Research Article
BAD Ser128 Is Not Phosphorylated by c-Jun NH2-Terminal
Kinase for Promoting Apoptosis
Jiyan Zhang, Jing Liu, Chenfei Yu, and Anning Lin
Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois
Abstract
The phosphorylation and regulation of the proapoptotic Bcl-2
family protein BAD by c-Jun NH2-terminal kinase (JNK) is
controversial. JNK can suppress interleukin-3 withdrawalinduced apoptosis via phosphorylation of BAD at Thr201.
However, it has also been reported that JNK promotes
apoptosis through phosphorylation of BAD at Ser128. Here,
we report that JNK is not a BAD Ser128 kinase. JNK
phosphorylates murine BAD (mBAD), but not human BAD
(hBAD), in which Ser91 is equivalent to Ser128 in mBAD. In
contrast, Cdc2, which phosphorylates Ser128, phosphorylates
both mBAD and hBAD. Replacement of Ser128 by alanine has
no effects on BAD phosphorylation by JNK in vitro and in vivo.
Two-dimensional phosphopeptide mapping in combination
with phosphoamino acid analysis reveals that JNK does not
phosphorylate BAD at Ser128. Elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in
apoptosis induced by UV via JNK or growth factor withdrawal.
Thus, our results show that Ser128 is not phosphorylated by
JNK for promoting cell death. (Cancer Res 2005; 65(18): 8372-8)
Introduction
Apoptosis is a process of programmed cell death that is essential
to many biological processes in multicellular organisms, including
embryonic development, immune responses, tissue homeostasis,
and normal cell turnover (1–3). The Bcl-2 family, which consists of
both antiapoptotic proteins Bcl-2 and Bcl-xL and proapoptotic
proteins Bax, Bak, BAD, Bid, NOXA, and BIM, plays a critical role in
regulation of apoptosis (2). On stimulation by a death signal, the
proapoptotic Bcl-2 family proteins, such as Bid and BAD, undergo
proteolysis and dephosphorylation, respectively (2). Consequently,
BAD and the proteolytic product of Bid, tBid, translocates to the
outer membrane of mitochondria, where they inactivate the
antiapoptotic Bcl-2 family proteins Bcl-xL and Bcl-2 or activates
Bax (2). This results in increased permeability of the outer
membrane of mitochondria, release of cytochrome c, and ultimately
apoptotic cell death (2).
The phosphorylation of BAD provides an important connection
between the cellular survival signaling pathways and the apoptotic
death machinery. BAD is phosphorylated at several serine and/or
threonine residues (Ser112, Ser136, Ser155, or Thr201) by a group of
protein kinases [Akt/protein kinase B (PKB), protein kinase A
(PKA), Raf-1, Rsk2, CaMKII, and c-Jun NH2-terminal kinase (JNK)]
in response to growth/survival factors, such as interleukin-3 (IL-3)
and insulin-like growth factor-I (IGF-I; refs. 4–11). Phosphorylation
Requests for reprints: Anning Lin, Ben May Institute for Cancer Research,
University of Chicago, Room N516, 5841 South Maryland Avenue, Chicago, IL 60637.
Phone: 773-753-1408; Fax: 773-702-6260; E-mail: [email protected].
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-0576
Cancer Res 2005; 65: (18). September 15, 2005
at the serines sequesters BAD in the cytoplasm via promoting the
interaction between BAD and the cytoplasmic anchorage protein
14-3-3, thereby preventing the interaction of BAD with Bcl-xL/Bcl-2
on the mitochondrial membrane (11). Although phosphorylation of
BAD by JNK at Thr201 reduces the interaction between BAD and
Bcl-xL (10), the underlying mechanism has yet to be determined.
