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INTRODUCTION |
The process of apoptosis is executed by a distinct genetic pathway
that is apparently shared by all multicellular organisms. The family of
Bcl-2-related proteins constitutes a class of apoptosis-regulatory gene
products that act at the effector stage of apoptosis. In vertebrates,
two functional classes of Bcl-2-related proteins exist that share
highly conserved Bcl-2 homology
(BH)1 1, BH2, BH3, and BH4
domains: (a) antiapoptotic members, including Bcl-2,
that inhibit cell death; and (b) proapoptotic members, including Bax, that accelerate apoptosis (reviewed in Refs. 1-3). Several gene ablation studies confirm that in vertebrates, the balance
between death-promoting and death-repressing members of the Bcl-2
family does indeed contribute a critical checkpoint that determines a
cell's susceptibility to an apoptotic stimulus (reviewed in Ref.
2).
Beside regulating apoptosis, a number of recent studies demonstrate
that Bcl-2-related proteins can also modulate the entry of quiescent
cells into the cell cycle (reviewed in Ref. 2). For instance, when
constitutively expressed in lymphocytes, Bcl-2 significantly delays the
response of both B and T cells to a variety of mitogenic stimuli
(4-6), whereas Bax exerts an opposite effect (7). The molecular
mechanism by which Bcl-2 alters cell cycle entry is poorly understood,
but mutagenesis experiments demonstrated that the apoptosis-inhibitory
and antiproliferative effects of Bcl-2 can be genetically separated
(8). However, mutations in the BH1 or BH2 domains of Bcl-2 (5) or
removal of its membrane insertion domain (9) interferes with Bcl-2's
ability to affect cell cycle entry, arguing that intactness of its
presumed hydrophobic pocket (10, 11) and membrane integration are
required for its cell cycle-inhibitory function.
A major question in relation to controlling the function of Bcl-2
family members is their regulation by extracellular signals. Several
studies demonstrated the phosphorylation of Bcl-2 in a variety of cell
lines (12-18). Among these, cytokine-induced survival of an
IL-3-dependent myeloid cell line temporally correlated with serine phosphorylation of Bcl-2 (12), suggesting growth
factor-initiated modulation of its function. However, the role of this
post-translational modification on regulating Bcl-2's function is
controversial. For instance, whereas Bcl-2 phosphorylation correlated
with Taxol-induced apoptosis in some cell lines (14, 15, 17), in other
cells types, phosphorylation of Bcl-2 was suggested to correlate with its antiapoptotic function (12, 16, 18).
In light of the emerging dual role of Bcl-2 as both an apoptosis-
and cell cycle-inhibitory protein, we considered whether phosphorylation selectively alters one function of the molecule in
certain cell types. To examine the structural requirements for Bcl-2
phosphorylation and the temporal correlation of Bcl-2 phosphorylation
status and its function, we have utilized an IL-3-dependent lymphoid cell line, FL5.12, in which Bcl-2 blocks apoptosis after growth factor withdrawal (19). Of note, IL-3 promotes cell growth by
simultaneously stimulating cell proliferation and suppressing apoptosis. This effect is achieved by receptor engagement-initiated activation of several distinct signal transduction pathways that regulate effector components of these two major cell fates (20, 21).
Here we show that in the presence of IL-3, a fraction of constitutively
expressed Bcl-2 was phosphorylated on serine residue(s), and this
phosphorylated pool of Bcl-2 lost its capacity to heterodimerize with
Bax. Whereas the majority of Bcl-2 resided in mitochondria, phosphorylation involved a minor pool of total Bcl-2 that selectively partitioned into a soluble fraction. Cytosolic targeting of Bcl-2 by
deletion of its membrane insertion domain greatly increased its ratio
of phosphorylation. The reduced phosphorylation of Bcl-2 upon IL-3
deprivation and its delayed rate of phosphorylation upon cytokine
restimulation temporally correlated with the accelerated exit and
delayed reentry of Bcl-2-expressing cells into the cell cycle. Thus,
above a threshold level of Bcl-2 expression, IL-3-induced phosphorylation of a distinct pool of Bcl-2 may represent a selective inactivation mechanism of its antiproliferative function.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Apoptosis Induction--
The
IL-3-dependent murine cell line FL5.12, a lymphoid
progenitor clone, and all its derivatives were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) and 10% WEHI-3B conditional medium as a
source of IL-3 (19). To induce apoptosis, FL5.12 cells were washed
three times in serum-free medium to remove the growth factor and
cultured in the absence or presence of 25 IU/ml recombinant murine IL-3 (Genzyme).
