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Molecular and Cellular Biology, March 2000, p. 1886-1896, Vol. 20, No. 5
Molecular Cardiology, Department of Internal
Medicine IV, University of Frankfurt, Frankfurt, Germany
Received 8 October 1999/Accepted 3 December 1999
The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a
critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may
switch on the apoptotic pathway. In endothelial cells, tumor necrosis
factor alpha (TNF- Programmed cell death is critical
for the successful development of multiple tissues and the maintenance
of normal tissue homeostasis. The signaling pathways involved in
apoptosis have been intensively studied (reviewed in references
2, 3, 19, 21, 23, and 24). Key regulatory proteins
in apoptotic events are the Bcl-2 family of proteins, which can either
promote cell survival (Bcl-2, Bcl-XL, A1, Mcl-1, and Bcl-W) or promote
cell death (Bax, Bak, Bcl-XS, and Bok) (1). The relative
amounts or equilibrium between these pro- and antiapoptotic proteins
influences the susceptibility of cells to apoptosis.
Bcl-2 is the prototype member of a protein family that functions to
suppress apoptosis in a variety of cell systems. Bcl-2 is localized in
the endoplasmatic reticulum, nuclear envelope, and outer mitochondrial
membrane, whereby its NH2-terminal part is facing the
cytosol. Several mechanisms have been proposed to explain the
antiapoptotic function of Bcl-2. Bcl-2 might act as a regulator of
Ca2+ homeostasis (4) or as an antioxidant
(34). Bcl-2 forms heterodimers with the proapoptotic protein
Bax and might thereby neutralize its death effector properties
(46). In addition, Bcl-2 prevents the release of potent
mitochondrial activators of the cytosolic death effector proteases, the
caspase protease family, which mediates the intracellular proteolysis
that is characteristic of apoptosis (60). The association of
Bcl-2 with the mitochondrial apoptosis-activating factor Apaf1 and the
blockade of cytochrome c release may prevent the activation
of the two death proteases, caspases 9 and 3 (39, 61). Bcl-2
also acts by modulating the collapse of the mitochondrial transmembrane
potential that occurs during apoptosis (60). Whereas any one
or a combination of these potential functions of Bcl-2 may operate to
suppress apoptosis, the mechanism by which Bcl-2 may be regulated to
preserve mitochondrial integrity has not been identified.
The regulation of Bcl-2 on the transcriptional level seems to be a
critical factor in the development of cancer, as has been demonstrated
by enhanced expression of Bcl-2 protein in cancer tissues
(36). In contrast, Bcl-2 protein levels have been shown to
be reduced in endotoxic shock in vivo (26), in endothelial cells exposed to lipopolysaccharide (27), in neuronal cells treated with The ubiquitin-proteolytic pathway is a major system for selective
protein degradation in eukaryotic cells. One of the first steps in this
process includes selective modification of The objectives of this study were to get further insights into the
signal transduction stimulating Bcl-2 degradation. Bcl-2 contains
several consensus protein kinase sites, such as a protein kinase B/Akt
site (RXRXXS/T) at position 132, a protein kinase C Cell culture.
HUVEC were purchased from Cell
Systems/Clonetics, Solingen, Germany, and cultured in endothelial basal
medium supplemented with hydrocortisone (1 µg/ml), bovine brain
extract (3 µg/ml), gentamicin (50 µg/ml), amphotericin B (50 µg/ml), epidermal growth factor (10 µg/ml), and 10% fetal calf
serum (FCS) until the third passage. COS-7 and HeLa cells were cultured
in Dulbecco's modified Eagle's medium with 10% FCS and 2 mM
L-glutamine.
Plasmid constructs.
Human Bcl-2 and wild-type MAP kinase
phosphatase 3 (MKP-3) were amplified and subcloned into
pcDNA3.1( Transient-transfection system.
Transient transfections of
HUVEC and COS-7 cells were performed as described previously
(16). HeLa cells were transfected with plasmids by the
calcium phosphate method (53). The transfection of
oligonucleotides into HUVEC or HeLa cells was carried out by using the
Lipofectamine procedure (GIBCO BRL, Karlsruhe, Germany). Briefly, 1.5 µg of phosphorothiolated sense
(5'-GGGAAGGATGGCGCACGCTG-3') or antisense
(5'-CAGCGTGCGCCATCCTTCCC-3')
oligonucleotides (Roth, Karlsruhe, Germany) corresponding to the
Bcl-2 sequence (49) or phosphorothiolated sense
(5'-CCTGAGTTCCACTGAGTTCC-3') or antisense (5'-GGAACTCAGTGGAACTCAGG-3') oligonucleotides corresponding
to a highly conserved sequence within the MAP kinase-specific
phosphatases (31) was incubated in 100 µl of RPMI medium
in the presence of 5 µl of Lipofectamine (GIBCO BRL) for 30 min at
room temperature. HUVEC or HeLa cells (5 × 105 cells
in 6-cm2 wells) were washed with RPMI medium and incubated
with 2 ml of RPMI medium before adding the
Lipofectamine-oligonucleotide mixture. After further incubation for
5 h, 3 ml of complete RPMI medium was added and the cells were
again incubated for 18 h before the determination of apoptosis.
Western blot analysis and immunoprecipitation.
Cells were
lysed as described (16). Western blots were performed with
either anti-Bcl-2 antibody (Boehringer Mannheim, Mannheim, Germany) or
anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). For
the detection of phosphorylated forms of Bcl-2, immunoprecipitation was
carried out with either an anti-phosphoserine-specific antibody (clone
4A9; Alexis Corporation, Grünberg, Germany) or an anti-myc
antibody (Santa Cruz Biotechnology). Immunoprecipitates were resolved
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was carried out with anti-myc antibody or an anti-phosphoserine-specific antibody.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Posttranslational Modification of Bcl-2 Facilitates
Its Proteasome-Dependent Degradation: Molecular Characterization of the
Involved Signaling Pathway
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic
AMP-dependent protein kinase sites does not affect Bcl-2 stability. In
contrast, inactivation of the three consensus mitogen-activated protein
(MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and
subsequently degraded by the 26S proteasome. Inactivation of these
sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to
play a major role. A Ser-to-Ala substitution at this position results
in 50% degradation, whereas replacement of Thr74 with Ala leads to
25% degradation, as assessed by pulse-chase studies. We further
demonstrated that incubation with TNF- induces dephosphorylation of
Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers
phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation
was observed in the presence of MAP kinase-specific phosphatases or the
MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative
stress mediates TNF--stimulated proteolytic degradation of Bcl-2 by
reducing MAP kinase activity. Taken together, these results demonstrate
a direct protective role for Bcl-2 phosphorylation by MAP kinase
against apoptotic challenges to endothelial cells and other cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-amyloid (47), and in neurons following
cerebral ischemia (37), suggesting a potential role of Bcl-2
turnover under inflammatory conditions. Moreover, a potential role for Bcl-2 degradation is supported by our recent finding that, in human
umbilical vein endothelial cells (HUVEC), tumor necrosis factor alpha
(TNF-) induces dephosphorylation and subsequent ubiquitin-dependent
degradation of Bcl-2 leading to apoptosis (16).
-NH2 groups
of lysine residues in the corresponding protein by ubiquitination, which targets the protein for ubiquitin-dependent degradation by the
proteasome complex. Although the mechanisms that underlie this
multicatalytic process are very well characterized (reviewed in
references 13, 14, 29, and 30), the signals that
target proteins for ubiquitination and, therefore, determine their
stability, are often unclear. In some cases, different patterns of
phosphorylation, a partially conserved sequence motif, or specific
structural features (as shown for mitotic cyclins, other cell cycle
regulators, or transcription factors) are required (reviewed in
reference 29).