JNK, also known as stress-activated protein kinase (12), is a
member of the mitogen-activated protein kinase (MAPK) superfamily (13). JNK has two ubiquitously expressed isoforms, JNK1 and
JNK2, and a tissue-specific isoform, JNK3, with splicing forms (p54
and p46; ref. 14). Recent studies suggest that JNK1 is the main JNK
isoform that is activated by extracellular stimuli, whereas JNK2
may interfere with JNK1 activation (15). JNK has been implicated to
play a critical role in many cellular activities, from growth control
to programmed cell death (14). The role of JNK in apoptosis is
complex, as it has been reported to have proapoptotic or
antiapoptotic functions, or no role in the apoptotic process,
depending on cell type, death stimulus, duration of activation, and
activation of other signaling pathways (14). In the absence of
nuclear factor-nB (NF-nB) activation, tumor necrosis factor-a
(TNF-a) induces prolonged JNK activation, which contributes to
TNF-a-induced apoptosis (14–19). JNK is also involved in cell
survival (14). In IL-3-dependent hematopoietic cells, JNK is
activated by IL-3 and in turn phosphorylates BAD at Thr201,
thereby inhibiting the proapoptotic activity of BAD (10). However,
it has been reported that JNK can phosphorylate BAD at Ser128,
thereby promoting the proapoptotic activity of BAD in neurons
(20). It is paradox that phosphorylation of BAD by JNK at two
different sites could either promote or inhibit the proapoptotic
activity of BAD. Here, we report that JNK does not phosphorylate
Ser128 in vitro and in vivo. Furthermore, elimination of Ser128
phosphorylation does not affect the proapoptotic activity of BAD in
apoptosis induced by UV via JNK or growth factor withdrawal.
Thus, this work clarifies the confusions in the field (21–33).
Materials and Methods
Reagents and plasmids. pGEX-KG-murine BAD (mBAD) was a gift
from Dr. Stanley Korsmeyer (Harvard Medical School, Boston, MA). S128A
and T201A mutations were introduced using the QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). pcDNA3.1-Hygro(+) vectors
encoding wild-type (W T) M2-mBAD, M2-mBAD(S128A), and
M2-mBAD(T201A) were generated by addition of a NH2-terminal M2 tag
to mBAD. All mutations and subcloning were confirmed by DNA sequencing.
pGEX-human BAD (hBAD) was a gift from Dr. John C. Reed (The
Burnham Institute, La Jolla, CA). Glutathione S-transferase (GST)-JNK1
and GST-JNK1(APF), pSRa-hemagglutinin (HA)-JNKK2-JNK1, and pSRa-HAJNKK2(KM)-JNK1 have been described previously (10, 34, 35). pEGFP-C3mBAD was a gift from Dr. Richard J. Youle (National Institutes of Health,
Bethesda, MD). Antibodies against JNK1 (antibody 333; ref. 15) and BAD
were from PharMingen (San Diego, CA) and Cell Signaling (Beverly, MA),
respectively. Antibody against Cdc2 was a gift from Dr. Yuzuru Minemoto
(Japan). Antibodies against HA and Bcl-xL were from Santa Cruz (Santa Cruz,
CA). Hoechst (H33258) and purified PKA catalytic subunit were from
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JNK Is Not a BAD Ser128 Kinase
Sigma (St. Louis, MO). Nocodazole was from Fisher Scientific (Pittsburgh,
PA). [g-32P]ATP was from Dupont NEN (Boston, MA).
Cell culture and transfection. BAD / fibroblasts (a gift from Dr.