Antibodies--
mAbs 6C8 (a human Bcl-2-specific hamster mAb;
Ref. 19), 4D2 (a murine Bax-specific hamster mAb; Ref. 22), and 3F11 (a murine Bcl-2-specific hamster mAb; Ref. 23) were used. mAb 124 (a human
Bcl-2-specific murine mAb) was purchased from DAKO and used for Western
immunostaining at a dilution of 1:100.
Metabolic Labelings and Immunoprecipitation--
Before
metabolic labeling with [35S]methionine, cells were
washed three times in prewarmed, serum-free, methionine-free
Dulbecco's medium (Life Technologies, Inc.). Cells were resuspended at
3-5 × 106 cells/ml in methionine-free Dulbecco's
medium supplemented with 10% phosphate-buffered saline (PBS)-dialyzed
fetal calf serum with or without 50 IU/ml recombinant murine IL-3
(Genzyme). Metabolic labeling was performed with 40 uCi/ml
[35S]methionine and [35S]cysteine
(Translabel; ICN) for the indicated times before lysis. For
phosphorylation studies, cells were washed three times in prewarmed,
serum-free, phosphor-free RPMI 1640 medium (Life Technologies, Inc.).
Cells were resuspended at 3-5 × 106 cells/ml in
phosphor-free RPMI 1640 medium supplemented with 10% water-dialyzed
fetal calf serum with or without 50 IU/ml recombinant murine IL-3
(Genzyme). Metabolic labeling was performed with 100 uCi/ml
[32P]orthophosphoric acid (ICN) for the indicated times
before lysis. All steps of the subsequent immunoprecipitations were
carried out as described previously (22, 24), except that some gels containing 32P-labeled proteins were transferred on
polyvinylidine difluoride membrane and immunostained
(see below) before autoradiography.
Subcellular Fractionation--
Cells were metabolically labeled
as described above and lysed in hypotonic buffer (42.5 mM
KCl, 5 mM MgCl2, and 10 mM HEPES, pH 7.4) by passaging them four times through a 30-gauge needle. Isotonicity was reestablished by adding an equal volume of hypertonic buffer (242.5 mM KCl, 5 mM MgCl2,
and 10 mM HEPES, pH 7.4). Nuclei and unlysed cells were
pelleted twice at 200 × g for 10 min. The supernatant
was centrifuged at 10,000 × g for 10 min to collect the heavy membrane pellet. That supernatant was centrifuged at 100,000 × g for 60 min, and the final supernatant was
collected as the soluble fraction, and the pellet was collected as the
light membrane fraction. The heavy membrane pellet was washed twice in
H medium (0.25 mM mannitol, 0.075 M sucrose, 1 mM EGTA, 5 mM HEPES, pH 7.4, and 0.1% fatty
acid-free bovine serum albumin). Both heavy and light membrane pellets
were lysed in RIPA buffer and, together with the soluble fraction, were
immunoprecipitated with mAb 6C8 as described above.
Western Blotting and Immunostaining--
For immunoblots,
proteins were electrotransferred at 4 °C on polyvinylidine
difluoride membranes (Millipore). Filters were blocked for 2 h
with 3% non-fat milk in PBS. All additional immunostaining steps were
performed in PBS with 0.05% Tween-20 (PBS-T) at room temperature.
Filters were incubated with primary antibody and species-specific
biotinylated secondary mAb (1:300) for 2 h. Immunoblots were
reacted with horseradish peroxidase-streptavidin (1:1000; Pierce) for
1 h. Filters were washed in PBS-T four times for 5 min between
each step and developed with diazobenzidine (Bio-Rad) enhanced with
nickel chloride (0.03%).
Cell Cycle Analysis--
Cell cycle analysis was performed as
described previously (25). Briefly, 1 × 106 cells
were washed in PBS, and the pellet was gently resuspended in 1 ml of
hypotonic fluorochrome solution (3.4 mM sodium citrate, pH
7.8, 100 µg/ml propidium iodide, 180 units/ml RNase A, 0.1% Triton
X-100, and 30 mg/ml polyethylene glycol) and incubated for 20 min at
37 °C. The cell suspension was then supplemented with 1 ml of
hypertonic solution (356 mM NaCl, 100 µg/ml propidium iodide, 0.1% Triton X-100, and 30 mg/ml polyethylene glycol) and stored at least for 6 h at 4 °C before analysis. Propidium
iodide fluorescence of individual nuclei were measured and analyzed
using a FACScan flow cytometer (Becton Dickinson).