(PKC)
or cyclic AMP (cAMP)-dependent protein kinase (PKA) site
(RXS/T) at an evolutionarily conserved serine site (Ser70),
and several mitogen-activated protein (MAP) kinase sites for
extracellular signal-regulated kinases 1 and 2 (ERK1/2) (PXXS/TP) at positions 56, 74, and 87. The role of these different putative phosphorylation sites for facilitating selective Bcl-2 degradation was examined. We demonstrate here that
dephosphorylation of the MAP kinase site Ser87 within Bcl-2 appears to
play a major role as a signal for its ubiquitin-dependent degradation.
Furthermore, we show that oxidative stress mediates TNF--stimulated
proteolytic degradation of Bcl-2 by inactivation of the MAP kinases
ERK1/2. Thus, Bcl-2 represents an attractive target for
posttranslational modification in response to proapoptotic stimuli in
endothelial cells and other cells.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)MycHis or pcDNA3.1() (Invitrogen, Groningen, The
Netherlands) under the control of the cytomegalovirus immediate-early
gene as described recently (16). The MAP kinase phosphatase
MKP-4 was amplified by PCR with oligonucleotides containing
EcoRV and BamHI restriction sites and cloned into
the respective sites of pcDNA3.1()MycHis under the transcriptional
control of the cytomegalovirus promoter. Desired mutant Bcl-2
constructs and a dominant negative form of MKP-3 (Cys293 to Ser) were
obtained by site-directed mutagenesis (Stratagene, Heidelberg,
Germany). Sequences were determined by an ABI automated sequencer.
-induced degradation, cells were incubated for
the time periods indicated with 100 ng of TNF- per ml, and cell
extracts were obtained by lysis of cells in 10 mM Tris-HCl (pH 8), 1%
Triton X-100, and 0.32 M sucrose on ice for 20 min. Then, homogenates
were centrifuged and the resulting supernatant was used for Western
blotting. Proteins (30 µg/lane) were loaded onto SDS-12.5% PAGE
gels and probed with anti-Bcl-2 antibody (Boehringer Mannheim) or
anti-myc antibody (Santa Cruz Biotechnology), and enhanced
chemiluminescence was performed according to the instructions of the
manufacturer (Amersham).
Stability of proteins in vivo. HeLa cells were starved in Dulbecco's modified Eagle's medium without methionine and cysteine for 1 h and then were metabolically labeled with L-[35S]methionine and L-[35S]cysteine for 2 h. HeLa cells were then chased in nonradioactive medium for the time periods indicated. Cells were lysed (10 mM Tris-HCl, pH 8; 1% Triton X-100; 0.32 M sucrose) at 4°C for 20 min. Samples containing equal amounts of protein were immunoprecipitated with an anti-myc antibody. Immunocomplexes were collected with immobilized Protein A/G-plus Sepharose (Amersham) and resolved on SDS-12.5% PAGE. The gel was dried, and proteins were visualized by a PhosphorImager (Molecular Dynamics).
In vitro kinase assay.
For the detection of in vitro
phosphorylation of Bcl-2 by MAP kinase or Akt kinase, respectively,
HeLa cells were transiently transfected with various myc-tagged Bcl-2
constructs and overexpressed proteins were immunoprecipitated with
anti-myc antibody. Isolated immunoprecipitated myc-tagged Bcl-2
proteins were incubated at 30°C in a kinase reaction mixture
containing 25 mM Tris (pH 7.5), 5 mM -glycerolphosphate, 0.1 mM
Na3VO4, 2 mM dithiothreitol, 10 mM
MgCl2, 15 µM ATP, and 5 µCi of
[
-32P]ATP with activated MAP kinase ERK2 (New England
Biolabs, Schwabach, Germany) for 30 min. Active Akt was obtained by
immunoprecipitation of myc-tagged, constitutive active Akt (Thr308Asp
and Ser473Asp) from transiently transfected COS-7 cells as described
recently (17). Immunoprecipitated myc-tagged Akt kinase was
combined with immunoprecipitated myc-tagged Bcl-2, and in vitro kinase assays were carried out as described above. The reaction was terminated by the addition of SDS loading dye, and samples were subjected to
SDS-12.5% PAGE and analyzed by a PhosphorImager.
Cell death analysis.
DNA fragmentation was demonstrated and
quantified by morphological analysis of apoptotic nuclei after
fluorescence staining with 4',6'-diamidino-2-phenylindole (DAPI) as
described previously (16). The data were confirmed by an
enzyme-linked immunosorbent assay specific for histone-associated DNA
fragments as recently described (18). To determine the
influence of the various Bcl-2 constructs on apoptosis, HUVEC were
transiently cotransfected with -galactosidase reporter and test
plasmids. Cells were fixed in 4% formaldehyde, and transfected cells
were identified by -galactosidase staining for 12 h. The
percentage of morphologically altered cells typical for apoptotic cell
death was determined by phase-contrast microscopy and then represented
as a function of the total number of blue cells under each condition.
The number of dead versus viable cells was counted by two independent
investigators in a total number of 600 cells.
Northern blot.
Total RNA was prepared as described recently
(5) and 10 µg of total RNA was loaded on 0.8% formamide
agarose gels. RNA was blotted on nylon membranes, and the blots were
hybridized with a [-32P]dCTP-labeled DNA probe
specific for human MKP-3. After incubation for 24 h, blots were
washed (0.1% SDS, 0.2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate]) and exposed to X-ray films.
Statistics. Data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analysis was performed with analysis of variance followed by a modified least significant difference test (SPSS-Software).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bcl-2 stability is mainly regulated by phosphorylation of its MAP
kinase sites Thr74 and Ser87.
We previously showed that incubation
with TNF- stimulates ubiquitin-dependent Bcl-2 proteolysis involving
dephosphorylation of Bcl-2 as a prerequisite (16). Since
Bcl-2 contains several consensus protein kinase sites, we investigated
the involvement of these putative phosphorylation sites in the
regulation of Bcl-2 protein stability. The consensus Akt site at
position 132 was mutated by an Arg-to-Cys substitution at position 129 (Bcl-2C129). The potential PKC or PKA site at position 70 was changed
from Ser to Ala (Bcl-2A70) (Fig. 1). To
determine the influence of these mutations on Bcl-2 stability,
pulse-chase analyses of the corresponding constructs were carried out.
Neither inactivation of the potential Akt site nor inactivation of the
PKC or PKA site affected Bcl-2 stability (Fig.
2A and B). No significant difference in
Bcl-2 protein levels could be observed after a 3-h chase following metabolic labeling of HeLa cells (Fig. 2A to D). In contrast, simultaneous mutation of all MAP kinase sites at positions 56, 74, and
87 (Bcl-2A1-3) to the amino acid Ala resulted in a Bcl-2 protein that
was rapidly degraded by the 26S proteasome (Fig. 2A, 2B, and
3B; P < 0.001). While
the half-life of wild-type Bcl-2 was found to be more than 6 h,
inactivation of MAP kinase sites (Bcl-2A1-3) resulted in a half-life of
less than 1.5 h (Fig. 2C and D).
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-induced degradation, Thr56,
Thr74, and Ser87 were replaced with phosphate-mimetic aspartic acid
residues, which mimic continuous phosphorylation of the protein
(45). Mutation of the single site Thr56 or Thr74 did not
significantly affect TNF--induced degradation, whereas the Ser87Asp
Bcl-2 mutant profoundly reduced TNF--triggered Bcl-2 degradation in
vivo and in vitro (data not shown). Furthermore, the combination
Thr56Asp-Thr74Asp (Bcl-2D1D2) mutation slightly reduced
TNF--stimulated proteolysis, whereas both Thr56Asp-Ser87Asp
(Bcl-2D1D3) and Thr74Asp-Ser87Asp (Bcl-2D2D3) mutants, as well as a
triple Thr56Asp-Thr74Asp-Ser87Asp Bcl-2 construct (Bcl-2D1-3), were
resistant towards TNF--stimulated degradation in endothelial cells
(Fig. 1 and 3C). These data indicate that dephosphorylation of Ser87
appears to play a major role as a signal for Bcl-2 degradation.
