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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1768-1775
Phosphorylation of BCL-2 After Exposure of Human Leukemic Cells
to Retinoic Acid
By
Z.-B. Hu,
M.D. Minden, and
E.A. McCulloch
From The Ontario Cancer Institute and Princess Margaret Hospital,
Toronto, Canada.
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ABSTRACT |
Serine phosphorylation of bcl-2 has been reported after treatment of
cells with protein kinase C, okadaic acid, taxol, and other
chemotherapeutic agents that attack microtubules. We report here that
bcl-2 is phosphorylated on serine in acute myeloblastic leukemia (AML)
blasts exposed to all trans retinoic acid (ATRA). Two-dimension gels
(isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis [SDS-PAGE]) disclosed a novel acidic isoform of
bcl-2 in ATRA-treated blast cells from a continuous line and from two
AML patients; when the cell lysates were digested with  -phosphatase,
bcl-2 reverted to the control position, indicating that it was
phosphorylated. Metabolic labeling experiments using
32Pi showed that, while control bcl-2 was
labeled, incorporation was greatly increased when cells were treated
with ATRA. A comparison of bcl-2 from blasts treated with ATRA or taxol
showed that bcl-2 was phosphorylated on serine in cells treated with
either agent; however, both qualitative and quantitative differences
were seen. Qualitatively, the phosphorylated isoform from taxol-treated
cells was slightly larger than the native isoform and could be
distinguished on 10% to 20% SDS-polyacrylamide gradient gels, while
the phosphorylated bcl-2 after ATRA ran as a single band on gradient
gels at the same position as control bcl-2. Quantitatively, all bcl-2
from ATRA-treated cells was in the phosphorylated isoform, while after taxol, both phosphorylated and native bcl-2 was present; incorporation of 32Pi into bcl-2 was stimulated to greater
extent in ATRA-treated compared with taxol-treated cells. We used
immunoprecipitation experiments to ask if bcl-2 phosphorylated after
ATRA or taxol had altered capacity to dimerize with bax. No change in
dimerization was demonstrated. We conclude that: bcl-2 is
phosphorylated on serine after treatment of AML blasts with ATRA; bcl-2
phosphorylation after ATRA is different from that seen after taxol;
bcl-2 phosphorylated after either agent retains capacity to dimerize
with bax. The ATRA or taxol-induced phosphorylation of bcl-2 can also
be seen in blast cells obtained from AML patients.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CANCER CELLS RESPOND to many
chemotherapeutic agents by programmed cell death or
apoptosis.1-4 Apoptosis is regulated by a number of
proteins or phospholipids that either promote cell death or protect
against it.2,5,6 These regulators may affect the outcome of
chemotherapy by changing the probabilities that cells will recover or
complete apoptosis. Members of the bcl-2 family are prominent among the
regulators of apoptosis.7-9 The family contains members
that protect against cell death, such as bcl-210 and
bcl-xL11; others, such as bax12 and
bad13 promote apoptosis. Members of the bcl-2 family share
two homology regions, BH1 and BH2, that allow them readily to form homo
and heterodimers.14 Dimerization between bcl-2 family
members with life and death promoting functions has been proposed as a
mechanism for regulating apoptosis after injury, including exposure to
chemotherapeutic agents.9,12
Posttranslational modification of bcl-2 family members by
phosphorylation may also be important in the regulation of apoptosis. Haldar et al15 reported phosphorylation of bcl-2 after
treatment with okadaic acid and suggested that the function of the
protein was inhibited. Observations of bcl-2 phosphorylation after
taxol treatment16,17 provided a link with chemotherapy, as
inhibition of bcl-2 function might increase cell kill.
Phosphorylation may affect function of bcl-2 or bcl-xL by
altering the capacity of the proteins to form dimers or act
independently of dimerization. The three-dimensional structure of
bcl-xL18 provides support for the latter
possibility. The structure consists of two hydrophobic  -helices
surrounded by amphipathic helices. In addition to these highly
structured elements, both bcl-xL and bcl-2 have large
unstructured protein loops. Chang et al19 have shown that
deletion of this protein loop increases the antiapoptotic activity of
bcl-xL and blocks okadaic acid-induced phosphorylation; heterodimerization with bax is not changed. Ito et al20
have shown the phosphorylation of serine at position 70 of bcl-2 is required for its capacity to protect NSF/N1.H7 mouse myeloid cells from
death after interleukin-3 (IL-3) deprivation, but does not alter its
capacity to dimerize with bax. Thus, the studies of Chang et al and Ito
et al provide examples of regulation of bcl-2 family members that are
independent of dimerization.
