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Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, 171 77 Stockholm, Sweden
1Correspondence: Institute of Environmental Medicine, Division of Toxicology, Doktorsringen 16C, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail: bengt.fadeel{at}i mm.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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Key Words: Bcl-2 family • BH3-only • caspases • mitochondria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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. The aspartate-specific proteases termed caspases,
which are homologous to ced-3, are critically involved in the apoptotic
process in mammalian cells and serve to incapacitate specific
substrates, thereby leading to the disassembly of nuclear and
cytoskeletal structures, disabling of cell repair, and tagging of the
apoptotic cell for engulfment (2
, 3)
. Apoptosis may thus
be considered to occur by a thousand (and one) cuts and caspase
activation viewed as the sine qua non of apoptotic cell
death (4)
. Recent studies have emphasized the importance
of mitochondria as sensors and/or executioners of apoptosis
(5)
and the release of several mitochondrial intermembrane
space proteins such as cytochrome c, adenylate kinase-2,
apoptosis-inducing factor
(AIF)2, and heat shock protein (hsp) 60 has been documented to occur during
the apoptotic process (6
7
8
9)
. Members of the Bcl-2 family,
mammalian homologues of ced-9, play key roles in the regulation of cell
death and appear to govern the decision to die at multiple checkpoints,
acting both at the level of mitochondria and at the pre- and
postmitochondrial stages (10
, 11)
. This review will focus
on the regulation of the Bcl-2 family of apoptosis regulators. |
A FAMILY OF DEATH AGONISTS AND ANTAGONISTS |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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. Bcl-2 serves as a powerful antidote to
cell death and may countermand the effect of both caspase-dependent and
independent modes of cell death through manifold independent functions,
which have been the subject of several excellent reviews
(10
11
12
13)
. These functions include the abrogation of
cytochrome c release from mitochondria (14
, 15)
, the regulation of calcium homeostasis (16
, 17)
, the promotion of glutathione sequestration to the nucleus
(18)
, and the modulation of antioxidant pathways
(19)
, and represent examples of the multiple mechanisms
whereby cells are normally insulated against cell death. Particular
interest has focused on the pore-forming capacity of Bcl-2 and its
congeners (20
21
22)
, which may be related to the capacity
of these molecules to regulate mitochondrial events during apoptosis as
well as necrosis. However, the manner in which these functions are
regulated, both at the transcriptional and posttranslational level, has
not previously been elucidated.
Numerous studies indicate that the differential expression of
Bcl-2-like molecules can be regulated at the level of transcription. A
physiological example is provided by human neutrophils, which
constitute an important first line of defense against invading
microorganisms and are extremely short-lived; these cells are also
known to express abundant levels of Bax, yet are completely devoid of
Bcl-2 (23). The preponderance of proapoptotic molecules may in part
explain why these cells so readily undergo apoptosis in the absence of
specific triggers (24
, 25)
. In addition, differentiation
of myeloid leukemic cells transpires with a decrease in Bcl-2
(26)
. An up-regulation of Bcl-2 mRNA in growth
factor-dependent cells by the survival factors interleukin (IL) -2 or
IL-3 has also been observed (27)
. Treatment of some tumor
cells with chemotherapeutic agents may induce a p53-dependent
down-regulation of Bcl-2 and a concomitant up-regulation of Bax
(28
29
30)
. However, the level of expression of death
agonists vs. antagonists does not always correlate with the
susceptibility of tumors to apoptosis (31)
, suggesting
that these regulators of cell death may also be subject to
posttranslational modulation.
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SUBCELLULAR TARGETING OF Bcl-2 FAMILY MEMBERS |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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, 33)
. Similarly, evidence
has been provided that the regulation of apoptotic signals is dependent
on the specific subcellular localization of Bcl-2-like proteins. Bcl-2
was originally reported to be an inner mitochondrial membrane protein
(34)
, and recent studies conducted in rat liver
mitochondria appear to strengthen these conclusions (35)
.
