of Bax from the inactive to active conformation. This study shows that Bax interacts with humanin (HN), an anti-apoptotic
peptide of 24 amino acids encoded in mammalian genomes. HN
prevents the translocation of Bax from cytosol to mitochondria.
Conversely, reducing HN expression by small interfering RNAs
sensitizes cells to Bax and increases Bax translocation to membranes.
HN peptides also block Bax association with isolated
mitochondria, and suppress cytochrome c release in vitro. Notably,
the mitochondrial genome contains an identical open
reading frame, and the mitochondrial version of HN can also
bind and suppress Bax. It is speculated therefore that HN arose
from mitochondria and transferred to the nuclear genome,
providing a mechanism for protecting these organelles from Bax (Guo, 2003).
The tumor suppressor p53 exerts its versatile function to maintain the genomic integrity of a cell, and the life of cancerous cells with DNA damage is often terminated by induction of apoptosis. The role of Noxa, one of the transcriptional targets of p53 that encodes a proapoptotic protein of the Bcl-2 family, was studied by the gene-targeting approach. Mouse embryonic fibroblasts deficient in Noxa [Noxa-/- mouse embryonic fibroblasts (MEFs)] show notable resistance to oncogene-dependent apoptosis in response to DNA damage, which is further increased by introducing an additional null zygosity for Bax. These MEFs also show increased sensitivity to oncogene-induced cell transformation in vitro. Furthermore, Noxa is also involved in the oncogene-independent gradual apoptosis induced by severe genotoxic stresses, under which p53 activates both survival and apoptotic pathways through induction of p21WAF1/Cip1 and Noxa, respectively. Noxa-/- mice show resistance to X-ray irradiation-induced gastrointestinal death, accompanied with impaired apoptosis of the epithelial cells of small intestinal crypts, indicating the contribution of Noxa to the p53 response in vivo (Shibue, 2003).
The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete by means of dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. Interactive cloning was used to identify Bid , which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis reveals that an intact BH3 domain of BID is required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerizes with BCL-2 fails to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retains death promoting activity interacts with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX (Wang, 1996).
Bak has been shown to both promote apoptosis and to inhibit cell death; in contrast, two other members of
the Bcl-2 family of proteins, Bcl-XL and Bcl-2 delay apoptosis induced by various stimuli, including
chemotherapeutic agents. Clones with stable expression of Bak wild-type (wt) and Bak
with its BH3 (delta78-86) domain deleted (deltaBH3) were generated in FL5.12 cells or FL5.12 cells expressing either Bcl-XL or Bcl-2 to determine if Bak could accelerate apoptosis and antagonize the death repressor activity of Bcl-XL and Bcl-2 during chemotherapy-induced apoptosis. Bak accelerates
cell death in FL5.12 cells treated with either etoposide, fluorouracil or taxol. In FL5.12 cells expressing
Bcl-XL and Bak wt or Bak deltaBH3, both Bak wt and Bak deltaBH3 are able to antagonize the
protective effect of Bcl-XL when treated with etoposide or fluorouracil. Both Bak wt and Bak deltaBH3
are also able to abrogate the protective effect of Bcl-2 in cells expressing Bcl-2 and Bak wt or Bak
deltaBH3 when challenged by etoposide or fluorouracil. Immunoprecipitation studies reveal that
deletion of BH3 disrupts heterodimerization between Bak and Bcl-XL and that both Bak wt and Bak
deltaBH3 fail to interact with Bcl-2. These results demonstrate that Bak does not require its BH3
domain to promote apoptosis in stably transfected cells. Bak can accelerate
chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2 (Simonian, 1997).
Bcl-2 and close homologs such as Bcl-xL promote cell survival, while other relatives, such as Bax,
antagonize this function. Since only the pro-survival family members possess a conserved N-terminal
region (denoted BH4), the role of this amphipathic helix has been explored for its survival function and for
interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the
nematode Caenorhabditis elegans. The BH4 of Bcl-2 can be replaced by BH4 of Bcl-x without perturbing
function but this is not the case when it is replaced by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity is
reduced by substitutions in two of the ten conserved BH4 residues. Deletion of BH4 renders Bcl-2 (and
Bcl-xL) inactive but does not impair either Bcl-2 homodimerization or its ability to bind to Bax or five other
pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not
sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 loses the ability both to bind
CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain
of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent
apoptosis (Huang, 1998).