We report here that serine phosphorylation of bcl-2 is seen in acute
myeloblastic leukemia (AML) blast cells of the continuous line
OCI/AML-5 and blasts obtained from two patients with AML. The work was
undertaken because we had observed that treatment of AML blasts with
all trans retinoic acid (ATRA) often increases their sensitivity to
cytosine arabinoside (ara-C), a major chemotherapeutic drug used in the
treatment of AML.21-23 In a search for mechanism, we found
that ATRA decreased the expression of bcl-2 mRNA and the stability of
the protein in OCI/AML-2 and OCI/AML-5 cells.24,25
We asked if the reduced half-life of bcl-2 seen after ATRA treatment
might be associated with posttranslational modification of the protein.
Two dimensional gels of lysates from cells treated with ATRA disclosed
a novel acidic bcl-2 isoform. After digestion with -phosphatase, the
acidic isoform became indistinguishable from the native bcl-2 from
control cells, indicating that it was a phosphorylated form of the
protein. We compared phosphorylated bcl-2 from AML cells treated with
ATRA or taxol. Serine phosphorylation was seen after either agent, but
quantitative and qualitative differences were seen in the
phosphorylated bcl-2. Metabolic labeling with orthophosphate confirmed
that bcl-2 phosphorylation was increased in AML blasts cells treated
with either ATRA or taxol. Neither agent changed the capacity of bcl-2
to form dimers with bax, as measured in immunoprecipitation
experiments. The work provides a further example of posttranslational
modification of bcl-2 that is not associated with change in its
capacity for dimerization.
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MATERIALS AND METHODS |
Cell culture.
The human leukemia cell line OCI/AML-5 established in this laboratory
was used in the experiments. The cell line was grown in -minimal
essential medium (-MEM) (GIBCO, Burlington, Canada) supplemented
with 10% heat-inactivated fetal calf serum (FCS) (Sigma, St Louis, MO)
(growth medium) and 10% medium conditioned by the bladder carcinoma
line 5637 (5637-CM); the cells were incubated at 37°C in an
atmosphere of 5% CO2 in air. Cells were harvested in the
logarithmic growth phase for experiments. Cryopreserved blast cells
from two patients were used, selected because Western blot analysis
showed high bcl-2 levels. The cells were thawed and cultured in
suspension in 5637-CM for 48 hours, then recultured, and exposed to
either ATRA or taxol.
Reagents and antibodies.
ATRA (Sigma) was first dissolved in 100% ethanol at a concentration of
10 2 mol/L, and the solution further diluted to the
final concentrations in the culture. The monoclonal antibodies against
human bcl-2 used in the experiment are: (1) bcl-2 124 (DACO,
Mississauga, Ontario, Canada); (2) 6C8, a gift from Dr S.J. Korsmeyer
(Washington University, St Louis, MO). Rabbit polyclonal antibax (N-20)
was purchased from Santa Cruz (Santa Cruz, CA) or was a gift from Dr D.W. Andrews (McMaster University, Hamilton,
Ontario, Canada). 32Pi was
obtained from Dupont (Mississauga, Ontario, Canada).
Western blot and immunoprecipitation.