However, studies from other laboratories have demonstrated that Bcl-2
resides predominantly in the outer mitochondrial membrane, the
endoplasmic reticulum, and the nuclear membrane (36
, 37)
.
Though the reason for these discrepancies remains unresolved, it seems
clear that the antiapoptotic effect of Bcl-2 is largely dependent
on its membrane localization (38
, 39)
. On the other hand,
the function of Bcl-2 and Bcl-XL is not solely
due to the prevention of cytochrome c release from
mitochondria, as demonstrated in experiments with microinjected
cytochrome c (40
, 41)
. In addition, Rossé
et al. (42)
have shown that Bcl-2 protects cells from
apoptosis downstream of Bax-induced release of cytochrome c
from mitochondria.
Although Bax expression per se is not lethal to cells, the
translocation of Bax toward mitochondria during apoptosis was shown to
be important for the ability of Bax to trigger cell death
(43)
. Deletion of the carboxy-terminal hydrophobic region
of Bax prevents Bax redistribution and abrogates its
apoptosis-promoting activity, underscoring the importance of
organelle binding for activity of this molecule. Incidentally, the
observation that Bax is cytosolic in healthy cells suggests that the
hydrophobic tail may normally be hidden within the interior of the
protein or bound to other cytosolic factors, indicating that
conformational changes are likely to be involved in the targeting of
Bax to mitochondria (44)
. In addition, enforced
dimerization of Bax results in its translocation to the mitochondria
and the subsequent induction of mitochondrial dysfunction and apoptosis
(45)
. Furthermore, Bcl-2 and Bcl-XL
are present in the mitochondrial outer membrane of resting cells,
whereas the proapoptotic molecules Bax, Bad, and
Bcl-XS are targeted to these organelles during
tumor necrosis factor (TNF) -induced apoptosis (46)
.
Hence, it appears that the cell resets the ratio of mitochondrial
proapoptotic to antiapoptotic protein in response to apoptosis
triggers. Translocation of Bax to the nucleus during the apoptotic
process has also been reported, although the significance of these
findings is presently not well understood (47)
.
The lesson that emerges from the aforementioned studies is that
intracellular movement during conditions of cellular stress is a common
theme in the regulation of apoptosis regulators. In particular,
translocation of proapoptotic family members from the cytosol to the
mitochondria, the `apoptosis headquarters' within the cell, appears
to constitute an important initiating event in apoptosis. An additional
example is provided by the elegant studies conducted by Puthalakath et
al. (48)
, who recently reported that the BH3-only family
member, Bim, is sequestered to the microtubule-associated dynein motor
complex in healthy cells. On triggering of apoptosis, Bim dissociates
from the dynein motor complex and free Bim translocates to Bcl-2 and
serves to neutralize its antiapoptotic function. These findings again
underscore the importance of subcellular targeting of Bcl-2 family
members and implicate BH3-only proteins as sensors of cellular stress
that counteract the protective effect of Bcl-2-like proteins (discussed
below). However, several questions arise from these studies: Is
cleavage involved in the liberation of Bim or do other proteins
displace Bim on triggering of apoptosis? Do additional death-promoting
members of the Bcl-2 family bind to components of the cytoskeleton? Are
chemotherapeutic agents that compromise the integrity of microtubules,
such as colchicine, vincristine, and vinblastine, capable of disrupting
the association between Bcl-2 family members and cytoskeletal
structures? In this context, it warrants some consideration that
mitochondria are also transported along the microtubules, in a manner
similar to cellular hitchhikers, rather than acting as free-floating
constituents within the cytosol (49
, 50)
. In fact, the
translocation and subsequent clustering of mitochondria around the
nucleus was recently demonstrated in cells undergoing TNF- and
Fas-triggered apoptosis (51
, 52)
. It is therefore
conceivable that mitochondria, and hence mitochondrial membrane
components such as Bcl-2 and Bcl-XL, may come in
close proximity of and interact with proapoptotic Bcl-2 family members
sequestered by the microtubules in cells that have sustained an
apoptotic assault. Another related question is whether the movement of
mitochondria is part of the killing process, and, if so, which signals
regulate these events. Inhibitors of microtubule organization suppress
the closure of the mitochondrial permeability transition pore (PTP), a
putative regulator of the apoptotic process, indicating that the
attachment of these organelles to the microtubular network may be
essential for PTP regulation (53)
.