MAP kinase site phosphate-mimetic Bcl-2 constructs inhibit
TNF--induced apoptosis.
We previously showed that a reduction
of Bcl-2 protein levels preceded the induction of apoptosis
(16). To establish that TNF--induced dephosphorylation of
Bcl-2 at its MAP kinase sites signals degradation of Bcl-2 and,
therefore, stimulates apoptosis, Bcl-2 constructs carrying
phosphate-mimetic amino acid substitutions at the corresponding MAP
kinase sites were transfected into HUVEC. TNF--induced apoptosis was
examined by detecting the morphological change of
-galactosidase-stained nuclei. According to the results illustrated
above, resistance to TNF--induced apoptosis paralleled the
inhibition of degradation of various Bcl-2 mutants, in which different
MAP kinase sites were mutated. TNF--induced apoptosis was
significantly reduced in cells expressing Bcl-2 constructs with the
relevant phospho-mimetic amino acid substitutions (Fig. 4A). A Bcl-2 construct with substitutions
of Asp for Thr56 and Thr74 (Bcl-2D1D2) showed significantly less
resistance to TNF--induced apoptosis compared to Bcl-2 constructs
with substitutions of Asp for both Thr56 and Ser87 (Bcl-2D1D3) or Thr74
and Ser87 (Bcl-2D2D3) (Fig. 4A).
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The MAP kinase sites Thr74 and Ser87 within Bcl-2 are dominantly
phosphorylated by activated MAP kinase in vitro.
To determine if
all three MAP kinase sites within Bcl-2 are potential targets of
activated MAP kinase, in vitro kinase assays were performed. Bcl-2
constructs containing only one intact MAP kinase site, either Thr56
(Bcl-2D2D3), Thr74 (Bcl-2D1D3), or Ser87 (Bcl-2D1D2), were transiently
expressed in HeLa cells and isolated by immunoprecipitation with an
anti-myc antibody. These immunocomplexes were added as substrates to a
MAP kinase assay. In agreement with the data described above, the
phosphorylation efficiency of the single MAP kinase sites correlated
with their functional role in regulating Bcl-2 stability. Activated MAP
kinase dominantly phosphorylated Bcl-2 proteins containing the intact
MAP kinase site Thr74 (Bcl-2D1D3) or Ser87 (Bcl-2D1D2), whereas the MAP
kinase site Thr56 (Bcl-2D2D3) was only slightly phosphorylated (Fig. 5, lanes 3 to 5). Bcl-2wt served as a
control for MAP kinase phosphorylation activity (Fig. 5, lane 2). A
Bcl-2 mutant in which all MAP kinase sites were inactivated could not
be phosphorylated by MAP kinase in vitro (16) (data not
shown).
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Incubation with TNF- induces dephosphorylation of Ser87 within
Bcl-2.
Our results suggested that dephosphorylation of Ser87 plays
a predominant role as a signal for ubiquitin-dependent degradation of
Bcl-2. To demonstrate directly that TNF- indeed triggers
dephosphorylation of Ser87, we transiently expressed myc-tagged Bcl-2
constructs in HUVEC. Cells were lysed following treatment with or
without TNF-. To determine Ser87 phosphorylation,
immunoprecipitation was carried out with an anti-phosphoserine-specific
antibody, and Western blot analysis was performed with an anti-myc
antibody. Bcl-2 degradation in consequence to dephosphorylation was
prevented by using degradation-resistant, lysine-free Bcl-2 constructs. As shown in Fig. 5, TNF- stimulation completely reduced Ser87 phosphorylation of Bcl-2. The same experiment was performed with a
lysine-free Bcl-2 construct, in which all MAP kinase sites were inactivated to prevent phosphorylation at these sites. No serine phosphorylation of this mutated Bcl-2 protein was determined, which
demonstrates the specificity for Ser87 phosphorylation (Bcl-2mtA1-3) (Fig. 5). Taken together, these data provide compelling evidence that
Ser87 phosphorylation is directly affected by TNF-.
The MAP kinase ERK2 induces phosphorylation of Bcl-2 in
vivo.
To show that activated ERK phosphorylates Bcl-2 in vivo, we
transiently cotransfected HeLa cells with ERK2 and a
degradation-resistant myc-tagged form of Bcl-2 (Bcl-2mt). myc-tagged
Bcl-2 was immunoprecipitated with an anti-myc antibody, and Western
blot analysis was performed with an anti-phosphoserine antibody. An
increased amount of phosphorylated Bcl-2 protein was observed in the
presence of activated ERK2, whereas less phosphorylated Bcl-2 was
detectable in the absence of ERK2 (Fig.
6A). In addition, the MAP kinase-specific
inhibitor PD98059 significantly reduced the amount of
serine-phosphorylated Bcl-2 compared with untreated cells (Fig. 6B).
Thus, Bcl-2 is phosphorylated by ERK2 in vivo.
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ERK-specific phosphatases induce Bcl-2 dephosphorylation and its
subsequent degradation.
MAP kinase phosphorylation is a reversible
process, indicating that protein phosphatases play a crucial role in
controlling cellular activities. An emerging class of dual-specificity
phosphatases has been shown to regulate directly and specifically MAP
kinase family members. The dual-specificity phosphatase family that
mainly dephosphorylates MAP kinases at both the Tyr and Thr residues necessary for enzymatic activity comprises MAP kinase phosphatase 1 (MKP-1), MKP-2, MKP-3, and MKP-4 (12, 43, 44). Transfection of HUVEC with MKP antisense oligonucleotides directed against a
conserved domain within the MKP family completely inhibited TNF--induced degradation of Bcl-2 in vivo, whereas neither sense nor
scrambled oligonucleotides affected the degradation process (Fig. 6C).
Furthermore, pretreatment with MKP antisense oligonucleotides completely prevented ERK dephosphorylation after 6 h of TNF- stimulation, whereas MKP sense oligonucleotides did not affect ERK
activity (data not shown). This inhibition of ERK dephosphorylation was
associated with the complete inhibition of TNF--induced apoptosis of
HUVEC (P < 0.05; Fig. 6D) indicating a pivotal role
for the MAP kinase pathway to modulate TNF--mediated apoptotic processes.
stimulated the time-dependent transcription of
MKP-3 mRNA, as assessed by Northern blot analysis (Fig.
7A). Finally, a dominant negative MKP-3
mutant in which Cys293 was changed to Ser (8) significantly
inhibited TNF--induced Bcl-2 degradation (not shown) and apoptosis
in HUVEC (Fig. 7B). Taken together, these data demonstrate a pivotal
role for MKP-3, not only for the TNF--triggered degradation process of Bcl-2 but also for TNF--mediated apoptosis induction.
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Effect of antioxidants on TNF--stimulated Bcl-2
degradation.
The role of reactive oxygen species (ROS) in
TNF--stimulated cell death has been previously reported (22,
55, 58). Studies using mitochondrial inhibitors and
respiration-deficient cells have shown that TNF- cytotoxicity is
primarily mediated by ROS generated at the ubiquinone site of the
mitochondrial electron transport (55). Inhibition of the
electron flow to ubiquinone by respiratory chain inhibitors prevented
formation of superoxide anions and strongly reduced TNF--induced
cell death (55). To investigate whether oxygen radicals are
involved in the TNF--induced degradation of Bcl-2, we studied the
effects of a series of antioxidants on the Bcl-2 degradation process.
Figure 8A demonstrates that coincubation
with antioxidants, such as N-acetylcysteine (NAC), pyrrolidinedithiocarbamate, or a combination of vitamins E and C,
significantly inhibited stimulus-dependent degradation of Bcl-2 in
intact cells. To further demonstrate the physiological relevance of
ROS-mediated Bcl-2 degradation, the effects of antioxidants on
TNF--stimulated apoptosis in HUVEC were studied. As illustrated in
Fig. 8B, antioxidants profoundly inhibited TNF--induced apoptosis.