The Bcl-2 family proteins comprise pro-apoptotic as well as anti-apoptotic members. Heterodimerization between members of the Bcl-2 family proteins is a key
event in the regulation of apoptosis. Bcl-2 protein is selectively cleaved by active caspase-3-like proteases in CTLL-2 cell apoptosis in
response to interleukin-2 deprivation. Structural and functional analyses of the cleaved fragment revealed that the NH2-terminal region of Bcl-2 (1-34 amid acids)
is required for its anti-apoptotic activity and heterodimerization with pro-apoptotic Bax protein. Site-directed mutagenesis of the NH2-terminal region showed that
substitutions of hydrophobic residues of BH4 domain results in the loss of ability to form a heterodimer with Bax. Particularly instructive was that the V15E mutant
of Bcl-2, which completely lost the ability to form a heterodimer with Bax, fails to inhibit Bax- and staurosporine-induced apoptosis. These results suggest that the
BH4 domain of Bcl-2 is critical for its heterodimerization with Bax and for exhibiting anti-apoptotic activity. Therefore, agents interferring with the critical residues of
the BH4 domain may provide a new strategy in cancer therapy by impairing Bcl-2 function (Hirotani, 1999).
Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and
cell death receptors. Mitochondrial events are required for apoptosis
induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. The Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on
TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the
TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. These results show for the first time that
Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis (Deng, 2002).
The signaling events leading to apoptosis can be divided into two distinct pathways, involving either
mitochondria or death receptors. In the mitochondria pathway, death signals lead to changes in mitochondrial membrane permeability
and the subsequent release of pro-apoptotic factors involved in various aspects of apoptosis. The released factors include cytochrome c (cyto c), apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac/DIABLO), and
endonuclease G. Cytosolic cyto c forms an essential part of the apoptosis complex 'apoptosome,' which is composed of cyto c, Apaf-1, and procaspase-9. Formation of the apoptosome leads to the activation of caspase-9, which then processes
and activates other caspases to orchestrate the biochemical execution of cells. Smac/DIABLO is also released from the mitochondria
along with cyto c during apoptosis, and it functions to promote caspase activation by inhibiting IAP (inhibitor of apoptosis) family proteins (Deng, 2002).
The IAP family proteins negatively regulate apoptosis by inhibiting caspase activity directly. Six human IAPs have been discovered. They
regulate apoptosis by preventing the action of the central execution phase of apoptosis through direct inhibition of the effector caspase-3
and/or caspase-7. In addition, they prevent initiation of the intrinsic caspase activation
cascade by directly inhibiting the apical caspase-9. Structural and biochemical dissection of XIAP, a widely expressed IAP member,
reveals that the conserved BIR domains of XIAP mediate both its inhibitory activity on caspases and the protein-protein interaction with
Smac/DIABLO. Binding of Smac/DIABLO to XIAP antagonizes caspase-XIAP interaction, thereby promoting apoptosis. Recent studies have shown that XIAP is highly expressed in most human cancer cells and that high levels of
XIAP confer tumor resistance to chemotherapy or irradiation (Deng, 2002).
The key regulatory proteins of mitochondria-mediated apoptotosis are the Bcl-2 family of proteins, which can either promote cell survival,
as do Bcl-2 and Bcl-xl, or induce cell death, as do Bax and Bak. Bcl-2 and Bcl-xl appear to directly or indirectly preserve the integrity of
the outer mitochondrial membrane, thus preventing cyto c release and mitochondria-mediated cell death initiation, whereas the
pro-apoptotic proteins Bax and Bak promote cyto c release from mitochondria. Bax has been implicated in
apoptosis in many cell types under various conditions. More recently, studies using Bax-deficient human colon cancer cells have provided
direct evidence that Bax plays a key role in mediating apoptosis induced by certain anti-cancer agents. The Bax
protein exerts at least part of its activity by triggering cyto c release from mitochondria. Bax is in a predominantly cytosolic latent form in
healthy cells and translocates to mitochondria after death signal stimulation. Accumulating evidence suggests that
Bax translocation is required for its pro-apoptotic function and that regulation of Bax's association with the mitochondrial membrane
represents a critical step in the transduction of apoptotic signals (Deng, 2002).