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RESULTS |
Phosphorylation of Bcl-2 Is Independent of Its Capacity to
Heterodimerize with Bax--
Phosphorylation of Bcl-2 was previously
observed in selected cell lines (12-18). However, the physiologic role
of this post-translational modification is controversial (15, 16, 18).
Also, the structural features of Bcl-2 required for this
post-translational modification were not examined in detail.
Our studies utilized the IL-3-dependent early lymphoid
progenitor murine cell line FL5.12, whose viability and proliferation is maintained by a minimum of 25 IU/ml recombinant murine IL-3 (data
not shown). In the absence of IL-3, FL5.12 dies by apoptosis, but
overexpression of Bcl-2 significantly extends its survival without
maintaining its proliferation (19). The Bcl-2 expression level in these
FL5.12-Bcl-2 clones (26, 27) was approximately the same as that seen in
a cell line established from a patient with t(14;18)-bearing follicular
B-cell lymphoma or in pre-B cells (19), thus representing
physiologically relevant protein levels. Of note, FL5.12 cells do
express a significant amount of endogenous Bax as well as a low amount
of endogenous Bcl-2 that is insufficient to provide protection against
IL-3 deprivation-induced apoptosis (24).
To investigate whether phosphorylation of Bcl-2 is dependent on its
capacity to heterodimerize with Bax, FL5.12 clones stably expressing
either wild type human Bcl-2 (Bcl-2) or a BH1 substitution mutant of
human Bcl-2 (mI-3)(G145A) were examined. This mutant fails
to counter apoptosis in FL5.12 cells (26) and heterodimerize with Bax
in a yeast two-hybrid assay (22). Equal numbers of FL5.12-Bcl-2 and
FL5.12-Bcl-2 mI-3 cells were metabolically labeled for 6 h with
either [32P]orthophosphoric acid or
[35S]methionine in the presence of recombinant murine
IL-3 and subsequently immunoprecipitated with the human Bcl-2-specific
6C8 mAb (19). FL5.12-Bcl-2 and FL5.12-Bcl-2 mI-3 cells contained a
comparable amount of [35S)]methionine-labeled (Fig.
1A, lanes 2 and 3)
and [32P]orthophosphoric acid-labeled (Fig. 1A,
lanes 1 and 4) proteins. Thus, phosphorylation of Bcl-2
is not dependent on its capacity to dimerize with Bax in FL5.12 cells.
Also, phosphoamino acid analysis of Bcl-2 confirmed the IL-3-induced
phosphorylation of Bcl-2 exclusively on serine residue(s) (Fig.
1B), as described previously (12).

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Fig. 1.
Analysis of metabolically labeled
immunoprecipitates. A, cell lysates of
[32P]orthophosphoric acid- and
[35S]methionine-labeled FL5.12-Bcl-2 or FL5.12-Bcl-2 mI-3
cells that were incubated in the presence of IL-3 and
immunoprecipitated for human Bcl-2 with the 6C8 mAb. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis. B, phosphoamino acid analysis of
immunoprecipitated Bcl-2 from A. Migration of free phosphate
(Pi), phosphoserine (P-Ser), phosphothreonine
(P-Thr), and phosphotyrosine (P-Tyr) markers is
indicated.
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The Native Conformation of Bcl-2 Is Altered by Its
Phosphorylation--
In FL5.12 cells, the antiapoptotic Bcl-2 molecule
resides predominantly in the mitochondria, whereas the majority of
proapoptotic Bax is located in the cytosol in monomeric form (28). Upon
apoptosis induction, cytosolic Bax undergoes a change in its
conformation and translocates to mitochondria (28-30). Here it can
heterodimerize with Bcl-2 (28, 31), presumably involving interactions
between their BH1, BH2, and BH3 domains (10, 11, 32). In the absence of
proapoptotic signals, nonionic detergents, such as Nonidet P-40, are
able to induce this conformational change of Bax and permit Bcl-2/Bax
dimerization in cell lysates (33, 34).