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-stimulated degradation of Bcl-2 and subsequent apoptosis, we
next examined the effect of H2O2 on Bcl-2
stability in vivo. Incubation with H2O2 for 6 to 12 h drastically reduced Bcl-2 levels in HUVEC (Fig. 8C) and
HeLa cells (not shown). Proteasome inhibitors, such as Z-LLL-H or ALLN,
completely inhibited ROS-triggered degradation of Bcl-2 (Fig. 8C),
demonstrating the involvement of the ubiquitin-proteasome pathway in
this proteolytic process. Furthermore, the stability of a lysine-free,
degradation-resistant Bcl-2 construct transiently transfected into
HUVEC was not affected by H2O2 (Fig. 8D). Thus, the degradation of Bcl-2 by ROS also requires activation of the ubiquitin-dependent proteasome complex.
Since previous studies demonstrated that prolonged incubation with
TNF- induced dephosphorylation and deactivation of ERK1/2 (16), we speculated whether ROS might influence the activity of these kinases. We first examined the long-term effect of
H2O2 on ERK1/2 activation. Transient activation
of ERK1/2 could be observed within the first 30 min of
H2O2 stimulation in HUVEC and HeLa cells.
However, after incubation of HUVEC with H2O2
for more than 1 h, phosphorylated active ERK was significantly
reduced (data not shown). Moreover, TNF--induced dephosphorylation
of ERK1/2 could be completely prevented by addition of antioxidants, such as vitamin C or NAC (Fig. 9A),
indicating an important role of ROS in TNF--triggered ERK1/2
deactivation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Under basal conditions, the antiapoptotic protein Bcl-2 is a
long-lived protein. Proapoptotic stimuli, such as TNF- or
H2O2, induce its degradation via the
ubiquitin-proteasome pathway both in vivo and in vitro. We have
previously demonstrated that the protein appears to be specifically
stabilized by phosphorylation processes (16). To further
dissect the mechanisms involved in Bcl-2 degradation, the present study
aimed to identify the crucial phosphorylation site(s) within Bcl-2 and
to determine its (or their) specificity for Bcl-2 stability. The
results of this study reveal the following novel findings: destruction
of the three putative MAP kinase sites at positions 56, 74, and 87 results in ubiquitination and subsequent degradation of the protein.
Progressive inactivation of these MAP kinase sites revealed that Bcl-2
stability is mainly regulated by phosphorylation at Thr74 and Ser87,
with Ser87 phosphorylation playing a predominant role. TNF- or the MAP kinase-specific inhibitor PD98059 diminishes Ser87 phosphorylation of Bcl-2 in vivo, while activated ERK2 induces phosphorylation of Bcl-2
in vivo and in vitro. In addition, phosphorylation of Bcl-2 was
demonstrated in 32P-labeled HUVEC transiently transfected
with wild-type Bcl-2 (data not shown). In particular, the 60-amino-acid
loop domain of Bcl-2 has been suggested to play a significant role for
Bcl-2 phosphorylation (9, 20). However, the functional
consequences of this phosphorylation event are discussed
controversially (9, 20). The present data indicate that
signaling through the MAP kinase pathway by phosphorylation of Thr74
and Ser87 located within or in close proximity to the loop domain plays
a critical role in the maintenance of Bcl-2 stability and, as a
consequence, apoptosis susceptibility. Overexpression of
degradation-resistant Bcl-2 proteins such as lysine-free or
phospho-mimetic Bcl-2 proteins significantly inhibits TNF--induced
apoptosis in HUVEC (16) (Fig. 4A), supporting the important
physiological role of Bcl-2 protein stability for cell survival.
Appropriate subcellular targeting of phospho-mimetic and lysine-free
Bcl-2 proteins with inactivated MAP kinase sites was confirmed by
immunolocalization experiments (data not shown). These data suggest
that mutations of MAP kinase sites within Bcl-2 mainly affect its
phosphorylation and, thus, Bcl-2 stability, whereas potential other
effects, such as alteration of protein conformation or of subcellular
localization, appear to be less important.
It has been proposed that caspases are capable of directly cleaving
Bcl-2. This caspase-mediated cleavage was suggested to be, at least in
part, responsible for the inability of Bcl-2 to block interleukin-3
withdrawal- or Fas-induced apoptosis in Jurkat or Ba/F3 cells
(11). However, Johnson and Boise (35) recently demonstrated that Bcl-2 is incapable of inhibiting TNF--induced cell
death in the murine pro-B cell line FL5.12 and cleavage-defective Bcl-2
proteins do not block TNF--induced apoptosis. In accordance with
this report, we have previously shown that Bcl-2 mutants which are
resistant to caspase-mediated cleavage (Asp32Ala and Asp34Ala) are
avidly degraded by the proteasome complex following TNF- stimulation
(16). Therefore, caspase-mediated Bcl-2 cleavage does not
appear to be important for TNF--induced apoptosis in endothelial cells.
MAP kinases ERK1/2 are at the center of many signal transduction pathways in eukaryotic cells (for review, see reference 50). These cascades are composed of a trio of sequentially acting protein kinases that transmit an extracellular physiological signal to the targets that orchestrate the appropriate cellular response. ERK1/2 phosphorylate a number of substrates participating in cell cycle regulation, including transcription factors (42). In these cases, ERK1/2 translocate to the nucleus upon activation and activate immediate-early gene transcription. Those periodically occurring, temporal events are essential for the maintenance of normal cell growth. In contrast, involvement of the ERK MAP kinase pathway in Bcl-2 stability appears to be a single, irreversible incident, as Bcl-2 dephosphorylation induces its degradation and then subsequent cell death. This implicates that long-term down-regulation of MAP kinases causes cytosolic signaling events, which lead to irreversible effects. There are other examples for cytosolic substrates of MAP kinases. They can phosphorylate and, thus, activate cytoplasmic phospholipase A2 in vitro (40) or the gap junction protein connexin 43 (59). However, the biological significance of these events has yet to be determined.
Protein phosphorylation is a reversible process involving protein
phosphatases as key regulators in cellular activities. MAP kinase
members are directly regulated by dual-specific phosphatases. MKP-1,
MKP-2, MKP-3, and MKP-4 have been previously implicated in the in vivo
inactivation of ERK1/2 (8, 12, 25, 36, 39, 40). As
demonstrated in the present study, Bcl-2 degradation was either
inhibited by overall suppression of MKP expression or triggered by
overexpression of MKP-3 or MKP-4, respectively. Compared with MKP-4,
MKP-3 induced a more pronounced Bcl-2 dephosphorylation at Ser87. This
correlates with the minor effect of MKP-4 on Bcl-2 protein stability
compared with the stronger effect of MKP-3 on Bcl-2 degradation.
Therefore, we postulate that MKP-3 is the major phosphatase involved in
TNF--induced Bcl-2 degradation by dephosphorylation of ERK. This
conclusion is also supported by the finding that TNF- induces MKP-3
expression. Moreover, we have previously shown that overexpression of
MKP-3 induces apoptosis in HUVEC (16), whereas
overexpression of a dominant negative form of MKP-3 dramatically inhibits TNF--induced Bcl-2 degradation and TNF--induced
apoptosis in HUVEC. It is of note that MKP-1 is strictly localized to
the nucleus, whereas MKP-4 can be found in the cytoplasm and in the nucleus. MKP-3 appears exceptional insofar that it is exclusively cytosolic and, in contrast to other dual specificity phosphatases, highly selective towards ERK inactivation (8). In this
respect, MKP-3 appears to play a superior role in the TNF--induced
signaling cascade of Bcl-2 degradation. Such compartmentalized
regulation of ERK may be of fundamental importance in molecular
processes underlying apoptosis induction.