Western blots were used to measure bcl-2 protein. Briefly, cells at a
concentration of 5 × 106 cells/mL were lysed in lysis
buffer (10 mmol/L tris, pH 7.4, 0.15 mol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 2 mg/mL aprotinin, 1%
Triton X-100) for 1 hour at 4°C with freshly added protease
inhibitors. The nuclei were removed by centrifugation. A total of 100 µg protein from each sample was solubilized with sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer,
and electrophoresed through 12.5% SDS-polyacrylamide gel for about 2 hours at room temperature. For immunoblotting, the proteins separated
by SDS-PAGE were electrotransferred onto nitrocellulose filters
(Amersham, Oakville, Canada). The filters with the proteins were
blocked with phosphate-buffered saline (PBS) containing 2% nonfat milk and 0.1% Tween-20 (Sigma) and incubated with primary antibody for 2 hours. The filters were then incubated with horseradish peroxidase
(HRP)-labeled secondary antibodies (Amersham) for
another 2 hours. The filters were developed using enhanced
chemiluminescent (ECL) detection systems (Amersham). For
immunoprecipitation, the lysates from untreated and treated cells were
precleared by adding 2 µg hamster IgG for anti-bcl-2 or rabbit IgG
for anti-bax; 20 µL 50% (vol/vol) protein G-agarose was added for 1 hour at 4°C, followed by centrifugation to remove the protein
G-agarose pellets. Specific antibody against human bcl-2 (6C8) or
against bax (N-20) was added to the lysate and incubated for 2 hours,
followed by incubation with 20 µL of 50% protein G-agarose for
another 2 hours to capture the immunoprecipitates. The
immunoprecipitates were then separated by 12.5% SDS-PAGE and
electrotransferred onto nitrocellulose filters (Amersham). The bcl-2
protein on the filters was detected by the immunostaining method as
described above for Western blotting. For gradient gels, equivalent
amounts of protein (100 µg) from each cell lysate were separated by
10% to 20% gradient SDS-PAGE. After electrophoresis, the proteins
were transferred to nitrocellulose filters, and Western blotting was
performed as described above.
Two-dimensional (2D) gel analysis of bcl-2 protein.
2D gel electrophoresis was used to study the heterogeneity in bcl-2
protein from the leukemia cells. Proteins were first separated on the
basis of charge by isoelectric focusing and then by size, using
SDS-PAGE. Briefly, lysates from the cells treated under a number of
conditions were separated by isoelectric focusing (IEF) using tube gels
(4% acrylamide, 9.2 mol/L urea, 5% ampholyte, pH 4-8, 2% NP-40)
(Resolyte, from BDH, Ltd, Poole, UK), with a Mini 2-D Electrophoresis
Cell from Bio-Rad (Bio-Rad Laboratories Ltd, Mississauga, Ontario,
Canada). IEF ran at 500 V for 6 hours. After the first-dimension IEF,
the tube gels were removed from the glass tubes and loaded onto slab
gels (12.5% SDS-polyacrylamide gel) for electrophoresis in the second
dimension at 100 V for 2 hours. The proteins separated by SDS-PAGE were
electrotransferred onto nitrocellulose filters (Amersham) and bcl-2
protein was detected by immunostaining as described for Western
blotting. The 2D gels were reproducible for each lysate, but small
variations were observed between lysates. Therefore, in every
experiment, bacterial-synthesized bcl-2 protein isolated from
recombinant Escherichia coli cells as a carboxy terminus
truncated human bcl-2 cloned into the expression vector pSPGEX
(pSPGEXbcl-2), obtained from Dr D. Andrews (McMaster University,
Hamilton, Ontario, Canada) was added (2.5 µL of cell lysate) to serve
as a marker. The marker helped in the orientation of bcl-2 isoforms and
facilitated comparison between lysates. The identification of
individual isoforms was proven by demonstrating their presence in
mixtures with lysates containing other isoforms.
Phosphatase treatment.
Recombinant phosphatase (-PPase) (New England Biolabs, Beverly,
MA) was used to release phosphate groups from serine, threonine, and
tyrosine residues. Lysates containing 200 µg protein from each sample
were diluted with -PPase buffer (50 mmol/L Tris-HCl [pH8.0], 5 mmol/L dithiothreitol [DTT], 2 mmol/L
MnCl2, and 100 µg/mL bovine serum albumin [BSA]) to a
final volume of 100 µL. A total of 1,000 U of -PPase was added to
each reaction for 2 hours at 30°C and the samples were separated
subsequently by 2D gel.
Metabolic labeling.
For in vivo metabolic labeling with 32Pi
OCI/AML-5 cells were cultured in -MEM supplemented with 10%
heat-inactivated FCS and 10% 5637-CM. One hour before labeling, medium
was removed and the cells were washed twice with Tris-buffered saline
(TBS). The cells were resuspended in phosphate-free MEM (Sigma)
supplemented with 5% dialyzed FCS at a concentration of 1 × 106 cells/mL and incubated at 37°C for 30 minutes.