To test whether the native conformation of Bcl-2 is altered by its
phosphorylation, nonionic detergent-induced dimerization between Bcl-2
and Bax was examined using co-immunoprecipitation experiments on
Nonidet P-40 lysates of FL5.12-Bcl-2 cells. To compare the relative
efficiency of co-immunoprecipitations with human Bcl-2-specific mAb 6C8
and murine Bax-specific mAb 4D2, [35S]methionine labeling
was performed. Parallel co-immunoprecipitations on Nonidet P-40 lysates
of FL5.12-Bcl-2 cells revealed a reduced amount of Bcl-2 in anti-Bax
4D2 immunoprecipitates compared with the total amount of Bcl-2
precipitated through the human Bcl-2-specific 6C8 mAb (Fig.
2A).

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Fig. 2.
Phosphorylated Bcl-2 does not heterodimerize
with Bax. A and B, cell lysates of (A)
[35S]methionine-labeled FL5.12-Bcl-2 cells and
(B) [32P]orthophosphoric acid-labeled FL5.12
Bcl-2 or FL5.12-NeoR cells were immunoprecipitated with the
4D2 mAb for murine Bax or the 6C8 mAb for human Bcl-2. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis.
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To examine anti-Bcl-2 and anti-Bax mAb immunoprecipitates of
[32P]orthophosphoric acid-labeled Nonidet P-40 cell
lysates, gels were transferred onto polyvinylidine difluoride membranes
and immunostained with human Bcl-2-specific mAb 124. This
immunostaining demonstrated human Bcl-2 expression in FL5.12-Bcl-2
cells in a ratio comparable to that seen in
[35S]methionine-labeled samples (data not shown).
Autoradiography of the same membrane revealed the expected ~25-kDa
phosphorylated Bcl-2 in the anti-Bcl-2 immunoprecipitates of
[32P]orthophosphoric acid-labeled FL5.12-Bcl-2 lysates.
In contrast, no corresponding protein could be immunoprecipitated with
the anti-Bax 4D2 mAb. The faint ~25-kDa protein band visible in this lane is likely to represent the [32P]orthophosphoric
acid-contaminated light chain of the anti-Bax mAb utilized in the
experiment (Fig. 2B). Thus, Nonidet P-40 is unable to induce
dimerization between phosphorylated Bcl-2 and Bax, suggesting a
conformational change of Bcl-2 after its phosphorylation.
Phosphorylated Bcl-2 Resides in a Soluble Subcellular Pool--
In
FL5.12 cells, the majority of Bcl-2 resides in the mitochondria as an
integral membrane protein (19, 28). A possible consequence of a
phosphorylation-induced change in Bcl-2's conformation is a
modification of the molecule's intracellular targeting.
To explore this idea, subcellular fractionations of FL5.12-Bcl-2 cells
were performed as described previously (19, 28). As an internal
control, we used a C-terminally truncated human Bcl-2 mutant (Bcl-2
TM), in which the last 22 amino acids of the molecule that comprises
its membrane insertion domain were removed (27, 35). Of note, deletion
of the membrane insertion domain of Bcl-2 shifts its localization from
a membrane-bound form to the cytosol, as shown by both subcellular
fractionation (27, 36, 37) and immunofluorescent localization (36, 38, 39) studies. Briefly, cells were labeled with
[35S]methionine or [32P]orthophosphoric
acid in the presence of IL-3 for 6 h and, after the removal of
nuclear fractions and unlysed cells, were subfractionated to heavy
membrane, light membrane, and soluble cytosolic fractions by
differential ultracentrifugation. Heavy membrane fractions enriched in
mitochondria and the fraction containing light membranes enriched in
endoplasmic reticulum were solubilized with RIPA lysis buffer. These
lysates and the remaining soluble S100 fraction representing the
cytosol were immunoprecipitated with anti-Bcl-2 6C8 mAb, and the
subcellular localization patterns of [35S]methionine- and
[32P]orthophosphoric acid-labeled Bcl-2 were compared.
The majority of [35S]methionine-labeled Bcl-2 localized
to the heavy membrane fraction, with only minor amounts observed in the
light membrane and S100 fractions, whereas Bcl-2
TM localized to the
S100 fraction (Fig. 3A, left
panels), as described previously (27, 36, 37). However,
phosphorylated Bcl-2 and Bcl-2
TM were both found almost exclusively
in the S100 fraction (Fig. 3A, right panels). We conclude
that whereas the majority of Bcl-2 is localized to the heavy membrane
fraction in FL5.12-Bcl-2 cells, the membrane integration of
phosphorylated Bcl-2 is compromised.