Alternatively, regulation of Bcl-2 stability might be directly affected
by yet-unknown phosphatases, which could dephosphorylate Bcl-2 on Thr74
and Ser87. While we do not know the nature of the protein phosphatase
responsible for direct Bcl-2 dephosphorylation, several phosphatases,
such as the protein phosphatase 2A (PP2A) or the Ca2+
calmodulin serine/threonine phosphatase PP2B/calcineurin, have been
implicated to interact directly with Bcl-2 in murine factor-dependent myeloid cells (15), baby hamster kidney cells, and Jurkat
cells and in the SU-DHL-4B-cell lymphoma cell line (56).
However, there is no evidence that the binding of either PP2A or PP2B
is involved in the regulation of Bcl-2 degradation. An intact Ser70 site within Bcl-2 is required for the reversible association of PP2A
with Bcl-2 (15). In our studies, neither an intact nor an
inactive Ser70 site influenced Bcl-2 stability and, furthermore, the
potent PP2A and PP2B inhibitor okadaic acid did not influence TNF--induced Bcl-2 degradation (data not shown). The interaction between PP2B and Bcl-2 apparently facilitates T-cell survival by
preventing the nuclear localization of the transcription factor NF-AT
(56). Therefore, PP2A and PP2B may have different functional consequences indicating the potential versatility of Bcl-2 in interacting with and regulating other components involved in apoptosis. In addition, these intrinsic differences might be due to the different cell lines used for the studies mentioned above.
The results of the present study further demonstrate that degradation
of Bcl-2 by the ubiquitin-dependent proteasome complex is regulated by
oxidative stress. Scavenging of ROS by antioxidants prevents
TNF--induced Bcl-2 degradation in HUVEC, suggesting the involvement
of ROS for TNF--stimulated Bcl-2 proteolysis. In addition, the
treatment of HUVEC with exogenous H2O2 also
stimulated Bcl-2 degradation. However, the intracellular target(s) of
the ROS is not yet clear. A direct interaction resulting in activation of the ubiquitin-dependent proteasome complex seems rather unlikely, since the proteasome-dependent degradation of I
B induced by TNF- (51) was not ameliorated by antioxidants (data not
shown). This demonstrates clearly the specificity of the contribution of ROS in TNF--stimulated degradation of Bcl-2. Otherwise, several kinases and phosphatases have been shown to be redox regulated (22, 32). This is supported by our findings that the
stimulation of HUVEC and HeLa cells with H2O2
leads, after a short-term initial activation, to a time-dependent ERK
inactivation after 1 h. Moreover, dephosphorylation and,
therefore, inactivation of ERK1/2 was completely inhibited in the
presence of various antioxidants, indicating a central role of ROS in
TNF--triggered ERK1/2 deactivation. Finally, stimulation of
apoptosis by antisense oligonucleotides against Bcl-2 was not affected
by antioxidants. These data clearly indicate that antioxidants
interfere with the apoptosis signal transduction leading to Bcl-2
degradation. However, further studies are required to investigate in
detail the ROS-modulated ERK1/2 deactivation.
The involvement of ROS in TNF--stimulated cell death has previously
been reported (22, 55, 58) and is supported by the present
data, which demonstrate that antioxidants abrogate endothelial cell
apoptosis induced by TNF-. The proteolysis of Bcl-2 seems to play an
important role in apoptosis signal transduction, since proteasome
complex inhibitors or overexpression of degradation-resistant Bcl-2
mutants lacking the ubiquitin-acceptor amino acids prevent TNF--triggered cell death (16).
Inhibition of apoptosis correlates with the prevention of the proteasome complex-mediated proteolytic degradation of Bcl-2, which has been shown to be activated by ROS. The degradation of Bcl-2 might directly induce apoptosis or render the endothelial cells more susceptible for proapoptotic stimuli. Therefore, the inhibition of endothelial cell apoptosis and Bcl-2 degradation by antioxidants might importantly contribute to the functional integrity of the endothelial monolayer and thereby inhibit damage to the vessel wall, which is a key event for the initiation of atherosclerotic lesion development.
Taken together, our study suggests an important, regulatory role of Bcl-2 stability for cell survival. In response to death signals, posttranslational modifications like dephosphorylation result in the inactivation of the antiapoptotic function of Bcl-2 by diminishing the Bcl-2 protein level via ubiquitin-proteolytic degradation. In light of the ambivalent reports about the role of Bcl-2 phosphorylation as both an apoptosis promoting and an apoptosis inhibitory process (10, 15, 28, 33, 52, 57) or in cell cycle progression (41, 48, 54), our data demonstrate a defined function of Bcl-2 phosphorylation and dephosphorylation in determining its stability. Activation of the Bcl-2 degradation process might therefore alter the balance between pro- and antiapoptotic proteins within a cell and, moreover, may initiate the apoptotic machinery. Thus, phosphorylation of Bcl-2 at Thr74 and Ser87 might be of substantial importance for the maintenance of cell survival.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant for young research scientists from the Faculty of Medicine, University of Frankfurt, and by the Sonderforschungsbereich SFB-553(C2).
We thank Alexandra Bittner, Christine Goebel, and Susanne Ficcus for expert technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Internal Medicine IV, Division of Cardiology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-6301-7440. Fax: 49-69-6301-7113. E-mail: Dimmeler{at}em.uni-frankfurt.de.
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REFERENCES |
|---|
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Adams, J. M., and S. Cory.
1998.
The Bcl-2 protein family: arbiters of cell survival.
Science
281:1322-1325 |
| 2. |
Arch, R. H.,
R. W. Gedrich, and C. B. Thompson.
1998.
Tumor necrosis factor receptor-associated factors (TRAFs) a family of adapter proteins that regulates life and death.
Genes Dev.
12:2821-2830 |
| 3. |
Ashkenazi, A., and V. M. Dixit.
1998.
Death receptors: signaling and modulation.
Science
281:1305-1308 |
| 4. |
Baffy, G.,
T. Miyashita,
J. R. Williamson, and J. C. Reed.
1993.
Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hemapoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production.
J. Biol. Chem.
268:6511-6519 |
| 5. | Batt, D. B., G. G. Carmichael, and Z. Liu. 1998. An improved rapid method of isolating RNA from cultured cells. Methods Mol. Biol. 86:15-17[Medline]. |
| 6. | Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254[CrossRef][Medline]. |
| 7. | Breitschopf, K., E. Bengal, T. Ziv, A. Admon, and A. Ciechanover. 1998. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J. 20:5964-5973[CrossRef]. |
| 8. |
Camps, M.,
A. Nichols,
C. Gillieron,
B. Antonsson,
M. Muda,
C. Chabert,
U. Boschert, and S. Arkinstall.
1998.
Catalytic activation of the phosphatase MKP-3 by ERK2-activated protein kinase.
Science
280:1262-1265 |
| 9. | Chang, B. S., A. J. Minn, S. W. Muchmore, S. W. Fesik, and C. B. Thompson. 1997. Identification of a novel regulatory domain in Bcl-2-xL and Bcl-2. EMBO J. 16:968-977[CrossRef][Medline]. |
| 10. |
Chen, C.-Y., and D. V. Faller.
1996.
Phosphorylation of Bcl-2 protein and association with p21Ras in Ras-induced apoptosis.
J. Biol. Chem.
271:2376-2379 |
| 11. |
Cheng, E. H.-Y.,
D. G. Kirsch,
R. J. Clem,
R. Ravi,
M. B. Kastan,
A. Bedi,
K. Ueno, and J. M. Hardwick.
1997.
Conversion of Bcl-2 to a Bax-like death effector by caspases.
Science
278:1966-1968 |
| 12. |
Chu, Y.,
P. A. Solski,
R. Khosravi-Far,
C. J. Der, and K. Kelly.
1996.
The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation.
J. Biol. Chem.
271:6497-6501 |
| 13. |
Ciechanover, A., and A. L. Schwartz.
1998.
The ubiquitin-proteasome pathway: the complexity and myriad functions of protein death.