Stanley Korsmeyer) and HeLa cells were cultured in DMEM supplemented
with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 units/mL
penicillin, and 100 Ag/mL streptomycin. Transfection was done by using
ExGen500 (MBI Fermentas, Canada) according to the manufacturer’s
protocol. FL5.12 Bcl-xL cells (a gift from Dr. Charles Rudin, Johns Hopkins
University, Baltimore, MD) and HCD57 cells (a gift from Dr. Amittha
Wickrema, University of Chicago, Chicago, IL) were cultured in Iscove’s
medium supplemented with 2 mmol/L glutamine, 100 units/mL penicillin,
and 100 Ag/mL streptomycin, 5% FBS or 25% FBS, and 10% Wehi
supernatant or 2 units/mL erythropoietin, respectively. Transfections were
done by electroporation with Bio-Rad (Hercules, CA) Gene Pulser at
960 AF/250 V or 960 AF/220 V.
Protein kinase assays and immunoblotting. Immune complex kinase
assays were done and quantitated as described previously (36). Briefly,
kinase assays were carried out for 60 minutes at 30jC in 20 mmol/L HEPES
(pH 7.6), 20 mmol/L MgCl2, 1 mmol/L DTT, 10 Amol/L nonradioactive ATP,
and 10 ACi [g-32P]ATP. GST-BAD or GST-c-Jun (2-4 Ag) was used as
substrate as indicated in the figure legends. Reactions were terminated by
the addition of 4 SDS sample buffer and heated at 95jC for 5 minutes. The
proteins were separated by SDS-PAGE and visualized by Coomassie brilliant
blue staining. Phosphorylated proteins were detected and quantitated by
PhosphorImager (Molecular Dynamics, Pittsburgh, PA). Active JNK1 or
active Cdc2 was immunoprecipitated from the lysates prepared from
cells stimulated by UV (60 J/m2, 20 minutes) or nocodazole (100 ng/mL,
12 hours), respectively, using anti-JNK1 antibody (antibody 333; ref. 15) or
anti-Cdc2 antibody, respectively. Purified PKA catalytic subunit is
constitutively active. Active GST-JNK1 was prepared by in vitro activation,
in which purified GST-JNK1 proteins (2 Ag) were incubated in a kinase
reaction with cell extracts containing transfected HA-DMEKK1, which has
constitutive kinase activity and then purified with glutathione column
(10, 36). GST-JNK1(APF), in which the phosphorylation sites Thr183 and
Tyr185 have been replaced by alanine and tryptophan, respectively, and
thereby cannot be phosphorylated and activated (10), were used as control.
Immunoblotting was done as described (37).
Two-dimensional tryptic phosphopeptide mapping and phosphoamino acid analysis. Two-dimensional tryptic phosphopeptide mapping or
phosphoamino acid analysis of phosphorylated GST-mBAD proteins was
done as described (10, 36, 38). Briefly, GST-mBAD proteins phosphorylated
by active JNK1 in vitro were separated by 10% SDS-PAGE, transferred to
nitrocellulose membrane, and visualized by autoradiograph. Phosphorylated
GST-mBAD proteins were excised, eluted out, and subjected to trypsin
digestion. Tryptic digests were applied to thin-layer cellulose plates for
two-dimensional peptide mapping by electrophoresis at pH 1.9 for
30 minutes at 1.5 kV in the first dimension and then by ascending
chromatography in the second dimension. The phosphoamino acid
composition of GST-mBAD proteins was determined by two-dimensional
electrophoresis of a partial acid hydrolysate of the tryptic digests mentioned
above. The two-dimensional electrophoresis was done by electrophoresis at
pH 1.9 for 30 minutes at 1.5 kV in the first dimension and by electrophoresis
at pH 3.5 for 20 minutes at 1.3 kV in the second dimension.
Apoptosis assays. Cells were stained with Hoechst and nuclear
condensation and DNA fragmentation were visualized by fluorescence
microscopy as described (10, 19, 37).
Results
c-Jun NH2-terminal kinase phosphorylates murine BAD but
not human BAD. The role of JNK in regulation of the proapoptotic
activity of BAD is controversial. JNK phosphorylates BAD at Thr201
and thereby inhibits its proapoptotic activity (10). However, it has
been reported that JNK phosphorylates BAD at Ser128 and
consequently enhances its proapoptotic activity (20). To address
this paradox, we analyzed phosphorylation of mBAD or hBAD by
JNK. As a proline-directed MAPK, JNK typically phosphorylates
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Figure 1. JNK phosphorylates mBAD but not hBAD. A, sequence alignment of
mBAD and hBAD. In hBAD, Ser91 is a Ser128 equivalent (* ) in mBAD. hBAD
does not have a threonine that is Thr201 equivalent (*). B, HeLa cells were
treated with UV (60 J/m2, 20 minutes) or nocodazole (100 ng/mL, 12 hours) or
left untreated. Active JNK1 or active Cdc2 was isolated by anti-JNK1 antibody
(antibody 333) or anti-Cdc2 antibody, respectively. Phosphorylation of GST-BAD
by active JNK1 or active Cdc2 was measured by immune complex kinase
assays as described previously (10, 36). KA, kinase assays; CCB, Coomassie
brilliant blue staining.