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Fig. 3.
Subcellular distribution of phosphorylated
Bcl-2. A, [35S]methionine-labeled (left
panels) or [32P]orthophosphoric acid-labeled
(right panels) FL5.12-Bcl-2 and FL5.12-Bcl-2 TM cells
were subfractionated to heavy membrane (HM), light membrane
(LM), and soluble S100 (S) fractions. Fractions
were immunoprecipitated with the 6C8 mAb for human Bcl-2. B,
lysates of [35S]methionine-labeled (top panel)
or [32P])orthophosphoric acid-labeled (bottom
panel) FL5.12-Bcl-2, FL5.12-Bcl-2 mI-3, and FL5.12-Bcl-2 TM
cells were immunoprecipitated with the 6C8 mAb for human Bcl-2.
Immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis.
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Cytosolic Targeting Substantially Enhances Bcl-2
Phosphorylation--
Because phosphorylated Bcl-2 localized to the
S100 cytosolic fraction, we next asked whether targeting Bcl-2 to the
cytosol by removal of its membrane insertion domain enhances its rate of phosphorylation. To this end, FL5.12-Bcl-2, FL5.12-Bcl-2 mI-3, and
FL5.12-Bcl-2
TM cells were labeled with
[35S]methionine or [32P]orthophosphoric
acid in the presence of IL-3 for 6 h, lysed in RIPA lysis buffer,
and immunoprecipitated with the human Bcl-2-specific 6C8 mAb.
Immunoprecipitates of [35S]methionine-labeled cell
lysates revealed a comparable amount of Bcl-2 expression in all three
cell lines (Fig. 3B, top panel). However, Bcl-2
TM proved
~50× more phosphorylated than the mostly membrane-bound Bcl-2 and
Bcl-2 mI-3 proteins (Fig. 3B, bottom panel). Thus, targeting
Bcl-2 into the cytosol by preventing its membrane association
substantially enhances its level of phosphorylation.
Bcl-2 Delays IL-3-induced Cell Proliferation in FL5.12
Cells--
Constitutively expressed Bcl-2 prevents the apoptosis of
FL5.12 cells upon IL-3 withdrawal (19, 24). As only a minor S100 fraction of Bcl-2 is phosphorylated in the presence of IL-3, the physiologic role of this post-translational modification may not directly relate to the molecule's apoptosis-inhibitory function in
these cells. Besides inhibiting apoptosis, several studies demonstrated
that Bcl-2 can also delay the entry of cells into the cell cycle
(reviewed in Ref. 2). Thus, we wished to examine the potential
relationship between Bcl-2's phosphorylation status and its
antiproliferative effect in these cells.
First, the cell cycle status of FL5.12-NeoR and two
FL5.12-Bcl-2 clones was tested after transient IL-3 deprivation and
subsequent IL-3 restimulation, in a manner similar to that described
previously (40). Before IL-3 deprivation, the apparent rate of cell
proliferation and the ratio of cells in S phase + G2/M
phase were essentially identical in all clones (Fig.
4A, 0hr), although
the proportion of Bcl-2-expressing cells in S phase was somewhat lower
than that in FL5.12-NeoR cells (Fig. 4B,
0hr). Upon transient IL-3 withdrawal, Bcl-2-expressing clones started to accumulate in the G1 phase of the cell
cycle faster than NeoR cells, and the ratio of
Bcl-2-expressing cells in G2/M phase also increased.
Because control FL5.12-NeoR cells remain fully viable for
only 12 h after cytokine deprivation (24), IL-3 was added back to
all clones at this time. 12 h after the readdition of IL-3, a
lower proportion of FL5.12-Bcl-2 cells was in the S phase + G2/M phase of the cell cycle compared with FL5.12-NeoR cells (Fig. 4A, + 12hr).
At this time, FL5.12-NeoR cells demonstrated a synchronous
entry of cells into S phase, which was delayed in FL5.12-Bcl-2 cells
(Fig. 4B, + 12hr). By 24 h after the
readdition of IL-3, this transient difference had disappeared (Fig. 4,
A and B). These data demonstrate that similarly to that seen before (40), Bcl-2 is able to provoke a temporary refractoriness to IL-3-stimulated cell proliferation in FL5.12 cells.