Proc. Natl. Acad. Sci. USA
95:2727-2730 |
| 14. | Coux, O., K. Tanaka, and A. L. Goldberg. 1996. Structure and function of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65:801-847[CrossRef][Medline]. |
| 15. |
Deng, X.,
T. Ito,
B. Carr,
M. Mumby, and W. S. May, Jr.
1998.
Reversible phosphorylation of Bcl2 following interleukin 3 or byrostatin 1 is mediated by direct interaction with protein phosphatase 2A.
J. Biol. Chem.
273:34157-34163 |
| 16. |
Dimmeler, S.,
K. Breitschopf,
J. Haendeler, and A. M. Zeiher.
1999.
Dephosphorylation targets Bcl-2 for ubiquitin-dependent degradation: a link between the apoptosome and the proteasome pathway.
J. Exp. Med.
189:1815-1822 |
| 17. | Dimmeler, S., I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, and A. M. Zeiher. 1999. A novel Ca2+-independent pathway for NOS activation in endothelial cells via Akt-dependent phosphorylation. Nature 399:601-605[CrossRef][Medline]. |
| 18. |
Dimmeler, S.,
J. Haendeler,
M. Nehls, and A. M. Zeiher.
1997.
Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like protease.
J. Exp. Med.
185:601-607 |
| 19. |
Evan, G., and T. Littlewood.
1998.
A matter of life and death.
Science
281:1317-1321 |
| 20. |
Fang, G.,
B. S. Chang,
C. N. Kim,
C. Perkins,
C. B. Thompson, and K. N. Bhalla.
1998.
"Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis.
Cancer Res.
58:3202-3208 |
| 21. | Gaiewski, T. F., and C. B. Thompson. 1996. Apoptosis meets signal transduction: elimination of a BAD influence. Cell 87:589-592[CrossRef][Medline]. |
| 22. |
Gotoh, Y., and J. A. Cooper.
1998.
Reactive oxygen species- and dimerization-induced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor- signal transduction.
J. Biol. Chem.
273:17477-17482 |
| 23. | Green, D. R. 1998. Apoptotic pathways: the roads to ruin. Cell 94:695-698[CrossRef][Medline]. |
| 24. |
Green, D. R., and J. C. Reed.
1998.
Mitochondria and apoptosis.
Science
281:1309-1316 |
| 25. |
Guan, K. L., and E. Butch.
1995.
Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase.
J. Biol. Chem.
270:7197-7203 |
| 26. | Haendeler, J., U. K. Messmer, B. Brune, E. Neugebauer, and S. Dimmeler. 1996. Endotoxic shock leads to apoptosis in vivo and reduces Bcl-2. Shock 6:405-409[Medline]. |
| 27. | Haendeler, J., A. M. Zeiher, and S. Dimmeler. 1996. Vitamin C and E prevent lipopolysaccharide-induced apoptosis in human endothelial cells by modulation of Bcl-2 and Bax. Eur. J. Pharmacol. 317:407-411[CrossRef][Medline]. |
| 28. |
Haldar, S.,
N. Jena, and C. M. Croce.
1995.
Inactivation of Bcl-2 by phosphorylation.
Proc. Natl. Acad. Sci. USA
92:4507-4511 |
| 29. | Hershko, A., and A. Ciechanover. 1998. The ubiquitin system. Annu. Rev. Biochem. 67:425-479[CrossRef][Medline]. |
| 30. | Hochstrasser, M. 1996. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30:405-439[CrossRef][Medline]. |
| 31. |
Horiuchi, M.,
W. Hayashida,
T. Kambe,
T. Yamada, and V. J. Dzau.
1997.
Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating mitogen-activated protein kinase phosphatase-1 and induces apoptosis.
J. Biol. Chem.
272:19022-19026 |
| 32. |
Huot, J.,
F. Houle,
F. Marceau, and J. Landry.
1997.
Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells.
Circ. Res.
80:383-392 |
| 33. |
Ito, T.,
X. Deng,
B. Carr, and W. S. May.
1997.
Bcl-2 phosphorylation required for anti-apoptosis function.
J. Biol. Chem.
272:11671-11673 |
| 34. | Jacobson, M. D., and M. C. Raff. 1995. Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374:814-816[CrossRef][Medline]. |
| 35. |
Johnson, B. W., and L. H. Boise.
1999.
Bcl-2 and caspase inhibition cooperate to inhibit tumor necrosis factor--induced cell death in a Bcl-2 cleavage-independent fashion.
J. Biol. Chem.
274:18552-18558 |
| 36. | Krajewski, S., S. Bodrug, R. Gascoyne, K. Berean, M. Krajewska, and J. C. Reed. 1994. Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes. Am. J. Pathol. 145:515-525[Abstract]. |
| 37. |
Krajewski, S.,
J. K. Mai,
M. Krajewska,
M. Sikorska,
M. J. Mossakowski, and J. C. Reed.
1995.
Upregulation of bax protein levels in neurons following cerebral ischemia.
J. Neurosci.
15:6364-6376 |
| 38. |
Kwak, S. P., and J. E. Dixon.
1995.
Multiple dual specificity protein tyrosine phosphatases are expressed and regulated differentially in liver cell lines.
J. Biol. Chem.
270:1156-1160 |
| 39. | Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489[CrossRef][Medline]. |
| 40. | Lin, L. L., M. Wartmann, A. Y. Lin, J. L. Knopf, A. Seth, and R. J. Davis. 1993. CPLA2 is phosphorylated and activated by MAP kinase. Cell 72:269-278[CrossRef][Medline]. |
| 41. |
Ling, Y.-H.,
C. Tornos, and R. Perez-Soler.
1998.
Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis.
J. Biol. Chem.
273:18984-18991 |
| 42. | Marshall, C. J. 1994. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr. Opin. Genet. Dev. 4:82-89[CrossRef][Medline]. |
| 43. |
Muda, M.,
U. Boschert,
R. Dickinson,
J.-C. Martinou,
I. Martinou,
M. Camps,
W. Schlegel, and S. Arkinstall.
1996.
MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new mitogen-activated protein kinase phosphatase.
J. Biol. Chem.
271:4319-4326 |
| 44. |
Muda, M.,
U. Boschert,
A. Smith,
B. Antonsson,
C. Gillieron,
C. Chabert,
M. Camps,
I. Martinou,
A. Ashworth, and S. Arkinstall.
1997.
Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.
J. Biol. Chem.
272:5141-5151 |
| 45. |
Musti, A. M.,
M. Treier, and D. Bohmann.
1997.
Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases.
Science
275:400-402 |
| 46. | Oltvai, Z. N., C. L. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609-619[CrossRef][Medline]. |
| 47. |
Paradis, E.,
H. Douillard,
M. Koutroumanis,
C. Goodyer, and A. LeBlanc.
1996.
Amyloid beta peptide of Alzheimer's disease downregulates Bcl-2 and upregulates Bax expression in human neurons.
J. Neurosci.
16:7533-7539 |
| 48. |
Poommipanit, P. B.,
B. Chen, and Z. N. Oltvai.
1999.
Interleukin-3 induces the phosphorylation of a distinct fraction of Bcl-2.
J. Biol. Chem.
274:1033-1039 |
| 49. |
Reed, J. C.,
C. Stein,
C. Subasinghe,
S. Haldar,
C. M. Croce,
S. Yum, and J. Cohen.
1990.
Antisense-mediated inhibition of Bcl2 protooncogene expression and leukemic cell growth and survival: comparisons of phosphodiester and phosphorothioate oligodeoxynucleotides.
Cancer Res.
50:6565-6570 |
| 50. | Robinson, M. J., and M. H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9:180-186[CrossRef][Medline]. |
| 51. |
Roff, M.,
J. Thompson,
M. S. Rodriguez,
J. M. Jacque,
F. Baleux,
F. Arenzana-Seisdedos, and R. T. Hay.
1996.