its substrates at either serine or threonine residue, which is
immediately followed by a proline residue (13). Whereas mBAD
has both Ser128 and Thr201 residues that are followed by proline,
hBAD contains Ser91 that is equivalent to Ser128 in mBAD but does
not have the Thr201 equivalent (Fig. 1A). Immune complex kinase
assays showed that active JNK1 immunoprecipitated by the antiJNK1 antibody 333 (15) significantly phosphorylated GST-mBAD
(Fig. 1B) as reported previously (10). Under the same conditions,
phosphorylation of hBAD by active JNK1 was undetectable (Fig. 1B).
The differential phosphorylation of mBAD and hBAD by JNK1 was
not the result of the difference between the amount of GST-mBAD
and GST-hBAD fusion proteins used in the kinase reactions
(Fig. 1B). The inability of active JNK1 to phosphorylate GST-hBAD
was also not due to possibly poor quality of GST-hBAD proteins.
Active Cdc2, which can phosphorylate mBAD at Ser128 and hBAD
at Ser91 (ref. 39; data not shown), phosphorylated GST-hBAD and
GST-mBAD to a similar extent (Fig. 1B). Because JNK does not
phosphorylate hBAD, in which Ser91 is equivalent to Ser128 in mBAD,
it is unlikely that Ser128 is phosphorylated by JNK.
Replacement of Thr201, but not Ser128, by alanine in BAD
abolished BAD phosphorylation by c-Jun NH2-terminal kinase.
To directly determine whether BAD Ser128 can be phosphorylated
by JNK, Ser128 or Thr201 was replaced by nonphosphorylatable
alanine in the full-length GST-BAD using site-directed mutagenesis.
Immune complex kinase assays showed that replacement of Ser128
by alanine did not affect phosphorylation of GST-BAD by active
JNK1 isolated from UV-irradiated HeLa cells (Fig. 2A). In contrast,
phosphorylation of GST-BAD by active JNK1 was abolished when
Thr201 was replaced by alanine (Fig. 2A). Because all three forms of
GST-BAD proteins can be phosphorylated by active PKA to a
similar extent (Fig. 2B), the inability of active JNK1 to phosphorylate GST-BAD(T201A) was not caused by potential changes in the
global structure of the protein (Fig. 2B). These results showed that
Thr201 is the major JNK phosphorylation site, whereas Ser128 is not.
It is possible that recombinant JNK, which was reported to
phosphorylate BAD at Ser128 (20), might act differently from
active JNK isolated from cells (10). To test this scenario, purified
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WT GST-JNK1 or kinase-deficient GST-JNK1(APF), in which Thr183
and Tyr185 have been replaced by nonphosphorylatable alanine and
tryptophan, respectively, and therefore can no longer be activated,
was modified in an in vitro kinase reaction containing nonradioactive ATP and an aliquot of extracts from cells transfected with a
constitutively active MEKK1 (10, 36). Under these conditions, WT
GST-JNK1 was activated, as it significantly phosphorylated GSTc-Jun (data not shown; refs. 10, 36). As expected, GST-JNK1(APF)
did not have detectable activity (ref. 10; data not shown). Although
active GST-JNK1 phosphorylated GST-BAD and GST-BAD(S128A)
to a similar extent, it failed to phosphorylate GST-BAD(T201A)
(Fig. 2C). Thus, mutation of Ser128 to alanine does not affect
phosphorylation of BAD by recombinant JNK.
Phosphorylation of BAD by JNK in vivo resulted in the ‘‘up-shift’’
of BAD proteins in SDS-PAGE (10). To determine whether Ser128 is
phosphorylated by JNK in cells, FL5.12 Bcl-xL cells were transfected
with the constitutively active JNKK2-JNK1 or its kinase-deficient
Figure 3. Two-dimensional tryptic phosphopeptide mapping and phosphoamino
acid analysis of WT GST-mBAD and GST-mBAD(S128A) phosphorylated by
active JNK1 and Cdc2. A and B, two-dimensional tryptic phosphopeptide
mapping of WT GST-mBAD and GST-mBAD(S128A) phosphorylated by active
JNK1 (A ) or active Cdc2 (B). C, phosphoamino acid analysis of WT GST-mBAD,
GST-mBAD(S128A), and GST-mBAD(T201A) phosphorylated by active JNK1.