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PROTEIN–PROTEIN INTERACTIONS: THE LIFE-DEATH RHEOSTAT |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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showed that the Bcl-2
molecule can heterodimerize with the proapoptotic protein Bax. Based on
these and similar findings, which pointed to the delicate equipoise
between Bcl-2 family members as an important determinant in the
decision of whether a cell should live or die, the `rheostat' concept
was formulated. This pervasive concept, expounded in recent years by
Korsmeyer and others, implies that the sensitivity to death stimuli is
determined by the relative ratio of agonist and antagonist homo- to
heterodimers (55
, 56)
. Mutagenesis experiments established
that the Bcl-2 homology (BH) domains 1 to 3 strongly influence homo-
and heterodimerization (57
, 58)
. However, it is clear that
Bcl-2 and Bax also have intrinsic functions independent of dimerization
(13)
. Moreover, Bcl-2 can bind nonhomologous proteins such
as the protein kinase Raf-1, the molecular chaperone regulator BAG-1,
the p53-binding protein p53-BP2, and calcineurin, a calcium- and
calmodulin-dependent protein phosphatase (13)
. The BH4
domain, present in all antiapoptotic family members but absent from
nearly all proapoptotic counterparts, is needed for interaction with
several of these seemingly unrelated proteins, including Raf-1, BAG-1,
and calcineurin (59
60
61)
. The latter is of particular
interest, considering the role of calcineurin binding for activation of
the transcription factor NF-AT, and suggests that Bcl-2 may play a role
in cell cycle regulation (61
, 62)
.
Several recent studies have addressed the role of Bcl-2-like proteins
in the assembly and regulation of the multicomponent cytoplasmic
complex termed the apoptosome, which is believed to consist of
multimeric APAF-1 (apoptotic protease-activating factor-1), a human
ced-4 homologue, in association with procaspase-9, dATP, and cytochrome
c (63)
. Physical interactions between the
nematode proteins ced-4 and ced-9 have been demonstrated and appear to
correlate with the ability of ced-9 to suppress ced-4-mediated
activation of the nematode caspase, ced-3 (64
65
66)
.
Similarly, a tertiary complex is formed between procaspase-9, APAF-1,
and the antiapoptotic protein Bcl-XL (67
, 68)
. Another Bcl-2 family member, Diva (Boo), is also capable of
forming a complex with APAF-1, and it was suggested that Diva and
Bcl-XL compete for binding to the apoptosome,
with Bcl-XL presumably inhibiting and Diva
enabling APAF-1-mediated activation of caspases (69)
. On
the other hand, Boo was shown in another study to inhibit apoptosis,
and the proapoptotic homologues Bak and Bik were found to disrupt the
association between Boo and APAF-1 (70). Finally, in view of the
apparent redundancy of mammalian proteins homologous to the other key
players in the nematode, ced-3 and ced-9, it seems prudent to assume
the existence of additional ced-4 (APAF-1)-like adaptor molecules that
may serve as platforms for caspase activation and as targets for
regulation by distinct Bcl-2 family members. It seems clear that pro-
and antiapoptotic members of the Bcl-2 family can regulate each other,
and hence the fate of the cell, not only at the mitochondrial membrane
but also at the level of the apoptosome.