32Pi (Dupont) was added to the culture at a final
radioactivity of 1 mCi/mL in the presence of 10-7 mol/L
ATRA or 106 mol/L taxol. The cells were incubated at
37°C for another 15 hours. The orthophosphate-labeled cells were
washed three times with TBS and lysed with lysis buffer. Radiolabeled
lysate containing 1 mg protein from each sample was immunoprecipitated
with anti-bcl-2 antibody (6C8), and the precipitates were separated by
12.5% SDS-PAGE and subsequently electrotransferred onto polyvinylidene
difluoride (PVDF) filters (Dupont, Boston, MA). The filters with
radiolabeled bcl-2 protein were first exposed to a phosphor imaging
screen (Molecular Dynamics, Sunnyvale, CA) for detection of phosphorus and then stained with anti Bcl-2 antibody (bcl-2 124; DACO,
Mississauga, Ontario, Canada). The bcl-2 protein on the filters was
then detected by immunostaining as described above for Western
blotting. For in vitro metabolic labeling, cells were treated with
either ATRA or taxol for 24 hours, harvested, lysed in lysis buffer
with freshly added protease inhibitors, and immunoprecipitated using
specific hamster antihuman bcl-2 (6C8) antibody for 2 hours, followed
by incubation with 20 µL of 50% protein G-agarose for another 2 hours to capture the immunoprecipitates. For the kinase reaction, the immunoprecipitates were washed, resuspended in 50 µL buffer
containing 30 mmol/L Tris-Cl pH7.5, 10 mmol/L MgCl2, 2 mmol/L MnCl2, 50 µCi/mL  -32P-adenosine
triphosphate (ATP) (3,000 Ci/mmol/L; Amersham) and incubated at
30°C for 30 minutes. The reactions were terminated by adding 50 µL SDS-sample loading buffer and heating at
100°C for 5 minutes. The precipitates were then
electrophoresed through 12.5% SDS-polyacrylamide gel and
electrotransferred onto PVDF filters for autoradiography. In each
procedure, to control for loading, after autoradiography, the filters
were stained with anti-bcl-2 antibody.
Analysis of phosphorylation of amino acids.
We used phosphoamino acid analysis26,27 to determine which
amino acids are phosphorylated in AML cells exposed to ATRA or taxol.
The cells were incubated overnight in phosphate-free medium with 5%
dialyzed FCS and 32Pi (1 mCi/mL) in the
presence of ATRA (107 mol/L) or taxol
(106 mol/L). The cells were lysed and the lysates
were immunoprecipitated with anti-bcl-2 antibody (6C8) and the
immunoprecipitates separated by SDS-PAGE. The separated proteins were
transferred to a PVDF membrane. After autoradiography, the radiolabeled
bcl-2 band was excised, digested with N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK)-trypsin, lyophilized, and treated with 6 N
HCl for 1 hour at 110°C and dried. Residual HCl was removed with
water by lyophilization. The phosphoamino acids were then identified by
2D electrophoresis first in buffer N-tosyl-L-phenylalanine chloromethyl
ketone (TPCK) pH 1.9 and then in buffer pH 3.5 in a thin-layer
chromatography plate; radioactive amino acids were visualized by
autoradiography. These were compared with the position of
ninhydrin-stained control phosphoamino acids. The same methods were
used to detect phosphorylation of amino acids obtained by in vitro
labeling (see above).
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RESULTS |
Phosphorylation of bcl-2 in ATRA-treated OCI/AML-5 cells.
Bcl-2 in OCI/AML-5 cells treated with ATRA becomes less
stable.24 We used 2D gel electrophoresis to determine
whether this change in stability was associated with a
posttranslational modification of the protein. OCI/AML-5 cells were
grown in 107 mol/L ATRA for 24 hours. The cells were
lysed and the protein separated by IEF, followed by size separation, as
described in Materials and Methods; the results of a replicated
experiment are shown in Fig 1. The
bacterially synthesized protein serves as a marker and is indicated in
all gels by an arrow. Comparing the result of control lysate to
ATRA-treated lysate, there is an apparent shift of bcl-2 to a more
acidic isoform. This shift was confirmed by mixing lysates of control
and ATRA-treated cells (data not shown). Phosphorylation is one form of
posttranslational modification that can produce such a shift. To
determine whether phosphorylation was responsible for the shift, lysate
from ATRA-exposed cells, was treated with -phosphatase, and analyzed
by 2D electrophoresis. The acidic isoform was not evident in
phosphatase-treated cells; bcl-2 protein was in the same position as
the native protein (Fig 1). The difference in isoelectric point of the
phosphatase-treated and untreated samples was confirmed by mixing
lysate from ATRA-treated cells with an aliquot of the same lysate that
had been digested with -phosphatase; the 2D gel of the mixture is
shown at the bottom of Fig 1. Three distinct bcl-2 reacting spots are
seen; that shown by the arrow is the bacterially synthesized protein. The spot just to the left of this marker is from the
phosphatase-treated lysate and is in the same position as bcl-2 from
control cells. The most acidic bcl-2 isoform is derived from the
ATRA-treated lysate. Together, these studies verify that ATRA treatment
induces a change in bcl-2 protein and that this change is due to
phosphorylation.