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Fig. 4.
Cell cycle status of FL5.12 clones after IL-3
deprivation and subsequent IL-3 restimulation. The percentage of
FL5.12-NeoR, FL5.12-Bcl-2-1, and FL5.12-Bcl-2-2 clones in S
phase + G2/M phase (A) or S phase (B)
before (0hr) and at the designated time points after IL-3
deprivation ( ) or after the IL-3 readdition after 12 h of
cytokine deprivation (+). Cell numbers are shown as the mean ± S.D. from triplicate samples. Each clone was tested three times with
similar results.
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Phosphorylation of Bcl-2 Is Dependent on the Presence of
Interleukin-3--
To determine the temporal correlation of Bcl-2's
phosphorylation status and its antiproliferative function, FL5.12-Bcl-2
and FL5.12-NeoR cells were metabolically labeled with
either [32P]orthophosphoric acid or
[35S]methionine in the presence or absence of recombinant
murine IL-3 and solubilized with RIPA lysis buffer at distinct time
points thereafter.
When lysates of FL5.12-NeoR cells were immunoprecipitated
with the endogenous murine Bcl-2-specific mAb 3F11, no phosphorylation of endogenous Bcl-2 was detected during the first 8 h of
[32P]orthophosphoric acid labeling (Fig.
5A, top panel) in either the
presence (+) or absence (
) of IL-3. Similarly, lysates of FL5.12-NeoR (or FL5.12-Bcl-2) cells did not demonstrate any
phosphorylation of Bax when precipitated with the anti-Bax mAb within
the same time frame (Fig. 5A, middle panel). In contrast,
efficient phosphorylation of constitutively expressed Bcl-2 was
detected in the presence of IL-3 when it was immunoprecipitated with
the human Bcl-2-specific 6C8 mAb (Fig. 5A, bottom panel, +).
However, in the absence of IL-3 during metabolic labeling, a
significant reduction in the amount of phosphorylated human Bcl-2 was
observed (Fig. 5A, bottom panel,
).

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Fig. 5.
Reduced phosphorylation of Bcl-2 during IL-3
deprivation. A and B, cell lysates of
(A) [32P]orthophosphoric acid-labeled and
(B) [35S]methionine-labeled
FL5.12-NeoR or FL5.12-Bcl-2 cells incubated in the presence
(+) or absence ( ) of IL-3 were prepared at the indicated times and
immunoprecipitated for endogenous murine Bcl-2 with the 3F11 mAb
(top panels), for endogenous murine Bax with the 4D2 mAb
(middle panels), and for overexpressed human Bcl-2 with the
6C8 mAb (bottom panels). The immunoprecipitates were
analyzed by SDS-polyacrylamide gel electrophoresis. Autoradiographies
were performed for various lengths of time to equalize the visible
signals.
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To ascertain that the differences seen in the phosphorylation of
overexpressed Bcl-2 in the presence or absence of IL-3 were not due
to differences in the rate of its de novo protein synthesis, [35S]methionine labeling was performed within the same
time course. Of note, the amount of endogenous murine Bcl-2 is about
10% compared with the amount of overexpressed human Bcl-2 (19),
whereas endogenous Bax levels are comparable to that of overexpressed
Bcl-2 (24). Both endogenous Bcl-2 and Bax incorporated a detectable
amount of radiolabeled methionine at the same rate in the presence or absence of IL-3 (Fig. 5B, top and middle panels).
Similarly, metabolic labeling demonstrated identical amount of newly
synthesized constitutively expressed Bcl-2 in the presence or absence
of the cytokine (Fig. 5B, bottom panel). We conclude that
above a threshold level of protein expression, a distinct pool of Bcl-2
is phosphorylated in the presence of IL-3, but upon cytokine
deprivation, Bcl-2 is either dephosphorylated or its phosphorylation is inefficient.
IL-3-induced Bcl-2 Phosphorylation Correlates with Reentry of
FL5.12 Cells into the Cell Cycle--
To further examine the temporal
correlation of Bcl-2's phosphorylation status and its cell cycle
inhibitory function, we determined the phosphorylation status of Bcl-2
after transient IL-3 deprivation and subsequent IL-3 restimulation. To
this end, FL5.12-Bcl-2 cells were first transiently deprived of IL-3
for 12 h. These cells were then metabolically labeled with
[32P]orthophosphoric acid or
[35S]methionine in the presence of recombinant IL-3 for
3, 6, and 12 h (Fig. 6, IL-3
Depr.). As controls, identical metabolic labeling was performed on
FL5.12-Bcl-2 cells that were incubated in the continuous presence of
IL-3 (Fig. 6, Control). Samples were lysed in RIPA lysis
buffer and immunoprecipitated with the human Bcl-2-specific 6C8 mAb at
the indicated time points.