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PROTEIN PHOSPHORYLATION OF Bcl-2 FAMILY MEMBERS |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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, 72)
. For instance,
mitogen-activated kinase (MAPK) signaling may suppress Fas-mediated
apoptosis (73)
. Moreover, the direct phosphorylation of
caspases has been demonstrated, furnishing an additional level of
control of these killer proteases (74
, 75)
. Bcl-2 family
members can also be phosphorylated, although the outcome of this
posttranslational modification appears to depend on the specific
stimulus. Taxol and other chemotherapeutic agents that target
microtubules induce serine phosphorylation of Bcl-2, which is
associated with an abrogation of its antiapoptotic function
(76
77
78
79)
. In fact, it was recently proposed that Bcl-2 may
serve essentially as a `guardian of microtubule integrity'
(80)
. Bcl-XL is also phosphorylated
after microtubule disruption (81)
. Exposure of cells to
all-trans retinoic acid (ATRA) induces phosphorylation of
Bcl-2, and this was suggested to result in a shortened half-life of the
protein perhaps by targeting of Bcl-2 for degradation
(82)
. In contrast, other studies have suggested that
phosphorylation of Bcl-2 may be required for its antiapoptotic function
in growth factor-dependent cell lines (83
84
85)
, and
Poommipanit et al. (86)
have demonstrated that
phosphorylation of Bcl-2 may contribute to the inactivation of the
antiproliferative function of Bcl-2. Indeed, the functional
consequences of Bcl-2 phosphorylation may be associated with mitotic
arrest rather than the induction of apoptosis (87
, 88)
.
Serine phosphorylation of the proapoptotic member, Bad, in response to
IL-3 treatment may also occur; these findings have provided an
illustrative example of signal transduction pathways triggered by
extracellular survival signals (89)
. Dephosphorylated Bad
is associated with Bcl-XL whereas phosphorylated
Bad translocates to the cytosol and binds to 14–3-3. This appears to
sequester Bad away from Bcl-XL at the
mitochondrial membrane, thereby allowing Bcl-XL
to exert its antiapoptotic effect. An additional report demonstrated
that overexpression of Bcl-2 can induce the localization of the protein
kinase Raf-1 to mitochondria where Raf-1 participates, most likely
indirectly, in the phosphorylation of Bad (59)
. The
serine-threonine kinase Akt may also serve to couple survival signals
to the regulation of Bad (90
, 91)
. Moreover, Harada et al.
(92)
reported that membrane-bound, cAMP-dependent protein
kinase acts as a specific Bad kinase. This cAMP-dependent protein
kinase is tethered to the mitochondrial membrane through its
association with a protein kinase A-anchoring protein, and these
findings illustrate the inactivation of a proapoptotic factor at its
specific target organelle in response to a survival factor.
A sustained elevation of intracellular calcium is known to trigger
apoptosis in various cell types (93)
, and Bcl-2 may exert
its antiapoptotic effect in part through the regulation of the
compartmentalization of calcium in the endoplasmic reticulum and the
nucleus (16
, 17)
. Binding of Bcl-2 to the
calcium-activated, serine-threonine phosphatase calcineurin prevents
calcineurin-mediated dephosphorylation and activation of the
transcription factor NF-AT (61)
. Moreover, calcineurin can
induce apoptosis through dephosphorylation of Bad, thereby promoting
the translocation of Bad from the cytosol to the mitochondria and
enhancing heterodimerization between Bad and
Bcl-XL (94)
. These data thus provide
a mechanism for calcium-triggered apoptosis induction whereby the
phosphorylation state and localization, and hence the activity, of a
proapoptotic Bcl-2 family member are regulated.
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CLEAVAGE (AND CONVERSION) OF Bcl-2 FAMILY MEMBERS |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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, who demonstrated that the HIV protease cleaved
Bcl-2, resulting in activation of NF
B, a transcription factor
required for viral replication, and cell death. In addition, Sindbis
virus infection may trigger the cleavage of Bcl-2 by endogenous
caspases (96)
. These findings were followed by additional
reports of caspase-dependent cleavage of the antiapoptotic molecules
Bcl-2 and Bcl-XL in response to numerous
apoptotic triggers, including growth factor withdrawal, Fas ligation,
and etoposide (97
98
99
100
101)
. In addition, cleavage of the
BH3-only protein, Bid, with the generation of an active fragment that
promotes the redistribution of cytochrome c has also been
documented (discussed above). Cleavage of Bcl-2 was found to occur in
the variable loop region, resulting in loss of the BH4 domain and
consequently in the exposure of the BH3 domain, converting this
antiapoptotic molecule to a proapoptotic fragment that accelerated cell
death (96
, 100)
. Evidence suggests that treatment of cells
with chemotherapeutic agents that damage DNA preferentially results in
the cleavage of Bcl-2 (100, 101), whereas agents that cause derangement
of the cytoskeleton induce phosphorylation of Bcl-2 (79, 80). In both
cases, these posttranslational modifications are predicted to
inactivate the antiapoptotic function of Bcl-2. Moreover, the
C. elegans homologue of Bcl-2, ced-9, is proteolytically
processed by ced-3, indicating that caspase-mediated cleavage of
Bcl-2-like molecules, like so many other features of the apoptotic
program, is conserved throughout evolution (102)
.