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| Fig 1.
2D gels; upper left is the bacterially synthesized marker
protein, shown throughout the figures as an arrow. Upper right is a 2D
gel of a control lysate from OCI/AML-5 cells. Middle left is 2D gel of
a lysate from ATRA-treated cells, showing the acidic isoform. Middle
right is a 2D gel of an aliquot of lysate from ATRA-treated cells,
digested with -phosphatase. It may be compared with the 2D control
gel directly above it in the figure. The 2D gel at the bottom of the
figure was made from a mixture of the two lysates above it; it shows
the marker, a spot at the position of the native protein, but derived
from -phosphatase-digested lysate from ATRA-treated cells and an
acidic isoform, derived from the ATRA-treated cells.
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Comparison of phosphorylation induced by ATRA or taxol.
Taxol and other drugs acting on microtubules have been reported to
induce phosphorylation of bcl-2; the phosphorylated bcl-2 isoform from
taxol-treated cells is recognized as a slower migrating band in 10% to
20% gradient polyacrylamide gels.15-17,28 To
determine whether ATRA-induced phosphorylation is seen in such gels,
OCI/AML-5 cells were treated with ATRA or taxol for 24 hours and the
lysates separated on 10% to 20% gradient gels and probed for bcl-2
(Fig 2A). A slower mobility band was
evident in the taxol-treated cells, but not the ATRA-treated or control
cells. We confirmed that the shift induced by taxol was due to
phosphorylation by treating lysates with -phosphatase. As expected,
this resulted in loss of the slow mobility form of bcl-2.
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| Fig 2.
(A) At the top is a 10% to 20% gradient gel showing the
presence of a slower-moving isoform of bcl-2 from cells treated with
taxol (middle lane). The control (left lane) and lysate from
ATRA-treated cells show only a single band (right lane). The bottom is
2D gels from taxol-treated cell lysate (left) and the lysate after
digestion with -phosphatase. These conditions permit the
demonstration of the slightly larger than control molecular weight of
the phosphorylated bcl-2 isoform seen after taxol. (B) Lysates from
ATRA- and taxol-treated cells (above) were mixed and the mixture run as
a 2D gel (below). The experiment shown is representative of two
replicates.
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These results indicate that a difference may exist between
phosphorylation of bcl-2 after treatment by ATRA or taxol. To determine whether this is the case, we compared taxol- and ATRA-induced phosphorylation of bcl-2 by 2D gel analysis. Bcl-2 from taxol-treated cells appeared as two spots in addition to the marker, one at the
location of the native protein and a second, more acidic, and slightly
larger in size; this latter spot was lost after treatment of the lysate
with phosphatase (Fig 2B). The position of the novel isoform in
taxol-treated cells appears to be less acidic than in ATRA-treated
cells. This was confirmed by mixing lysates from ATRA- and
taxol-treated cells and then separating the proteins on a 2D gel (Fig
2A). Four spots, representing bcl-2 isoforms, are seen. The most basic
form is the bacterially expressed bcl-2 (denoted by an arrow). The two
isoforms just to the left of the marker are derived from the taxol
lysate and represent the native isoform and a phosphorylated isoform.
The most acidic isoform is derived from the ATRA-treated cells. Taken
together, these experiments indicate that there is both a qualitative
and quantitative difference in the phosphorylation of bcl-2 induced by
taxol compared with ATRA.
Phosphorylation of bcl-2 in primary AML blast cells.