PS, phosphoserine; PT, phosphothreonine.
Figure 2. Replacement of Thr201, but not Ser128, by alanine abolishes
phosphorylation of mBAD by JNK. A, replacement of Ser128 by alanine did not
affect phosphorylation of mBAD by active JNK1 isolated from UV-irradiated HeLa
cells, whereas replacement of Thr201 by alanine abolished the phosphorylation.
B, mBAD(S128A) and mBAD(T201A) mutants were still phosphorylated by
purified PKA catalytic unit. C, phosphorylation of GST-mBAD by activated
recombinant GST-JNK1 in vitro . Inactive GST-JNK1(APF) was used as a
control. D, replacement of Thr201, but not Ser128, by alanine affects JNK
phosphorylation-induced modification of M2-mBAD in FL5.12 Bcl-xL cells.
FL5.12 Bcl-xL cells were transfected with expression vectors encoding various
M2-mBAD constructs (WT, S128A, and T201A, 40 Ag each), along with
HA-JNKK2-JNK1, or the kinase-deficient HA-JNKK2(K149M)-JNK1 (40 Ag
each). After 24 hours, cells were deprived of IL-3 for 2 hours. Expression and
modification (the up-shift) of various mBAD proteins were analyzed by
SDS-PAGE in combination with immunoblotting using anti-BAD antibody.
Cancer Res 2005; 65: (18). September 15, 2005
JNKK2(K149M)-JNK1 mutant, alone with BAD WT, S128A, or
T201A mutants. Immunoblotting analysis revealed that cotransfection with JNKK2-JNK1, but not JNKK2(K149M)-JNK1 mutant,
resulted in up-shift of WT BAD (Fig. 2D). The BAD(S128A) mutant,
which can be phosphorylated by JNK to the same extent as WT
BAD in vitro, was still up-shifted when cotransfected with the
active JNKK2-JNK1 (Fig. 2D). In contrast, the BAD(T201A) mutant,
which can no longer be phosphorylated by JNK, was not up-shifted
(Fig. 2D). These data showed that Ser128 is not a phosphoracceptor for JNK in vivo.
c-Jun NH2-terminal kinase phosphorylates BAD at Thr201
but not Ser128. It is possible that Ser128 is only a minor JNK
phosphorylation site in BAD; its mutation may not have a profound
effect on the overall phosphorylation of BAD by JNK. To test this
possibility, we compared JNK1-phosphorylated sites in WT BAD
and BAD(S128A). Two-dimensional tryptic phosphopeptide mapping revealed that JNK1-phosphorylated WT GST-BAD and GSTBAD(S128A) proteins had the same patterns (Fig. 3A). There was no
detectable difference even the autoradiograms were overexposed
(data not shown). In parallel, two-dimensional tryptic phosphopeptide mapping revealed that replacement of Ser128 by alanine
specifically eliminated the phosphopeptide 5 in Cdc2-phosphorylated GST-BAD(S128A) when compared with Cdc2-phosphorylated
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JNK Is Not a BAD Ser128 Kinase
WT GST-BAD (Fig. 3B). Therefore, the tryptic phosphopeptide
containing phosphorylated Ser128 could be detectable by the twodimensional tryptic phosphopeptide mapping as long as it was
phosphorylated. The fact that there was no detectable difference
between the pattern of JNK1-phosphorylation sites in WT GST-BAD
and GST-BAD(S128A) (Fig. 3A) suggested that Ser128 is even not a
minor JNK phosphorylation site. Consistently, phosphoamino acid
analysis revealed that JNK1-phosphorylated WT GST-BAD and
GST-BAD(S128A) mutant contained both phosphoserine and
phosphothreonine (Fig. 3C). In contrast, JNK1-phosphorylated
GST-BAD(T201A) mutant contained only phosphoserine (Fig. 3C).
Thus, JNK does not phosphorylate BAD at Ser128.