A primary role of Bcl-2 has been proposed to be the prevention of
cytochrome c leakage from the mitochondrial intermembrane
space during apoptosis, resulting in the amputation of APAF-1-mediated
caspase activation in the cytosol (14
, 15)
. Cleavage of
Bcl-2, a relatively early event in the cascade of apoptotic events
(101)
, may therefore be envisioned to yield an accelerated
redistribution of cytochrome c from mitochondria to cytosol.
In essence, Bcl-2 cleavage could act as a feed-forward amplification
loop of apoptosis, thus serving to seal the fate of those cells which
are sentenced to die. Indeed, nitric oxide may inhibit the positive
feedback amplification of caspase activation by preventing Bcl-2
cleavage and release of cytochrome c (103)
.
When cells are transfected with an uncleavable Bcl-2 mutant, TNF- and
actinomycin D-triggered cytochrome c release and the
subsequent increase in cytosolic caspase-3-like activity are more
efficiently prevented (103)
. Finally, even though no
correlation has been found between the antiapoptotic and
antiproliferative effect of Bcl-2 (104, 105), these dichotomous roles
of Bcl-2 are dependent on an intact BH4 domain, and it would therefore
be of interest to see whether cleavage of Bcl-2 impinges on its
function in cell cycle regulation.
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AT THE SCENE OF THE CRIME: PROTEASES IMPLICATED IN THE CLEAVAGE OF Bcl-2 |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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, evidence was
provided for the in vitro cleavage of Bcl-2 by recombinant
caspase-3, and a recent study has demonstrated that recombinant
caspases belonging to all three caspase subgroups are capable of
cleaving Bcl-2 in vitro into a similar 23 kDa fragment
(103)
. On the other hand, Grandgirard et al.
(97)
appeared to exclude caspase-3 and caspase-7, at least
in the case of alphavirus-triggered Bcl-2 cleavage. Both caspase-1 and
caspase-3 can cleave the related Bcl-2 family member
Bcl-XL (98)
, although the importance
of caspase-1 activation for apoptosis remains questionable. However,
Fujita et al. (99)
have demonstrated that
Bcl-XL cleavage is blocked by inhibitors of
caspase-3-like, but not caspase-1-like, enzymes. Moreover, Bcl-2
cleavage apparently may result from noncaspase protease activation
(106)
. We favor the involvement of caspase-9 or related
enzymes, since inhibitors of type III caspases (i.e., caspase-6, -8,
-9), but not type II caspases (caspase-2, -3, -7), were effective in
preventing cleavage of Bcl-2 in etoposide-treated cells
(101)
. Also unresolved is the possible contribution of
caspases sequestered in subcellular compartments such as the
mitochondria and endoplasmic reticulum (107
108
109
110
111)
. Our
studies have indicated that Bcl-2 is preferentially cleaved in the
mitochondrial fraction of etoposide-treated cells (101)
,
and the role of cytosolic vs. mitochondrial caspases in this event is
currently under investigation.