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Fig. 6.
Delayed phosphorylation of Bcl-2 after
transient IL-3 deprivation. Cell lysates of
[32P]orthophosphoric acid-labeled (top panels)
and [35S]methionine-labeled FL5.12-Bcl-2 cells
(bottom panels) incubated for the indicated time in the
presence of IL-3, with (IL-3 Depr.) or without
(Control) 12 h of transient cytokine deprivation.
Samples were immunoprecipitated for human Bcl-2 with the 6C8 mAb. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis.
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Compared with control cells, the amount of phosphorylated Bcl-2 was
reduced in transiently IL-3-deprived cells at all time points tested.
This difference was most pronounced at 3 h after the initiation of
metabolic labeling. However, at 6 h, but not at 12 h, the
phosphorylation level of Bcl-2 was still weaker in the transiently
IL-3-deprived cells than in control cells (Fig. 6, top
panel). To ascertain that the differences seen in Bcl-2's level
of phosphorylation were not due to differences in the rate of its
de novo protein synthesis, [35S]methionine
labeling was performed within the same time course. As shown in Fig. 6,
bottom panel, both transiently IL-3-deprived and control
cells incorporated radiolabeled methionine at similar rates at all time
points. Thus, after transient cytokine withdrawal, the delayed rate of
IL-3-stimulated phosphorylation of Bcl-2 (Fig. 6) correlated to a
certain degree (3 and 6 h) with the reduced sensitivity of
FL5.12-Bcl-2 cells to IL-3-stimulated cell proliferation (Fig.
4).
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DISCUSSION |
In vertebrates, death-promoting and death-repressing members of
the Bcl-2-related proteins are important regulators of the effector
stage of apoptosis. The regulation of their biologic activity is poorly
understood, but differential subcellular targeting of these molecules
is clearly involved. For instance, Bcl-2 is an integral membrane
protein that resides in mitochondria, endoplasmic reticulum, and
nuclear membranes (19, 41, 42). In contrast, the majority of
proapoptotic Bax is located in the cytosol in monomeric inactive form
(28, 29). Upon apoptosis induction, cytosolic Bax translocates from the
cytosol to the mitochondria, where it displays its apoptosis-inducing
function (28-30), which perhaps involves its heptamerization (43).
Alternatively, mitochondrial Bax can heterodimerize with Bcl-2 (28,
31), an association that is likely to involve interactions between
their BH1, BH2, and BH3 domains. NMR and x-ray crystallography
structure of Bcl-xL monomer (10) and of a
Bcl-xL-Bak BH3 peptide complex (32) revealed both
hydrophobic and electrostatic interactions between the BH3 amphipathic
-helix and the Bcl-xL hydrophobic pocket formed by its
BH1, BH2 and BH3 domains (32). Selective BH1 mutations that abolish
Bcl-2's heterodimerization capacity with Bax in a yeast two-hybrid
assay (22) can also reduce its antiapoptotic function in mammalian
cells (26, 44), further underlying the functional significance of this interaction.
Another type of functional regulation of Bcl-2 family members involves
their post-translational modification by phosphorylation. For instance,
phosphorylation of proapoptotic Bad (45) on two serine residues in
response to IL-3 stimulation promotes its cytosolic targeting and
association with the 14-3-3 family of proteins (46). Similarly, a
number of studies demonstrated the phosphorylation of Bcl-2 in a
variety of cell lines, but the functional consequence of this
post-translational modification remains unclear. In some studies,
chemotherapeutic-induced apoptosis correlated with concomitant phosphorylation of Bcl-2 (14, 15, 17, 47, 48), whereas in others
phosphorylation of Bcl-2 correlated with its antiapoptotic function
(12, 16, 18). Among these, cytokine-induced survival of an
IL-3-dependent myeloid cell line temporally correlated with serine phosphorylation of Bcl-2 (12), suggesting growth
factor-initiated modulation of its function.