The above experiments were performed with the cell line OCI/AML-5;
identical results were seen in a second cell line, OCI/AML-2 (data not
shown). To show that the ATRA- and taxol-induced phosphorylation of
bcl-2 is not restricted to cell lines, we cultured the cryopreserved AML blast cells from two patients for 48 hours in growth medium and
5637-CM. The cells were then treated with taxol (106
mol/L) or ATRA (107 mol/L) for 24 hours, lysates
prepared, and analyzed by 2D gel electrophoresis. As evident in
Fig 3, the changes in bcl-2, seen in the
primary blast cells, are the same as those seen in the leukemic cell
lines.
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| Fig 3.
2D gels of lysates from blast cells from an AML patient.
Control is seen at the top; the middle shows gels from cells treated
with ATRA (left) and taxol (right). At the bottom is the 2D gel of a
mixture of these two lysates. Similar results were obtained using the
blast cells from a second patient.
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Metabolic labeling.
We looked for direct evidence of bcl-2 phosphorylation by measuring
incorporation of radiophosphorus into the protein, either in vivo or in
vitro as described in Materials and Methods. The labeled
immunoprecipitates were separated by 12.5% PAGE, transferred to a PVDF
filter, and visualized by radioautography.
Figure 4 shows the radioautographs; some
phosphorylation of native bcl-2 was seen, but this increased in cells
treated with either ATRA or taxol. The 26 kD
phosphoprotein was identified as bcl-2 by comparison with Western
blots. Both in vivo and in vitro, bcl-2 from ATRA-treated cells was
more highly phosphorylated than bcl-2 from taxol-treated cells. A
second 30-kD phosphorylated protein is observed in in vivo labeled,
ATRA-treated cells, but not in controls or taxol-treated cells.
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| Fig 4.
Metabolic labeling of bcl-2 from OCI/AML-5 cells in vivo
(left) and in vitro (right). Radioautographs of labeled protein from
control, ATRA-treated and taxol-treated cells, separated by 12.5%
SDS-PAGE are shown at the top. Densitometer readings are shown. At the
bottom, Western blots of the lysates, stained with anti-bcl-2, are
shown. Such Western blots, prepared after 12.5% SDS-PAGE, show bcl-2
from taxol-treated cells only as a single band.
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Phosphoamino acid analysis.
32Pi labeling of bcl-2 both in vivo and in
vitro was used to identify the phosphorylated amino acids, as described
in Materials and Methods. The results of the in vivo labeling are shown
in Fig 5 as 2D electrophoresis on thin
layer chromatography with the label detected by autoradiography. The
positions of three phosphoamino acids (P-serine, P-threonine, and
P-tyrosine), as detected by ninhydrin staining, are shown. Lysates were
prepared from controls and cells treated either with ATRA or taxol. In all three conditions, only phosphorylation of serine was observed. The
same result was obtained when bcl-2 was labeled in vitro (data not
shown). The finding that serine is phosphorylated after taxol is in
agreement with results in the literature.28
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| Fig 5.
Phosphoamino acid analysis to show phosphorylation of
bcl-2 on serine. The figure shows 2D electrophoresis on thin layer
chromatography with the label detected by autoradiography. The cells
were incubated overnight with 32Pi as controls
or with ATRA or taxol. Cell lysates were prepared and
immunoprecipitated with anti-bcl-2 antibody (6C8) and the
immunoprecipitates were separated by SDS-PAGE and transferred to PVDF
membrane. After autoradiography, the radiolabeled bcl-2 band was
excised, digested with TPCK-trypsin, lyophilized, treated with 6 N HCl
for 1 hour at 110°C and dried. The phosphoamino acids were then
identified by 2D electrophoresis first at pH 1.9 and then at pH 3.5. The positions of the radioactive amino acids were compared with
ninhydrin-stained control amino acids, as indicated in each panel of
the figure. The same methods were used to detect phosphorylation of
amino acids obtained by in vitro labeling. Using both methods, only
serine phosphorylation was detected.
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Dimerization of bcl-2 and bax.
Haldar et al28 found that bcl-2 phosphorylated after
treatment with ATRA did not dimerize with bax. Based on this, they proposed that the function of bcl-2 was inhibited. We took two different approaches to ask whether phosphorylation of bcl-2 affects dimerization with bax. First, lysates from controls, taxol-, or ATRA-treated cells were immunoprecipitated with antibody against bax.