Bcl-2 cleavage occurs in activated human T lymphocytes after induction
of apoptosis by etoposide, staurosporine, and anti-Fas antibodies
(112)
. In fact, proteolysis of Bcl-2 is a more specific
marker of apoptosis in these cells than cleavage of the `classical'
caspase-3 substrate poly(ADP-ribose) polymerase (PARP), since Bcl-2
cleavage is detected only on triggering of apoptosis whereas the
emergence of the 85 kDa PARP cleavage fragment is evident also in
nonapoptotic, activated T lymphocytes (B. Fadeel, unpublished results).
These findings agree with the observation that caspase-3-like enzymes
can indeed be activated in T lymphocytes in the absence of other
characteristic features of apoptosis (113
, 114)
. Moreover,
our findings argue against a critical role for caspase-3-like enzymes
in the cleavage of Bcl-2, at least in apoptotic T lymphocytes, and are
thus at variance with the in vitro evidence presented by
Cheng et al. (96)
. The latter findings perhaps should be
interpreted with caution since data concerning substrate cleavage in
cell-free systems may not always mimic the apoptotic events within
intact cells (115)
.
The proapoptotic family member, Bax, can be cleaved both by caspases
(97)
and the calcium-activated protease calpain
(116)
. However, the cleavage of substrates by both
caspases and calpains is not without precedent. We and others have
demonstrated that the cytoskeletal protein fodrin (117
, 118)
, the endogenous calpain inhibitor calpastatin (119
, 120)
, as well as caspases themselves (D. H. Burgess and S.
Orrenius, unpublished results) are all cleaved by both classes of
apoptotic proteases. Taken together, cleavage of Bcl-2 family members
by caspases and/or calpain, which may convert antiapoptotic molecules
to proapoptotic molecules (and possibly vice versa), has
emerged as an important mechanism for the interception and regulation
of cell death instigated by various stimuli.
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BH3-ONLY FAMILY MEMBERS: EMISSARIES OF DEATH |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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.
A subfamily of proapoptotic proteins consisting of Bad, Bid, Bik, Bim,
Blk, and Hrk, which share homology only in the latter domain, are
therefore referred to as BH3-only proteins. These molecules are closely
related to the C. elegans protein egl-1, a cell death
activator critical for developmental death in the nematode and thought
to act genetically upstream of ced-9 (122). Two examples of the role of
BH3-only proteins and how they may be subject to regulation have been
discussed and include the demonstration that Bim, which is normally
sequestered (or hijacked) by the dynein motor complex, translocates to
mitochondria on apoptosis triggering and antagonizes the effect of
Bcl-2 (48) and the finding that the activity of Bad is controlled by
phosphorylation and dephosphorylation in response to extracellular
stimuli (59
, 89
90
91
, 94)
. A third example will be provided
and relates to the role of Bid.
Two principal pathways of activation of downstream caspases have been
described (3)
. Signaling through the surface receptor Fas
results in recruitment of the adaptor molecule FADD and activation of
procaspase-8, the apical proteolytic activity in this pathway, which in
turn cleaves and activates downstream caspases such as procaspase-3,
-6, and -7. An alternative pathway is initiated upon release of
cytochrome c from mitochondria and assembly of the cytosolic
apoptosome complex that leads to the activation of procaspase-9 and
subsequent activation of the downstream caspases. The relative
contribution of these two pathways in Fas-mediated death may also vary
between different cell types, depending on the efficiency of
FADD-mediated recruitment of procaspase-8 at the plasma membrane
(123)
. These authors demonstrated that Fas signaling may
proceed through the triggering of cytochrome c release in
certain cell types, although the mechanism for caspase-8-induced
mitochondrial perturbation was not defined. Recent data have now
provided a bid for the missing link or, in the morbid parlance of Gross
and associates, a `linchpin' between cell surface receptors and the
mitochondria (52
, 124
, 125)
. Hence, procaspase-8 is
capable of cleaving Bid, a BH3-only protein present in the cytosol,
thereby releasing fragments 15 and 11 kDa in size (52
, 124)
. The 15 kDa Bid fragment then translocates to mitochondria,
where it promotes the release of cytochrome c, which
associates with the apoptosome complex and serves to activate
procaspase-9. Bid cleavage thus serves to amplify caspase activation in
cells undergoing apoptosis and bridges the activation of cell surface
receptors and mitochondria. Han et al. (126)
have also
identified a cytochrome c efflux-inducing factor in
cytosolic extracts from apoptotic cells and have found that this
factor, which is activated by caspases and antagonized by Bcl-2, is
identical to Bid.