Role of iB ubiquitination in signal-induced activation of NFB in vivo.
J. Biol. Chem.
271:7844-7850 |
| 52. |
Ruvolo, P. P.,
X. Deng,
B. K. Carr, and W. S. May.
1998.
A functional role for mitochondrial protein kinase C in Bcl2 phosphorylation and suppression of apoptosis.
J. Biol. Chem.
273:25436-25442 |
| 53. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 54. |
Scatena, C. D.,
Z. A. Stewart,
D. Mays,
L. J. Tang,
C. J. Keefer,
S. D. Leach, and J. A. Pietenpol.
1998.
Mitotic phosphorylation of Bcl-2 during normal cell cycle progression and taxol-induced growth arrest.
J. Biol. Chem.
273:30777-30784 |
| 55. | Schulze-Osthoff, K., P. H. Krammer, and W. Dröge. 1994. Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J. 13:4587-4596[Medline]. |
| 56. | Shibasaki, F., E. Kondo, T. Akagi, and F. McKeon. 1997. Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386:728-731[CrossRef][Medline]. |
| 57. |
Shrivastava, R. K.,
A. R. Srivastava,
S. J. Korsmeyer,
M. Nesterova,
Y. S. Cho-Chung, and D. L. Longo.
1998.
Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase.
Mol. Cell. Biol.
18:3509-3517 |
| 58. |
Sidon-de Fraisse, C.,
V. Rincheval,
Y. Risler,
B. Mignotte, and J. L. Vayssiere.
1998.
TNF activates at least two apoptotic signaling cascades.
Oncogene
17:1639-1651[CrossRef][Medline].
|
| 59. |
Warn-Cramer, B. J.,
P. D. Lampe,
W. E. Kurata,
M. Y. Kanemitsu,
L. W. Loo,
W. Eckhart, and A. F. Lau.
1996.
Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein.
J. Biol. Chem.
271:3779-3789 |
| 60. |
Yang, J.,
X. Liu,
K. Bhalla,
C. N. Kim,
A. M. Ibrado,
J. Cai,
T. I. Peng,
D. P. Jones, and X. Wang.
1997.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275:1129-1132 |
| 61. | Zou, H., W. J. Wenzel, X. Liu, A. Lutschg, and X. Wang. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:405-413[CrossRef][Medline]. |
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319: 1062-1069
[Abstract] [Full Text] -
Azad, N., Vallyathan, V., Wang, L., Tantishaiyakul, V., Stehlik, C., Leonard, S. S., Rojanasakul, Y.
(2006). S-Nitrosylation of Bcl-2 Inhibits Its Ubiquitin-Proteasomal Degradation: A NOVEL ANTIAPOPTOTIC MECHANISM THAT SUPPRESSES APOPTOSIS. J. Biol. Chem.
281: 34124-34134
[Abstract] [Full Text] -
Cottet, S., Michaut, L., Boisset, G., Schlecht, U., Gehring, W., Schorderet, D. F.
(2006). Biological characterization of gene response in Rpe65-/- mouse model of Leber's congenital amaurosis during progression of the disease. FASEB J.
20: 2036-2049
[Abstract] [Full Text] -
Chanvorachote, P., Nimmannit, U., Stehlik, C., Wang, L., Jiang, B.-H., Ongpipatanakul, B., Rojanasakul, Y.
(2006). Nitric Oxide Regulates Cell Sensitivity to Cisplatin-Induced Apoptosis through S-Nitrosylation and Inhibition of Bcl-2 Ubiquitination.. Cancer Res.
66: 6353-6360
[Abstract] [Full Text] -
Herold, M. J., Zeitz, J., Pelzer, C., Kraus, C., Peters, A., Wohlleben, G., Berberich, I.
(2006). The Stability and Anti-apoptotic Function of A1 Are Controlled by Its C Terminus. J. Biol. Chem.
281: 13663-13671
[Abstract] [Full Text] -
Choi, H., Kim, S.-H., Chun, Y.-S., Cho, Y.-S., Park, J.-W., Kim, M.-S.
(2006). In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2.. Exp. Biol. Med.
231: 463-472
[Abstract] [Full Text] -
Tatone, C., Carbone, M. C., Gallo, R., Delle Monache, S., Di Cola, M., Alesse, E., Amicarelli, F.
(2006). Age-Associated Changes in Mouse Oocytes During Postovulatory In Vitro Culture: Possible Role for Meiotic Kinases and Survival Factor BCL2. Biol. Reprod.
74: 395-402
[Abstract] [Full Text] -
Harada, C., Nakamura, K., Namekata, K., Okumura, A., Mitamura, Y., Iizuka, Y., Kashiwagi, K., Yoshida, K., Ohno, S., Matsuzawa, A., Tanaka, K., Ichijo, H., Harada, T.
(2006). Role of Apoptosis Signal-Regulating Kinase 1 in Stress-Induced Neural Cell Apoptosis in Vivo. Am. J. Pathol.
168: 261-269
[Abstract] [Full Text] -
Aouacheria, A., Brunet, F., Gouy, M.
(2005). Phylogenomics of Life-Or-Death Switches in Multicellular Animals: Bcl-2, BH3-Only, and BNip Families of Apoptotic Regulators. Mol Biol Evol
22: 2395-2416
[Abstract] [Full Text] -
Hoefnagel, J. J., Dijkman, R., Basso, K., Jansen, P. M., Hallermann, C., Willemze, R., Tensen, C. P., Vermeer, M. H.
(2005). Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling. Blood
105: 3671-3678
[Abstract] [Full Text] -
Shenoy, S. K., Lefkowitz, R. J.
(2005). Receptor-specific Ubiquitination of {beta}-Arrestin Directs Assembly and Targeting of Seven-transmembrane Receptor Signalosomes. J. Biol. Chem.
280: 15315-15324
[Abstract] [Full Text] -
Wu, H. M., Jin, M., Marsh, C. B.
(2005). Toward functional proteomics of alveolar macrophages. Am. J. Physiol. Lung Cell. Mol. Physiol.
288: L585-L595
[Abstract] [Full Text] -
Chen, J., St-Germain, J. R., Li, Q.
(2005). B56 Regulatory Subunit of Protein Phosphatase 2A Mediates Valproic Acid-Induced p300 Degradation. Mol. Cell. Biol.
25: 525-532
[Abstract] [Full Text] -
Marchetti, S., Gimond, C., Chambard, J.-C., Touboul, T., Roux, D., Pouyssegur, J., Pages, G.
(2005). Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation. Mol. Cell. Biol.
25: 854-864
[Abstract] [Full Text] -
Wei, H., Ahn, S., Barnes, W. G., Lefkowitz, R. J.
(2004). Stable Interaction between {beta}-Arrestin 2 and Angiotensin Type 1A Receptor Is Required for {beta}-Arrestin 2-mediated Activation of Extracellular Signal-regulated Kinases 1 and 2. J. Biol. Chem.
279: 48255-48261
[Abstract] [Full Text] -
Lee, J.-H., Jeon, M.-H., Seo, Y.-J., Lee, Y.-J., Ko, J. H., Tsujimoto, Y., Lee, J.-H.
(2004). CA Repeats in the 3'-Untranslated Region of bcl-2 mRNA Mediate Constitutive Decay of bcl-2 mRNA. J. Biol. Chem.
279: 42758-42764
[Abstract] [Full Text] -
Ahn, S., Shenoy, S. K., Wei, H., Lefkowitz, R. J.
(2004). Differential Kinetic and Spatial Patterns of {beta}-Arrestin and G Protein-mediated ERK Activation by the Angiotensin II Receptor. J. Biol. Chem.
279: 35518-35525
[Abstract] [Full Text] -
Sherrill, K. W., Byrd, M. P., Van Eden, M. E., Lloyd, R. E.
(2004). BCL-2 Translation Is Mediated via Internal Ribosome Entry during Cell Stress. J. Biol. Chem.
279: 29066-29074
[Abstract] [Full Text] -
Kanda, H., Miura, M.
(2004). Regulatory Roles of JNK in Programmed Cell Death. J Biochem
136: 1-6
[Abstract] [Full Text] -
Tejera, N., Gomez-Garre, D., Lazaro, A., Gallego-Delgado, J., Alonso, C., Blanco, J., Ortiz, A., Egido, J.
(2004). Persistent Proteinuria Up-Regulates Angiotensin II Type 2 Receptor and Induces Apoptosis in Proximal Tubular Cells. Am. J. Pathol.
164: 1817-1826
[Abstract] [Full Text] -
Ninomiya, Y., Kato, K., Takahashi, A., Ueoka, Y., Kamikihara, T., Arima, T., Matsuda, T., Kato, H., Nishida, J.-i., Wake, N.
(2004). K-Ras and H-Ras Activation Promote Distinct Consequences on Endometrial Cell Survival. Cancer Res.
64: 2759-2765
[Abstract] [Full Text] -
Deng, X., Gao, F., Flagg, T., May, W. S. Jr.
(2004). Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions. Proc. Natl. Acad. Sci. USA
101: 153-158
[Abstract] [Full Text] -
Harada, T., Harada, C., Wang, Y.-L., Osaka, H., Amanai, K., Tanaka, K., Takizawa, S., Setsuie, R., Sakurai, M., Sato, Y., Noda, M., Wada, K.
(2004). Role of Ubiquitin Carboxy Terminal Hydrolase-L1 in Neural Cell Apoptosis Induced by Ischemic Retinal Injury in Vivo. Am. J. Pathol.
164: 59-64
[Abstract] [Full Text] -
YANG, Y., YU, X.
(2003). Regulation of apoptosis: the ubiquitous way. FASEB J.
17: 790-799
[Abstract] [Full Text] -
Losman, J. A., Chen, X. P., Vuong, B. Q., Fay, S., Rothman, P. B.
(2003). Protein Phosphatase 2A Regulates the Stability of Pim Protein Kinases. J. Biol. Chem.
278: 4800-4805
[Abstract] [Full Text] -
Wall, N. R., O'Connor, D. S., Plescia, J., Pommier, Y., Altieri, D. C.
(2003). Suppression of Survivin Phosphorylation on Thr34 by Flavopiridol Enhances Tumor Cell Apoptosis. Cancer Res.
63: 230-235
[Abstract] [Full Text] -
Lei, K., Nimnual, A., Zong, W.-X., Kennedy, N. J., Flavell, R. A., Thompson, C. B., Bar-Sagi, D., Davis, R. J.
(2002). The Bax Subfamily of Bcl2-Related Proteins Is Essential for Apoptotic Signal Transduction by c-Jun NH2-Terminal Kinase. Mol. Cell. Biol.
22: 4929-4942
[Abstract] [Full Text] -
HUANG, S.-T. J., CIDLOWSKI, J. A.
(2002). Phosphorylation status modulates Bcl-2 function during glucocorticoid-induced apoptosis in T lymphocytes. FASEB J.
16: 825-832
[Abstract] [Full Text] -
Gordo, A. C., Rodrigues, P., Kurokawa, M., Jellerette, T., Exley, G. E., Warner, C., Fissore, R.
(2002). Intracellular Calcium Oscillations Signal Apoptosis Rather than Activation in In Vitro Aged Mouse Eggs. Biol. Reprod.
66: 1828-1837
[Abstract] [Full Text] -
Brouard, S., Berberat, P. O., Tobiasch, E., Seldon, M. P., Bach, F. H., Soares, M. P.
(2002). Heme Oxygenase-1-derived Carbon Monoxide Requires the Activation of Transcription Factor NF-kappa B to Protect Endothelial Cells from Tumor Necrosis Factor-alpha -mediated Apoptosis. J. Biol. Chem.
277: 17950-17961
[Abstract] [Full Text] -
Lapucci, A., Donnini, M., Papucci, L., Witort, E., Tempestini, A., Bevilacqua, A., Nicolin, A., Brewer, G., Schiavone, N., Capaccioli, S.
(2002). AUF1 Is a bcl-2 A + U-rich Element-binding Protein Involved in bcl-2 mRNA Destabilization during Apoptosis. J. Biol. Chem.
277: 16139-16146
[Abstract] [Full Text] -
Milella, M., Estrov, Z., Kornblau, S. M., Carter, B. Z., Konopleva, M., Tari, A., Schober, W. D., Harris, D., Leysath, C. E., Lopez-Berestein, G., Huang, Z., Andreeff, M.
(2002). Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia. Blood
99: 3461-3464
[Abstract] [Full Text] -
Dijkers, P. F., Birkenkamp, K. U., Lam, E. W.-F., Thomas, N. S. B., Lammers, J.-W. J., Koenderman, L., Coffer, P. J.
(2002). FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. JCB
156: 531-542
[Abstract] [Full Text] -
Stupack, D. G., Cheresh, D. A.
(2002). Get a ligand, get a life: integrins, signaling and cell survival. J. Cell Sci.
115: 3729-3738
[Abstract] [Full Text] -
Adams, M. L., Pierce, R. H., Vail, M. E., White, C. C., Tonge, R. P., Kavanagh, T. J., Fausto, N., Nelson, S. D., Bruschi, S. A.
(2001). Enhanced Acetaminophen Hepatotoxicity in Transgenic Mice Overexpressing BCL-2. Mol. Pharmacol.
60: 907-915
[Abstract] [Full Text] -
Torcia, M., De Chiara, G., Nencioni, L., Ammendola, S., Labardi, D., Lucibello, M., Rosini, P., Marlier, L. N. J. L., Bonini, P., Sbarba, P. D., Palamara, A. T., Zambrano, N., Russo, T., Garaci, E., Cozzolino, F.
(2001). Nerve Growth Factor Inhibits Apoptosis in Memory B Lymphocytes via Inactivation of p38 MAPK, Prevention of Bcl-2 Phosphorylation, and Cytochrome c Release. J. Biol. Chem.
276: 39027-39036
[Abstract] [Full Text] -
Jiang, A., Clark, E. A.
(2001). Involvement of Bik, a Proapoptotic Member of the Bcl-2 Family, in Surface IgM-Mediated B Cell Apoptosis. J. Immunol.
166: 6025-6033
[Abstract] [Full Text] -
Dimmeler, S., Zeiher, A. M.
(2000). Endothelial Cell Apoptosis in Angiogenesis and Vessel Regression. Circ. Res.
87: 434-439
[Abstract] [Full Text] -
Fleming, J. V., Wang, T. C.
(2000). Amino- and Carboxy-Terminal PEST Domains Mediate Gastrin Stabilization of Rat L-Histidine Decarboxylase Isoforms. Mol. Cell. Biol.
20: 4932-4947
[Abstract] [Full Text] -
Breitschopf, K., Zeiher, A. M., Dimmeler, S.
(2000). Ubiquitin-mediated Degradation of the Proapoptotic Active Form of Bid. A FUNCTIONAL CONSEQUENCE ON APOPTOSIS INDUCTION. J. Biol. Chem.
275: 21648-21652
[Abstract] [Full Text] -
Rossig, L., Haendeler, J., Hermann, C., Malchow, P., Urbich, C., Zeiher, A. M., Dimmeler, S.
(2000). Nitric Oxide Down-regulates MKP-3 mRNA Levels. INVOLVEMENT IN ENDOTHELIAL CELL PROTECTION FROM APOPTOSIS. J. Biol. Chem.
275: 25502-25507
[Abstract] [Full Text] -
Dijkers, P. F., Birkenkamp, K. U., Lam, E. W.-F., Thomas, N. S. B., Lammers, J.-W. J., Koenderman, L., Coffer, P. J.
(2002). FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. JCB
156: 531-542
[Abstract] [Full Text]
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