The immunoprecipitates were separated by electrophoresis on a gradient
gel and the resulting filters probed with antibody against bax or
bcl-2. Bcl-2 protein coprecipitated with bax in all three cases. In the
taxol-treated lysate, both isoforms of bcl-2 were brought down,
indicating that bax is dimerized with phosphorylated bcl-2
(Fig 6). In the ATRA-treated cells, we also infer that bax is dimerized with phosphorylated bcl-2, as by 2D gel
analysis, virtually all of the bcl-2 is in a phosphorylated form (Fig
1).
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| Fig 6.
Dimerization of bcl-2 and bax. Lysates from control cells
and cells treated with ATRA or taxol were immunoprecipitated with
anti-bcl-2 (A) or with anti-bax (B). The immunoprecipitates were
separated by 10% to 20% SDS-PAGE gel electrophoresis, transferred to
nitrocellulose, and stained with bcl-2. It is evident that both normal
and phosphorylated bcl-2 formed heterodimers with bax.
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Second, because this result differed from that of Haldar et al, we
tried to determine whether there was a quantitative difference in the
amount of bax associated with bcl-2. For these experiments, lysates
from control, taxol- or ATRA-treated cells were immunoprecipitated with
antibody against bcl-2. Protein from the pellet and the supernatant was
separated by SDS-PAGE and transferred to nitrocellulose. The resulting
membranes were then probed for the presence of bcl-2 in the pellet and
bax in the supernatant. All of the bcl-2 was present in the pellet from
samples after immunoprecipitation with anti-bcl-2; blc-2 protein could
not be demonstrated in the supernatants by Western blot (data not
shown). After precipitation with anti-blc-2, the amounts of bax in the
supernatants were similar, regardless of how the cells were treated
(Fig 7). These experiments demonstrate that
phosphorylation of bcl-2 by taxol or ATRA does not result in the
release of bax from bcl-2.
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| Fig 7.
10% to 20% gradient gels after bcl-2
immunoprecipitation of lysates from OCI/AML-5 cells, as controls or
after treatment with ATRA or taxol. The panel at the top shows the
immunoprecipitates, stained with anti-bcl-2. The bottom panels show
the supernatants, stained with anti-bax. It is seen that treatment of
the OCI/AML-5 cells did not change the amount of bax that did not
coprecipitate with bcl-2.
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| |
DISCUSSION |
There are two major findings described in this report. First, we report
that bcl-2 becomes phosphorylated on serine after treatment with ATRA.
Second, phosphorylation of bcl-2 after ATRA is different from that
observed in cells treated with taxol.
Phosphorylation of bcl-2 was shown using 2D gels of lysates from
ATRA-treated blast cells. After ATRA, bcl-2 became more acidic; after
digestion of lysates from ATRA-treated cells with -phosphatase, 2D
gels showed bcl-2 in the same position as the control native isoform.
This observation showed that the acidic bcl-2 isoform is
phosphorylated. Metabolic labeling of bcl-2 with
32Pi or -32P-ATP showed
increased incorporated isotope in bcl-2 of ATRA-treated cells compared
with controls, confirming directly that ATRA leads to the
phosphorylation of bcl-2.
Bcl-2 is phosphorylated in cells treated in culture with okadaic acid
or taxol.15,17 The phosphorylated isoform after such treatment can be detected in gradient gels as a novel band with decreased mobility. Similar bands are seen when bcl-2 is treated with
c-Jun N-terminal kinase (JNK) in vitro.29 Electrophoresis of lysates from ATRA-treated blast cells through 10% to 20%
polyacrylamide gels failed to show a second band. Because of this
difference between phosphorylation after ATRA and taxol, we undertook a
direct comparison of the effects of the two agents. As expected from the published results, taxol-induced phosphorylation increased the
apparent size of bcl-2 protein; the phosphorylated isoform was seen as
an extra band in gradient gels (Fig 3). This change in mobility was
shown to be due to phosphorylation indirectly by demonstrating the
sensitivity of the acidic isoform to -phosphatase and directly by
showing incorporation of phosphorus into the bcl-2 protein, although
stimulation of phosphorylation was less after taxol than after ATRA.