Phosphorylation of Ser128 does not contribute to the
proapoptotic activity of BAD in c-Jun NH2-terminal kinase–
dependent apoptosis. It has been postulated that BAD was
phosphorylated by JNK at Ser128 in JNK-dependent apoptosis (20). If
so, replacement of Ser128 by alanine should reduce the proapoptotic
activity of BAD, thereby inhibiting JNK-dependent apoptosis. To test
this possibility, BAD-null fibroblasts (Fig. 4A) and HeLa cells were
transfected with mammalian expression vectors encoding WT
GFP-BAD or GFP-BAD(S128A), respectively. Immunoblotting analysis showed that expression levels of GFP-BAD(S128A) were similar
to that of WT GFP-BAD (Fig. 4B). The susceptibility of the cells
to UV-induced apoptosis, which is JNK dependent (15, 40), was
analyzed by apoptotic death assays. Eighteen to 20 hours after
transfection, cells expressing GFP-BAD(S128A) had f22% (basal
level) apoptosis, similar to that in cells expressing WT GFP-BAD
(Fig. 4C and D). Furthermore, UV irradiation induced similar levels
of apoptotic cell death in cells expressing either GFP-BAD(S128A)
or WT GFP-BAD (Fig. 4C and D; 85% in BAD-null fibroblast and
76-78% in HeLa cells, respectively). These data suggest that
phosphorylation of Ser128 does not affect the proapoptotic activity
of BAD in JNK-dependent apoptosis.
Phosphorylation of BAD at Ser128 had no effect on growth
factor withdrawal–induced cell death. JNK is activated by a
variety of extracellular stimuli, including growth factors, such as
IL-3 (ref. 10; data not shown) and erythropoietin (data not shown).
The survival effect of IL-3 and erythropoietin is mediated, at least
Figure 4. Replacement of Ser128 by alanine in
mBAD does not inhibit the proapoptotic activity of
BAD in UV-induced JNK-dependent apoptosis.
A, characterization of BAD / fibroblasts. B, BAD /
or HeLa cells were transfected with expression vectors
encoding WT GFP-mBAD, GFP-mBAD(S128A), or
GFP (1.0 Ag each), respectively. Expression of
GFP-mBAD proteins was analyzed by immunoblotting
using anti-BAD antibody. C, UV-induced apoptotic cell
death of various transfected, GFP-positive BAD /
cells. Sixteen hours after transfection, cells were
treated with or without UV (60J/m2, 4 hours), and the
apoptotic cell death was measured by Hoechst
staining. D, schematic presentation of UV-induced
apoptotic cell death in BAD / and HeLa cells
expressing various BAD proteins. BAD / cells were
treated as described in (C ), whereas HeLa cells were
transfected with expression vectors encoding WT
GFP-mBAD, GFP-mBAD(S128A), or GFP (1.0 Ag
each). Sixteen hours after transfection, HeLa cells
were treated with or without UV (60 J/m2, 2 hours).
Data represent three independent experiments.
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Figure 5. Replacement of Ser128 by alanine in mBAD does not
inhibit the proapoptotic activity of BAD in growth factor
withdrawal–induced apoptosis. A, FL5.12 Bcl-xL cells were
transfected with expression vectors encoding WT GFP-mBAD
or GFP-mBAD(S128A) (10 Ag each), respectively. Expression
of GFP-mBAD proteins was analyzed by immunoblotting using
anti-BAD antibody. B, FL5.12 Bcl-xL cells transfected with WT
GFP-mBAD or GFP-mBAD (S128A) were deprived of IL-3 for
various times (24 and 48 hours). Apoptotic cell death of
GFP-positive cells was analyzed as described (10). Data
represent three independent experiments. C, HCD57 cells
were transfected with expression vectors encoding WT
GFP-mBAD or GFP-mBAD(S128A) (5 Ag each) along with
Bcl-xL (20 Ag), respectively. Expression of GFP-mBAD and
Bcl-xL proteins was analyzed by immunoblotting using
anti-BAD antibody and anti-Bcl-xL antibody, respectively.
D, HCD57 cells transfected with WT GFP-mBAD or
GFP-mBAD(S128A) were deprived of erythropoietin (EPO ) for
24 hours and apoptotic cell death of GFP-positive cells was
analyzed as described (10). Data represent three independent
experiments.
in part, by phosphorylation and consequent inactivation of the
proapoptotic molecule BAD through activation of a group of
protein kinases, such as Akt, PKA, and JNK (4–11). If JNK
phosphorylated BAD at Ser128 and enhanced its proapoptotic
activity, as suggested previously (20), replacement of Ser128 by
alanine would reduce the proapoptotic activity of BAD, thereby
suppressing growth factor withdrawal–induced cell death. To test
this, IL-3-dependent FL5.12 Bcl-xL cells were transfected with
mammalian expression vectors encoding WT GFP-BAD or GFPBAD(S128A), respectively. Immunoblotting analysis showed that
expression levels of GFP-BAD(S128A) were similar to that of WT
GFP-BAD (Fig. 5A). Withdrawal of IL-3 for 2 hours resulted in
inactivation of JNK in FL5.12 Bcl-xL cells as measured by immune
complex kinase assays (data not shown). Apoptotic death assays
revealed that there was no significant difference between cells
expressing GFP-BAD(S128A) or WT GFP-BAD in the presence of IL3 or on IL-3 withdrawal (Fig. 5B). Similar results were obtained in
erythropoietin-dependent HCD57 cells. There was no significant
difference between HCD57 cells expressing GFP-BAD(S128A) or
WT GFP-BAD (Fig. 5C) in the presence of erythropoietin or on
erythropoietin withdrawal (Fig. 5D). These data suggest that
phosphorylation of Ser128 does not affect the proapoptotic activity
of BAD in growth factor withdrawal–induced cell death.
Discussion
The proapoptotic Bcl-2 family protein BAD plays a central role
in the cross-talk between the growth/survival factor signaling
pathways and the intrinsic, mitochondria-dependent death machinery (2). In response to growth/survival factors, such as IL-3 or
IGF-I, BAD is phosphorylated at the regulatory serines (Ser112,
Ser136, or Ser155) by Akt/PKB, PKA, Raf-1, Rsk2, and CaMKII (4–9,
11). The phosphorylation promotes the interaction of BAD with
the cytoplasmic anchorage proteins 14-3-3, which sequesters BAD
in the cytoplasm (11). On withdrawal of growth/survival factors,
BAD is dephosphorylated and subsequently translocates to
Cancer Res 2005; 65: (18). September 15, 2005
mitochondria to inactivate the antiapoptotic Bcl-2 family proteins
Bcl-xL and Bcl-2, resulting in increased mitochondrial permeability,
release of cytochrome c, caspase activation, and ultimately
apoptotic cell death (2). In addition, BAD is phosphorylated by
JNK at Thr201 in response to the survival factor IL-3 in IL-3dependent hematopoietic cells (10). The Thr201 phosphorylation
also inhibits the proapoptotic activity of BAD by reducing the
interaction between BAD and Bcl-xL (10). However, it was reported
that BAD could be phosphorylated by JNK at Ser128, leading to the
enhancement of its proapoptotic activity (20). These contradictory
results regarding the role of JNK in regulating the proapoptotic
activity of BAD cause confusions in the field (21–33) and thereby
require further investigation. In this report, we show that Ser128 is
not phosphorylated by JNK and elimination of Ser128 phosphorylation does not affect the proapoptotic activity of BAD in
apoptosis induced by UV via JNK or growth factor withdrawal.
These conclusions are based on the following evidence.
First, JNK1 only phosphorylated mBAD, which has both Ser128
and Thr201, but not hBAD, which has Ser91 (Ser128 equivalent) but
not the Thr201 equivalent (Fig. 1B). In contrast, Cdc2, which is
known to phosphorylate Ser128 (39), phosphorylated both mBAD
and hBAD (Fig. 1B). Second, replacement of Thr201, but not Ser128,
by alanine abolished phosphorylation of BAD by active JNK1
in vitro and in vivo (Fig. 2A and D). Third, in vitro, purified active
recombinant JNK1 phosphorylated WT BAD and BAD(S128A) to a
similar extent, but not the BAD(T201) mutant (Fig. 2C). Fourth, site
mutagenesis in combination with two-dimensional phosphopeptide mapping and phosphoamino acid analysis showed that JNK1
only phosphorylated BAD at Thr201 but not Ser128 (Fig. 3). Fifth,
replacement of Ser128 by alanine did not reduce the proapoptotic
activity of BAD in apoptosis induced by UV via JNK1 or growth
factor withdrawal (Figs. 4 and 5). These observations were obtained
by using JNK1 because it is the major JNK isoform that is
significantly activated by extracellular stimuli (15). Taken together,
these results show that JNK only phosphorylates BAD at Thr201, but
not Ser128, and Ser128 phosphorylation is not required for the
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JNK Is Not a BAD Ser128 Kinase
proapoptotic activity of BAD in apoptosis induced by UV via JNK or
growth factor withdrawal.
JNK has proapoptotic or antiapoptotic functions depending on
the cell context and death stimulus (14). JNK contributes to TNF-ainduced apoptosis when NF-nB activation is blocked (14–19) or
UV-induced apoptosis (15, 40). However, the molecular target(s) of
JNK in apoptosis is still elusive. Our results showed that JNK does
not phosphorylate BAD at Ser128 to promote apoptosis. In contrast,
we have shown previously that JNK activation contributes to the
survival of IL-3-dependent hematopoietic cells (10). JNK is
activated by IL-3 and in turn phosphorylates BAD at Thr201, but
not Ser128, resulting in suppression of the proapoptotic activity of
BAD (10). Because JNK directly phosphorylates BAD at Thr201
(ref. 10; Fig. 3), this raises an important question that why JNK
contributes to, rather inhibits, apoptosis induced by death stimuli
like TNF-a and UV, if JNK can phosphorylate BAD at Thr201 and
inhibit the proapoptotic activity of BAD. One of the possibilities is
that the death stimuli that activate JNK, such as TNF-a and UV,
might simultaneously activate yet-to-be identified threonine
phosphatase(s) that could dephosphorylate JNK-phosphorylated
Thr201. Indeed, no significant BAD phosphorylation of Thr201 was
observed in cells treated with the death stimuli, such as UV and
TNF-a, which also activate JNK (data not shown). Another
possibility is that, unlike IL-3 withdrawal-induced apoptosis that
depends on dephosphorylation of BAD, dephosphorylation of BAD
may not be the mechanism by which other death stimuli trigger
apoptosis. Future studies are needed to explore these possibilities.
Our finding that JNK does not phosphorylate BAD at Ser128 is
contradictory to an earlier report, which showed that JNK
phosphorylated BAD at Ser128 (20). In this earlier report, there
was no evidence that it was JNK that phosphorylated BAD at Ser128
either in vitro or in vivo. First, purified recombinant JNK1a1
preparation from a commercial company was used as the source of
JNK in the in vitro kinase assay to phosphorylate BAD. However,
the kinase-deficient recombinant JNK1a1 mutant was not included
as a control to exclude the possibility that the preparation may be
contaminated by other protein kinases that phosphorylate BAD at
Ser128. In this case, the mass spectrometric analysis only showed
that BAD was phosphorylated at Ser128, but it did not prove that
Ser128 was phosphorylated by JNK. Second, the BAD(S128A) mutant
was not used to show that JNK indeed phosphorylated BAD at
Ser128. This is odd because the BAD(S128A) mutant was used to
show that Cdc2 phosphorylated BAD at Ser128 (39). Third,
anisomycin or transfected MEKK1 was used to show that Ser128
was phosphorylated by JNK in cells. However, both anisomycin and
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Acknowledgments
Received 2/18/2005; revised 7/8/2005; accepted 7/13/2005.
Grant support: NIH grants CA92650 and CA100460 (A. Lin).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Stanley Korsmeyer, John C. Reed, Richard J. Youle, Jialing Xiang,
Charles Rudin, Amittha Wickrema, and Yuzuru Minemoto for reagents that make this
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BAD Ser128 Is Not Phosphorylated by c-Jun NH2-Terminal
Kinase for Promoting Apoptosis
Jiyan Zhang, Jing Liu, Chenfei Yu, et al.
Cancer Res 2005;65:8372-8378.
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