Besides regulating apoptosis, Bcl-2-related proteins can also modulate
the entry of quiescent cells into the cell cycle (reviewed in Ref. 2),
suggesting a cell autonomous coordination between proliferation and
cell death. In light of the emerging dual role of Bcl-2 as both an
apoptosis- and cell cycle-inhibitory protein, we considered whether
phosphorylation of Bcl-2 may differentially alter just one function of
the molecule in certain cell types. To examine the temporal correlation
of Bcl-2's phosphorylation status and its antiproliferative function,
we have utilized an IL-3-dependent lymphoid cell line in
which Bcl-2 blocks apoptosis after growth factor withdrawal.
The data presented in this paper suggest that IL-3-induced
phosphorylation of Bcl-2 may temporally correlate better with
abrogation of its cell cycle-inhibitory effect than with regulation of
its apoptosis-inhibitory function. First, transient IL-3 deprivation resulted in a reduced level of Bcl-2 phosphorylation and, in time, correlated with the accelerated exit of Bcl-2-expressing cells from the
cell cycle (Figs. 4 and 5). Similarly, upon cytokine restimulation,
Bcl-2-expressing clones exhibited a temporary refractoriness to
IL-3-induced cell proliferation that correlated with the delayed rate
of Bcl-2 phosphorylation (Figs. 4 and 6). Thus, in both cases, the
phosphorylation status of Bcl-2 correlated with its antiproliferative effect. Also, in FL5.12 cells, Bcl-2 provides an extended protection from IL-3 deprivation-induced apoptosis (19). Taken together, these
data suggest that IL-3-induced phosphorylation of Bcl-2 may contribute
to the inactivation of its antiproliferative function rather than
altering Bcl-2's antiapoptotic effect. To directly test this
hypothesis, identification of IL-3-induced Bcl-2 phosphorylation sites
and the creation and testing of phosphorylation-deficient Bcl-2 mutants
will be needed.
Several considerations also suggest that in FL5.12 cells,
IL-3-stimulated Bcl-2 kinase may not affect the majority of
constitutively expressed Bcl-2. First, in FL5.12 cells in the presence
of IL-3, Bcl-2 predominantly localized to the mitochondria-rich heavy
membrane fraction, in accordance with that described previously (Refs. 19 and 28; Fig. 3A, left panel). However, almost all
phosphorylated Bcl-2 resided in the soluble S100 fraction (Fig.
3A, right panel). This demonstrates that
phosphorylation-induced conformational change of Bcl-2 results in the
loss of its capacity for firm membrane integration. Second, Bcl-2
TM, a Bcl-2 mutant that is predominantly cytosolic due to deletion
of its membrane insertion domain, was ~50× more phosphorylated than
membrane integrated Bcl-2 when both exhibited a similar overall protein
expression level (Fig. 3B). This suggests that
IL-3-stimulated Bcl-2 kinase is sufficiently active to phosphorylate
perhaps all intracellular Bcl-2. Consequently, the majority of Bcl-2 is
apparently shielded from this kinase activity and integrates to the
mitochondria in the presence of IL-3.
But what shields Bcl-2 from this kinase effect? Two separate scenarios
can be envisioned. First, IL-3-induced Bcl-2 kinase may require
sufficient time to phosphorylate cytosolic Bcl-2. Thus, rapid
mitochondrial integration of newly synthesized Bcl-2 may prevent such
an effect. However, slower integration to alternative sites, such as
endoplasmic reticulum membranes or selected mitochondrial subregions,
may allow sufficient time for phosphorylation to take place that
subsequently prevents Bcl-2's membrane integration (Fig.
7). Of note, in vitro
targeting experiments demonstrated that efficient insertion of Bcl-2
into the mitochondrial outer membrane is mechanistically different from
its comparatively low-affinity association with endoplasmic reticulum
that may not be dependent on its C-terminal membrane insertion domain
(35). In the second scenario, Bcl-2 that is already inserted in the
membrane may show a differential sensitivity to kinase activity
according to its site of integration. Thus, a small pool of
mitochondria- or endoplasmic reticulum-localized Bcl-2 with subtle
differences in its protein conformation may be selectively accessible
to IL-3-stimulated Bcl-2 kinase, whereas the majority of its
mitochondrial counterpart is shielded from it. Expression of Bcl-2
mutants targeted selectively to the endoplasmic reticulum or
mitochondrial subregions in FL5.12 cells will be needed to clarify this
issue.