In the death receptor pathway, the apoptotic events are initiated by engaging the tumor necrosis factor (TNF)-family receptors, including
TNFR1, Fas, DR-3, DR-4, and DR-5. Upon ligand binding or when overexpressed in cells, TNF receptor family members aggregate,
resulting in the recruitment of an adapter protein called FADD. The receptor-FADD complex then recruits procaspase-8. This allows
proteolytic processing and activation of the receptor-associated procaspase-8, thereby initiating the subsequent cascade of additional
processing and activation of downstream effector caspases (Deng, 2002).
TRAIL/Apo2L (TNF-related apoptosis-inducing ligand TRAIL or Apo2 ligand) is an apoptosis-inducing member of the TNF gene
superfamily. Unlike TNF-alpha and FasL, TRAIL appears to specifically kill
transformed and cancer cells while leaving normal cells intact. Preclinical experiments in mice and
nonhuman primates have shown that administration of TRAIL suppresses tumor growth without apparent systematic cytotoxicity. Therefore, TRAIL represents a promising anti-cancer agent. TRAIL interacts
with four cellular receptors that form a distinct subgroup within the TNFR superfamily. Most recent experiments have shown that FADD and procaspase-8 associate with the endogenous TRAIL receptors DR4
and DR5. FADD and caspase-8 are required for TRAIL-induced apoptosis. Thus, TRAIL/Apo2L and FasL appear to engage similar pathways to apoptosis (Deng, 2002).
Although the extrinsic pathway (through the death receptors) and the intrinsic pathway (through the mitochondria) for apoptosis are
capable of operating independently, accumulating evidence suggests that a cross-talk between the two pathways exists in cells. The link between death receptor signaling and the mitochondrial pathway comes from the finding that a
BH3-domain-only subfamily protein, Bid, is cleaved by active caspase-8. The truncated Bid (tBid) translocates to mitochondria and
triggers cyto c release. It has been proposed that tBid regulates cyto c release by inducing
the homo-oligomerization of pro-apoptotic family members Bak or Bax. Cells lacking both Bax and Bak, but not cells lacking just one of these components, are completely resistant to tBid-induced cyto c release and apoptosis (Deng, 2002).
Bid appears to link the intrinsic pathway to the cell death receptor-mediated apoptosis. However, the precise mitochondrial events
required for this cross-talk remain unclear. The mechanisms of TRAIL-induced apoptosis and the role of mitochondria in the cell death
receptor pathway also need further investigation. Using human colon cancer cells defective in Bax function, it has been shown that mitochondrial
events are required for TRAIL-induced apoptosis. The reason for this requirement is the presence of negative regulation of caspase cascade by XIAP. Activation of the mitochondrial pathway leads to the release of Smac/DIABLO, which removes
XIAP blockage of caspase activation. These results further show that release of Smac/DIABLO, not cyto c, is the key event mediating the contribution of the mitochondrial pathway to the death receptor-mediated apoptosis (Deng, 2002).
Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. The regulation of Bak was investigated and it was found that, in healthy cells, Bak associates with Mcl-1 (a close relative of Bcl-2) and Bcl-xL but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-xL (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also requires neutralization of Bcl-xL by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-xL and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family (Willis, 2005).
Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. This study demonstrates that an Escherichia coli toxin, MazF, inhibits protein synthesis by cleavage of cellular mRNA and induces apoptosis in mammalian cells. MazF-induced apoptosis requires proapoptotic BAK and its upstream regulator, the proapoptotic BH3-only protein NBK/BIK, but not BIM, PUMA, or NOXA. Interestingly, in response to MazF induction, NBK/BIK activates BAK by displacing it from anti-apoptotic proteins MCL-1 and BCL-XL that sequester BAK. Furthermore, NBK/BIK- or BAK-deficient cells are resistant to cell death induced by pharmacologic inhibition of translation and by virus-mediated shutoff of protein synthesis. Thus, the BH3-only protein NBK/BIK is the apical regulator of a BAK-dependent apoptotic pathway in response to shutoff of protein synthesis that functions to displace BAK from sequestration by MCL1 and BCL-XL. Although NBK/BIK is dispensable for development, it is the BH3-only protein targeted for inactivation by viruses, suggesting that it plays a role in pathogen/toxin response through apoptosis activation (Shimazu, 2007).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D. The Interactive Fly resides on the
death executioner Bcl-2 homologue:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.