The 3-dimensional structure of Bid was recently determined and
has provided additional insight into the regulation of this death
ligand (127
, 128)
. From this structure one may predict
that the BH3 domain of Bid is buried and hence inaccessible for
interaction with other Bcl-2 family members. However, on
caspase-8-mediated cleavage, a previously buried surface area becomes
exposed, suggesting that the marked changes in surface electrostatics
and hydrophobic exposure that occur upon cleavage may be the driving
force in the translocation of truncated Bid and subsequent membrane
insertion (128)
. However, Chou et al. (127)
could find no evidence for major conformational changes in Bid on
caspase-8 cleavage. As an alternative mode of action, these authors
propose that truncated Bid, which contains the structural motifs for
channel formation, may form selective ion channels similar to Bax and
promote apoptosis independent of its BH3 domain (127)
.
Nevertheless, one may predict that Bcl-2-like molecules that possess
`hidden' BH3 domains may be cleaved or undergo conformational changes
that lead to unmasking of the BH3 domain, thus converting them to
constitutively active death agonists. Griffiths et al.
(129)
showed that changes in the conformation, but not in
the location, of Bak occurred upon treatment of tumor cells with
staurosporine, etoposide, or dexamethasone and were associated with the
release of Bcl-XL from Bak. Ligation of cells via
the Fas molecule did not trigger these changes in conformation and
protein–protein interaction, suggesting that the signaling cascades
involved may differ depending on the death stimulus. Finally, in cells
undergoing staurosporine-triggered apoptosis, a Bid-induced
conformational change of Bax is responsible for the release of
cytochrome c, further emphasizing the role of BH3-only
family members as intracellular ligands or stress sensors that transmit
apoptotic signals to membrane-bound counterparts such as Bcl-2,
Bcl-XL, and Bax (130)
.
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SPECULATIONS ON THE REGULATION OF Bcl-2 REGULATION |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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. Deletion of the variable loop domain of Bcl-2 blocks
okadaic acid- and paclitaxel-induced phosphorylation, thereby
increasing the antiapoptotic activity of Bcl-2 (131, 132). We surmise
that phosphorylation within the loop region may also determine
susceptibility to cleavage, perhaps by altering the conformation of
Bcl-2, thus rendering the cleavage sites within the loop more
accessible to proteases. A case in point, the caspase-dependent
cleavage of IB-
and presenilin-2 is regulated by the
phosphorylation of these substrates (133
, 134)
. Cleavage
of Bcl-2 family members could also be regulated by subcellular
targeting. For example, Bax is more susceptible to proteolysis when
inserted into the mitochondrial membrane (135)
. |
CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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). Finally, these considerations bring us to the Gordian knot of
contemporary apoptosis research, which is how to harness apoptotic
processes for therapeutic benefit, for example, in cancer and
neurodegenerative disorders, while maintaining a healthy degree of
apoptosis in unaffected bystander tissues. A detailed knowledge of the
regulation of apoptosis regulators may aid in this endeavor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONA FAMILY OF DEATH...SUBCELLULAR TARGETING OF Bcl-2...PROTEIN-PROTEIN INTERACTIONS:...PROTEIN PHOSPHORYLATION OF Bcl-2...CLEAVAGE (AND CONVERSION) OF...AT THE SCENE OF...BH3-ONLY FAMILY MEMBERS:...SPECULATIONS ON THE REGULATION...CONCLUDING REMARKSREFERENCES |
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in association with suppression of apoptosis. J. Biol. Chem. 269,26865-26870-spectrin breakdown by calpain and interleukin 1ß-converting enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319,683-690
B- inhibits its cleavage by caspase CPP32 in vitro. J. Biol. Chem. 272,29419-29422This article has been cited by other articles:
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