Further, 2D gels of lysates from taxol-treated cells showed both the
native isoform and a novel acidic isoform, which was sensitive to
-phosphatase. Taken together these data show both qualitative
differences (comparison of size in gradient gels and 2D gels) and
quantitative differences (complete conversion of the native isoform to
the phosphorylated isoform after ATRA compared with retention of the
native isoform after taxol, and increased metabolic labeling in
ATRA-treated cells compared with taxol-treated cells). Parallel
phosphoamino acid analysis of ATRA- and taxol-treated
cells showed that both are phosphorylated on serine
(Fig 5). Therefore, the differences between phosphorylated bcl-2 in
ATRA-treated compared with taxol-treated cells cannot be explained by
phosphorylation of different amino acids; different sites or extent of
phosphorylation or both remain as possible reasons for the observed
differences. Experiments are in progress directed towards this
important issue.
The role of bcl-2 phosphorylation in the regulation of apoptosis
remains unclear. Conflicting reports are available concerning bcl-2
phosphorylation. First, descriptions of the influence of bcl-2
phosphorylation on its capacity to form dimers with other family
members are not consistent. Both of the reports of Zha et
al30 and Haldar et al17 show phosphorylation
modifying the function of bcl-2 family members by changing patterns of
dimerization. In contrast, Ito et al20 and Chang et
al19 found that phosphorylation of bcl-2 did not affect
dimerization. Our studies provide support for the latter in that we
found that bcl-2 phosphorylated after either ATRA or taxol has
unchanged capacity to form heterodimers with bax. The discrepancy
between our taxol result and that of Haldar et al may be explained by
differences in the cellular systems or immunoprecipitation methods used
in the two studies. Regardless, our studies provide a further example
of posttranslational modification of bcl-2 by phosphorylation that does
not change its capacity to form heterodimers with bax.
Second, controversy exists as to whether phosphorylation increases or
diminishes the antiapoptotic activity of bcl-2 family members. Mutants
of bad that abolish phosphorylation are very effective in promoting
apoptosis, as these are incapable of dimerization; mutants with
reduced, but not eliminated, phosphorylation sites show intermediate
apoptotic capacity.30 In contrast, Ito et al20
report that phosphorylation is required for antiapoptoic activity. We
have only correlative evidence for the consequence of ATRA-induced
phosphorylation. ATRA reduces clonogenicity in AML cell lines in
proportion to the presence of its receptor31; ATRA-treated
OCI/AML-5 and OCI/AML-2 cells are sensitized to ara-C after exposure to
ATRA.21 ATRA may have many effects on leukemic cells;
nonetheless, loss of clonogenicity and increased ara-C sensitivity
might be the consequence, in part, of decreased bcl-2 function. The
reduced stability of bcl-2 protein in ATRA-treated cells is consistent
with altered function. The bcl-2 phosphorylation after ATRA is a
posttranslational modification that is associated with a shortened
half-life and may contribute to a change in its function, perhaps
through degradation. If this view is correct, our data would agree with
those who propose that bcl-2 phosphorylated after taxol is less able to
prevent cell death.16,17 It remains a possibility that, if
phosphorylation is a regulator of bcl-2 function, its effect might be
different, depending on extent and sites of phosphorylation and upon
the cellular context.
ATRA is an important biologic agent in the treatment of promyelocytic
leukemia32,33 and is showing promise in the early results
of a randomized clinical trial as part of a chemotherapeutic regimen
for acute myeloblastic leukemia without the t(15;17).34 We
have shown that ATRA treatment led to phosphorylation of bcl-2 in cells
from two AML patients. The response of fresh cells to ATRA is
heterogeneous, when clonogenic cell survival or sensitization to ara-C
are the end-points; we expect that phosphorylation of bcl-2 after ATRA
will also vary from patient-to-patient. There exists an opportunity,
therefore, to test for an association between phosphorylation of bcl-2
and response in trials that include ATRA. If such an association were
found, it would provide convincing evidence of the functional
significance of bcl-2 phosphorylation and might help to select for
patients who would benefit from the addition of ATRA to a therapeutic
regimen.
| |
FOOTNOTES |
Submitted December 30, 1997;
accepted April 30, 1998.
Supported by the National Cancer Institute of Canada and the Medical
Research Council of Canada.
Address reprint requests to E.A. McCulloch, MD, 610 University Ave, Toronto, Ontario M5G 2M9, Canada; e-mail:
mcculloch{at}oci.utoronto.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract