Interference of BAD (Bcl-xL/Bcl-2-Associated Death Promoter)-Induced Apoptosis in Mammalian Cells by 143-3 Isoforms and P11
Sheau Yu Hsu,
Antti Kaipia,
Li Zhu and
Aaron J. W. Hsueh
Division of Reproductive Biology (S.Y.H., A.K., A.J.W.H.)
Department of Gynecology and Obstetrics Stanford University Medical
School Stanford, California 94305-5317
Division of
Molecular Biology Application (L.Z.) CLONTECH Lab, Inc. Palo
Alto, California 94303-4607
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ABSTRACT
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Apoptosis and survival of diverse
cell types are under hormonal control, but intracellular mechanisms
regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins
contains conserved Bcl-2 homology regions that mediate the formation of
homo- or heterodimers important for enhancing or suppressing apoptosis.
Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2
associated death promoter), a death enhancer, has no C-terminal
transmembrane domain for targeting to the outer mitochondrial membrane
and nuclear envelope. We hypothesized that BAD, in addition to binding
Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family.
Using the yeast two-hybrid system to search for BAD-binding proteins in
an ovarian fusion cDNA library, we identified multiple cDNA clones
encoding different isoforms of 143-3, a group of evolutionally
conserved proteins essential for signal transduction and cell cycle
progression. Point mutation of BAD in one (S137A), but not the other
(S113A), putative binding site found in diverse 143-3 interacting
proteins abolished the interaction between BAD and 143-3 without
affecting interactions between BAD and Bcl-2. Because the S137A BAD
mutant presumably resembles an underphosphorylated form of BAD, we used
this mutant to screen for additional BAD-interacting proteins in the
yeast two-hybrid system. P11, a nerve growth factor-induced neurite
extension factor and member of the calcium-binding S-100 protein
family, interacted strongly with the mutant BAD but less effectively
with the wild type protein. In Chinese hamster ovary (CHO) cells,
transient expression of wild type BAD or its mutants increased
apoptotic cell death, which was blocked by cotransfection with the
baculovirus-derived cysteine protease inhibitor, P35. Cotransfection
with 143-3 suppressed apoptosis induced by wild type or the S113A
mutant BAD but not by the S137A mutant incapable of binding 143-3.
Furthermore, cotransfection with P11 attenuated the proapoptotic effect
of both wild type BAD and the S137A mutant. For both 143-3 and P11,
direct binding to BAD was also demonstrated in vitro. These
results suggest that both 143-3 and P11 may function as BAD-binding
proteins to dampen its apoptotic activity. Because the 143-3 family
of proteins could interact with key signaling proteins including Raf-1
kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas
P11 is an early response gene induced by the neuronal survival factor,
nerve growth factor, the present findings suggest that BAD plays an
important role in mediating communication between different signal
transduction pathways regulated by hormonal signals and the apoptotic
mechanism controlled by Bcl-2 family members.
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INTRODUCTION
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For the maintenance of homeostasis in a
multicellular organism, a balance of cell division and cell death must
be reached. Apoptosis is an active form of cell death essential for the
elimination of superfluous cells during diverse physiological processes
in essentially all animal species (1, 2). Although regulation of
apoptosis by extracellular mediators is cell type-specific,
intracellular pathways leading to apoptosis are conserved from nematode
to mammals and include proteins of the
ced-3/interleukin-1ß-converting enzyme (ICE) and ced-9/bcl-2 gene
families (3, 4, 5, 6). While the ced-3/ICE family of protease function
to disintegrate important cellular proteins during apoptosis, the
ced-9/bcl-2 family members are regulators upstream of the ced-3/ICE
proteolytic cascade (1, 6). In Caenorhabditis elegans, the
ced-9 gene is essential for apoptosis repression during embryonic
development whereas its mammalian homologs form homo- and heterodimers
with some members (Bcl-2, Bcl-w, Bcl-xL, Mcl-1, and A1) acting as
suppressors of cell death and others [Bax, BAD
(Bcl-xL/Bcl-2-associated death promoter), Bak, Bid, and Bik] as death
promoters. However, little is known regarding the coordination of
apoptosis controlled by Bcl-2 family members and pathways involved in
signal transduction or cell cycle progression.
BAD is a new member of the Bcl-2 family; it shares the
conserved Bcl-2 homology (BH)1 and BH2 domains with other Bcl-2 family
members and counters the antiapoptotic effects of Bcl-xL in an
interleukin 3-dependent cell line (7). However, BAD does not have the
C-terminal transmembrane domain found in most Bcl-2 family members but
contains unique hydrophilic PEST motifs (7) postulated to be targets of
protease degradation (8). Based on the unique structural features of
BAD, we hypothesized that BAD may regulate apoptosis in a manner
different from other Bcl-2 family members and interact with cellular
proteins outside the Bcl-2 family.
In a recent report using expression cloning of an
embryonic cDNA library, the interaction between mouse BAD and 143-3
proteins was demonstrated (9). In addition, serine phosphorylation of
BAD was shown to be important for 143-3 binding and cell survival. We
used the yeast two-hybrid system to search for BAD-interacting proteins
and also found that different isoforms of the 143-3 protein bind
strongly to BAD through a putative 143-3 binding site. The highly
conserved ubiquitous 143-3 family of proteins is expressed in diverse
eukaryotic organisms (10). They are capable of binding to a variety of
protooncogenes and key enzymes important in different
intracellular signaling pathways including mitogen-activated cell
cycle progression, signal transduction mediated by protein kinase C
isoforms, and oncogenesis (11, 12, 13, 14, 15, 16, 17). We found that overexpression of
143-3 suppresses apoptosis induced by BAD in Chinese hamster ovary
(CHO) cells, indicating that interactions between them may allow
coordination of cell death and 143-3-regulated intracellular
signaling pathways. Using a BAD mutant (S137A BAD) not capable of
binding 143-3 as a bait in the yeast two-hybrid system, we further
identified another BAD-interacting protein P11. P11, also known as 42C
or calpactin I light chain, is an early response gene induced by nerve
growth factor (NGF) and found to be essential for neuronal cell
survival and neurite formation (18, 19). Overexpression of P11 was also
found to partially block BAD-induced apoptosis in CHO cells.
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RESULTS
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Specific Interaction between BAD and 143-3 Family of
Proteins: Serine to Alanine Mutation in One of the Two Putative 143-3
Binding Sites of BAD Abolishes BAD Binding to 143-3 in Yeast
Using BAD as the bait, screening of 1.5 million
independent transformants from a rat ovarian Matchmaker cDNA library
using the yeast two-hybrid system (20, 21) yielded more than 50 clones
positive for both GAL1-HIS and GAL4-lacZ reporter gene expression. DNA
sequencing of eight prominent positive clones identified them as the
full-length rat homologs of ß,
, and
forms of 143-3 cDNAs.
Sequence analysis also showed that one cDNA clone for the ß-form of
143-3 encodes a novel splicing variant with a shorter 3'-
untranslated region. Studies using yeast cells further indicated
comparable interactions between BAD and four 143-3 isoforms (ß,
,
, and
; Fig. 1A
; left
panel). In the same assay, neither Bcl-2 (Fig. 1A
; right
panel) nor Bax or lamin C (data not shown) interacted with any of
the 143-3 proteins, suggesting the interaction between BAD and
143-3 is specific. Independent verification of protein-protein
interactions was confirmed based on activation of the GAL4-lacZ
reporter expression (data not shown).

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Figure 1. Interaction between BAD and 143-3 in Yeast Cells
A, Different 143-3 isoforms interact with BAD but not with
Bcl-2. Left panel, Growth of yeast colonies expressing
BAD fused to the GAL4-binding domain together with different isoforms
of 143-3 (ß, , , and ) fused to the GAL4 activation
domain. Right panel, Lack of growth of yeast colonies
expressing Bcl-2 fused to the GAL4-binding domain together with
isoforms of 143-3 fused to the GAL4 activation domain. B,
Diagrammatic drawing of BAD sequence showing two putative
143-3-binding sites and mutant BAD constructs with point mutations
within the 143-3-binding motifs or with deletions. C, Interaction
between 143-3 and BAD mutants. Yeast cells were grown in plates with
30 mM 3-aminotriazole and without Trp, Leu, and His. +,
Positive for the HIS3 and GAL4 reporter gene expression in yeast
colonies expressing BAD or its mutants together with the ß-isoform of
143-3; , Absence of expression of both reporter genes. Ser to Ala
mutation in one of the two putative 143-3-binding sites of BAD
abolished interactions between BAD and 143-3. Furthermore, truncation
of residues 1139, but not 142, 161, or 189205 of BAD, disrupted
its interaction with 143-3, whereas BAD 141205 and BAD 1188
showed no interaction with Bcl-2.
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Analysis of deduced polypeptide from rat BAD cDNA revealed that
the BAD protein contains a BH3-like motif downstream of the BH1 domain
proposed earlier (7) and two consensus 143-3 binding motifs RxRSxSxP
(Fig. 1B
and Ref.16). To test the structural requirement for
interactions between BAD and 143-3, BAD mutants with single amino
acid changes within the putative 143-3 binding motifs or with
truncations were generated (Fig. 1B
and Ref.22). In yeast two-hybrid
analysis (Fig. 1C
), S137A, but not S113A, mutation in the binding motif
of BAD abolished interaction of BAD with 143-3. Likewise, double
mutations in both sites also abolished BAD binding to 143-3. The
effect of the S137A mutation is context-specific because this mutant,
like the wild type protein, still interacted with Bcl-2. Furthermore,
truncation of residues 1140, but not 142, 161, or 189205, in
BAD disrupted its interaction with 143-3 (Fig. 1C
).
Identification of P11 as a Binding Protein for Underphosphorylated
BAD: Preferential Binding of P11 to the S137A BAD Mutant
Phosphorylation of the second serine in the 143-3 binding
site is essential for interaction of 143-3 with different signaling
proteins (16). We hypothesized that the S137A BAD mutant not capable of
binding 143-3 resembles an underphosphorylated form of BAD and used
it as a bait to screen for an additional 1.5 million yeast
transformants expressing rat ovarian fusion cDNAs. Among 13
positive clones, eight encoded P11, also known as 42C or calpactin I
light chain. As shown in Fig. 2A
(left panel), yeast cells coexpressing P11 and the S137A BAD
mutant showed pronounced growth whereas minimal growth was found in
cells coexpressing P11 and the wild type BAD. Likewise, the S113A/S137A
BAD double mutant not capable of binding 143-3 also showed strong
interaction with P11. In the same assay, wild type BAD, but not its
S137A and S113A/S137A mutants, interacted with 143-3 (Fig. 2A
;
right panel). Independent verification of protein-protein
interactions between S137A BAD and P11 was confirmed based on
activation of the GAL4-lacZ reporter expression. Using the same assay,
no interaction of P11 with Bcl-2, BAX, or lamin C was observed (data
not shown). These data suggested that underphosphorylated S137A BAD
binds preferentially to P11.

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Figure 2. Preferential Binding of P11 to the S137A BAD Mutant
A, Protein-protein interactions were studied in yeast cells
coexpressing the GAL4-binding domain fused to wild type BAD or BAD
mutants, together with the GAL4 activation domain fused to P11.
Left panel, Pronounced growth of yeast cells was found
in colonies expressing P11 together with S137A or S113A/S137A BAD
mutants but minimal growth in cells expressing P11 and wild type BAD.
Right panel, Using the same assay, interactions between
143-3 and BAD or its mutants are shown for comparison. B, Interaction
between P11 and BAD mutants with truncation and/or S137A point
mutation. Assay of protein-protein interactions in yeast was conducted
as described in Fig. 1C .
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Further studies on interactions between P11 and different truncated
forms of the S137A BAD in the two-hybrid assay demonstrated that
truncation of residues 1140 or 189205, but not 161, in S137A BAD
disrupted its interaction with P11, indicating that the N-terminal
sequence of BAD is not essential for the interaction between P11 and
BAD (Fig. 2B
).
Induction of Cell Death after Overexpression of Wild Type and
Mutant BAD in CHO Cells and Blockage by Baculovirus Apoptosis Inhibitor
P35
To investigate the role of BAD and its interacting proteins on
cell survival, a ß-galactosidase cotransfection assay was used to
examine BAD activity (3, 23). CHO cells were transfected with various
expression vectors together with a 1/10 equivalent of the pCMV-ß-gal
plasmid. After 36 h, cells were stained with X-gal to identify
transfected blue cells for examination of morphological signs of cell
death. As shown in Fig. 3A
(left
panel), cells transfected with an empty pcDNA3 vector showed
normal spindle-shaped morphology with minimal cell death. In contrast,
most cells transfected with the BAD expression plasmid showed
characteristics of apoptosis including cell shrinkage, a round-up
shape, and cytoplasmic fragmentation (Fig. 3A
; right panel).
Immunocytochemical analysis (Fig. 3B
) showed BAD expression in both
normal and apoptotic cells transfected with the BAD expression vector
but not in cells transfected with the empty vector. Quantification of
cell death using the cotransfection assay indicated that >55% of
cells underwent apoptosis after BAD expression. Similar to wild type
BAD, overexpression of the S113A and S137A BAD mutants also increased
the percentage of cells undergoing apoptosis (P <
0.01) whereas cells transfected with a plasmid with BAD in reverse
orientation did not (Fig. 3C
). Western blot analysis further
demonstrated that wild type BAD migrated as two bands whereas only the
lower band was seen for cells expressing the mutants (Fig. 3C
, lower panel).

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Figure 3. Induction of Apoptosis after Overexpression of Wild
Type BAD or its Mutants in CHO Cells: Suppression of BAD Action by the
Caspase Inhibitor P35
A, Morphology of CHO cells transfected with empty or BAD expression
vector. CHO cells were transiently transfected with pcDNA3 expression
vector alone (left panel) or the vector containing BAD
cDNA (right panel; 2.5 µg DNA/35 mm dish). The
pCMV-ß-gal expression vector (0.25 µg/dish) was included to monitor
transfected cells. Apoptotic cells are indicated by
arrowheads. B, Immunocytochemical detection of BAD in
cells transfected with the vector alone (left panel) or
the vector containing BAD cDNA (right panel). Positive
staining is indicated by arrowheads. C, Quantification
of apoptosis induced by BAD or BAD mutants in transfected cells. The
percentage of ß-gal-expressing cells showing apoptotic morphology was
determined at 36 h after transfection. Data are expressed as the
percentage (mean ± SEM) of blue cells
exhibiting signs of apoptosis. A control group transfected with a
vector containing BAD in reverse orientation (Rev BAD) is also shown.
In the bottom panel, Western blot analysis of BAD
expression is shown. Aliquots of lysate from cells transfected with
different expression vectors were resolved on 13% SDS-PAGE and probed
with an anti-BAD rabbit polyclonal antibody. The higher molecular mass
band presumably represents BAD phosphorylated at both potential
phosphorylation sites as found for FL5.12 cells (9). D, Blockage of
BAD-induced apoptosis after cotransfection with baculovirus-derived
apoptosis inhibitor P35. Cells were cotransfected with wild type,
S113A, or S137A BAD with or without the P35 expression plasmid, and the
percentage of ß-gal staining cells showing apoptosis was determined.
In these experiments, cells were transfected with a total of 8.25 µg
plasmid DNA including 7.5 µg of pcDNA3 expression constructs and 0.75
µg of the pCMV-ß-gal reporter. In groups receiving two different
pcDNA3 expression plasmids, 3.75 µg of each were used. Data are
expressed as the percentage (mean ± SEM) of
blue cells exhibiting signs of apoptosis.
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To demonstrate that the observed apoptosis is mediated by the caspase
family of proteases, cells were cotransfected with plasmids encoding
BAD or its mutants with or without the baculovirus-derived serpin
inhibitor P35 (24, 25). As shown in Fig. 3D
, induction of apoptosis by
BAD or its S137A and S113A mutants was reduced after coexpression with
P35 (P < 0.01), suggesting the involvement of a
caspase-mediated proteolysis cascade. In contrast, transfection with
the P35 expression vector alone did not affect cell survival.
Suppression of BAD-Induced Apoptosis by 143-3 and P11 in CHO
Cells
To test the ability of 143-3 to modulate BAD-induced apoptosis,
CHO cells were cotransfected with vectors encoding BAD and the
ß-isoform of 143-3 (Fig. 4A
). The
ability of wild type or S113A BAD to induce apoptosis was reduced after
cotransfection with an equivalent amount of the 143-3 expression
vector (P < 0.01). In contrast, apoptosis induced by
the S137A BAD mutant was not affected by 143-3 coexpression,
consistent with its inability to bind 143-3. Also, transfection with
the 143-3 expression vector alone did not affect cell survival.
Western blot analysis of cell lysate showed the expression of BAD
proteins with apparent molecular masses of 27 and 28 kDa in cells
transfected with the expression plasmid encoding wild type BAD, while
the mutant proteins migrated as a single band of 27 kDa (Fig. 4A
, lower panel). The higher molecular mass band presumably
represents phosphorylated BAD. Of importance, the BAD antigen level did
not decrease in cells coexpressing BAD and 143-3, suggesting that the
observed attenuation of BAD action by 143-3 is not due to changes in
BAD expression. In addition, Western blot analysis using a
ß-isoform-specific 143-3 antibody indicated that cotransfection
with the 143-3 expression vector increased the level of this protein
in transfected cells (Fig. 4A
, bottom panel).
Immunoprecipitation analysis further confirmed direct interaction
between BAD and 143-3 in CHO cells. Incubation of lysate of cells
expressing wild type BAD, but not lysates from cells transfected with
the empty vector, with an anti-BAD antibody resulted in the
precipitation of 143-3 proteins (Fig. 4B
).

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Figure 4. Suppression of BAD-Induced Apoptosis after
Coexpression with 143-3
A, Overexpression of 143-3 attenuates apoptosis induced by wild type
or S113A BAD but not cell death induced by S137A BAD. CHO cells were
cotransfected with 143-3 (ß-isoform) and/or expression vectors
encoding wild type BAD and its mutants. Estimation of apoptosis was
determined as described in Fig. 3 legend. Lower panel
shows Western blot analysis of BAD and ß-isoform 143-3 in cells
expressing BAD or its mutants with or without 143-3. B,
Coprecipitation of wild type BAD and 143-3 in CHO cells. After
transfection with the BAD expression vector, cell lysate was
precipitated with BAD antibodies before SDS-PAGE analysis and
immunoblotting using anti-143-3 (K19; Santa Cruz Biotechnology).
Specific band for 143-3 found in BAD-transfected cells is indicated
by an arrow whereas a nonspecific band with higher
molecular mass is found in cells with or without BAD expression.
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To test the role of P11 in modulating BAD-induced apoptosis, CHO cells
were cotransfected with an expression plasmid encoding P11 together
with those encoding wild type BAD or the S137A mutant (Fig. 5A
). Although
overexpression of P11 alone did not affect the percentage of cells
undergoing apoptosis, coexpression of P11 decreased apoptosis induced
by either wild type BAD or the S137A BAD (P < 0.01).
Furthermore, Western blot analysis indicated that cotransfection with
P11 did not affect the amount of BAD antigen expressed (Fig. 5A
, lower panel), suggesting the blockage of BAD action by P11
was not due to uneven protein expression. To further substantiate the
interaction between P11 and BAD observed in yeast cells, a glutathione
S-transferase (GST) fusion protein system was used.
Bacteria-derived recombinant GST-BAD, a fusion protein of BAD and GST,
was immobilized onto glutathione-Sepharose beads and used to confirm
the interaction between P11 and BAD (Fig. 5B
). Recombinant P11 protein
tagged with a hemagglutinin epitope (HA-P11) bound to beads containing
the GST-BAD fusion protein but did not bind to beads containing GST
alone, further suggesting that P11 directly interacts with BAD without
posttranslational modification.

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Figure 5. Suppression of Apoptosis Induced by Wild Type or
S137A BAD when Coexpressed with P11
A, CHO cells were cotransfected with P11 and/or wild type or S137A BAD
expression vectors. Estimation of apoptosis was determined as described
in Fig. 3 legend. Lower panel shows Western blot
analysis of the BAD antigen in cells expressing the BAD mutant with or
without P11. Specific bands for BAD are indicated by
arrows. B, Direct binding between P11 and BAD in
vitro. Recombinant HA-P11, GST, and GST-BAD produced in E.
coli were purified by affinity column, and the levels and
purity of the fusion proteins were evaluated by SDS-PAGE. Equal
concentrations (1 µg) of recombinant GST and GST-BAD were immobilized
onto glutathione-Sepharose beads and incubated with recombinant HA-P11
(0.5 µg) in a binding buffer for 4 h at 4 C. After extensive
washing, proteins retained on the beads were extracted and analyzed by
SDS-PAGE and Western blotting. Immunoblot analysis using an anti-GST
polyclonal antibody showed that equal amounts of GST and GST-BAD were
immobilized on GST beads (top panel). Immunoblotting of
the same samples using an anti-HA antibody showed that HA-P11 protein
was specifically retained by the immobilized GST-BAD but not by the GST
protein (lower panel).
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DISCUSSION
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We have identified 143-3 family proteins and P11 as
binding proteins for the pro-apoptotic Bcl-2 family member BAD; 143-3
interacts with wild type BAD that is presumably phosphorylated at the
consensus 143-3 binding sites whereas P11 interacts preferentially
with underphosphorylated BAD. In CHO cells, BAD induces apoptosis,
whereas overexpression of 143-3 or P11 dampens cell death induced by
BAD. These results suggest that 143-3 and P11 may interact with BAD
to constrain its pro-apoptotic activity. Depending on its
phosphorylation status at the 143-3 binding sites, BAD induction of
apoptosis may be attenuated through preferential interaction with the
widely expressed 143-3 or the hormone-inducible P11, underlying the
importance of BAD in coordinating extracellular survival signals and
the Bcl-2-associated apoptotic machinery.
BAD was originally isolated as a Bcl-2-binding protein that blocks the
antiapoptotic effect of Bcl-xL in an interleukin 3-dependent cell line
(7). Because BAD interacts only with the antiapoptotic proteins Bcl-xL
and Bcl-2 and does not promote cell death by itself in the interleukin
3-dependent cell line, BAD was proposed to promote apoptosis by
dimerization with Bcl-xL and displace the apoptosis promoter Bax (7).
In this study, transient overexpression of BAD alone in CHO cells
induces apoptosis, thus providing a convenient system to study the role
of BAD and its binding proteins in modulating apoptosis. Because the
baculovirus serpin inhibitor P35 blocked BAD-induced apoptosis, the
pro-apoptotic action of BAD may involve a caspase-mediated proteolysis
cascade. Dimerization among different Bcl-2 family members is mediated
by the conserved BH1 and BH2 domains (26, 27). Although the exact Bcl-2
family member serving as the dimerization partner for BAD in CHO cells
is unclear, BAD could exert its pro-apoptotic action by blocking the
antiapoptotic effect of one of the survival genes in the Bcl-2 family.
A fundamental question in apoptosis research is the mechanisms by which
hormonal and other extracellular survival signals regulate the Bcl-2
and the downstream caspases. Identification of 143-3 and P11 as
binding proteins for BAD and the observation that 143-3 and P11 both
attenuate apoptosis induced by BAD suggest that the pro-apoptotic
activity of BAD could be modulated by multiple signaling proteins
outside the Bcl-2 family.
The highly conserved ubiquitous 143-3 proteins are expressed in
perhaps all eukaryotic organisms, including yeast, plants, insects, and
mammals, with at least seven different isoforms identified in mammalian
cells (10). Proteins of the 143-3 family bind diverse enzymes and
signaling molecules, including Raf-1 kinase, B-Raf, phosphatidyl
inositol 3 kinase, CDC25 phosphatases, Bcr, Cbl, and polyoma middle T
antigen (14). They are important in intracellular signaling, cell cycle
control, oncogenesis, and neurotransmitter biosynthesis in neuron (10).
Crystallographic studies revealed that different isoforms of 143-3
dimerize to generate a complex with two ligand-binding sites (28, 29),
thus allowing the assembly or anchoring of functional complexes
containing diverse signaling proteins and cytoskeletal elements
(15, 16, 17). Although the mechanisms by which 143-3 interferes with
BAD-induced apoptosis remain to be defined, it is possible that 143-3
proteins may bring BAD to the proximity of specific enzymes, allowing
cross-talks between BAD and different signaling pathways.
Alternatively, this interaction may serve to prevent BAD from
interacting with downstream effectors. After treatment with
interleukin-3, FL5.12 cells showed an increase in BAD phosphorylation.
Subsequent binding of 143-3 to phosphorylated BAD prevents BAD
interaction with the membrane-bound BclxL, thus freeing BclxL to
function as a survival protein (9). Our findings are consistent with
these studies and further suggest that overexpression of 143-3 could
suppress BAD-induced apoptosis. Because 143-3 proteins bind and
modulate the activity of Raf-1 (30, 31, 32, 33), interactions between 143-3
and BAD may allow coordinated regulation of cell cycle progression and
apoptosis.
Phosphorylation of the second serine in the 143-3 consensus-binding
site (RxRSxSxP) is essential for interaction between 143-3 and its
binding proteins (14, 16). Among the two potential 143-3-binding
sites in BAD (Fig. 1B
), the second shows complete consensus whereas the
first is less conserved. In our study, mutation of serine 137 in the
second consensus site, but not serine 113 in the first site, abolished
BAD binding in yeast cells. Likewise, overexpression of 143-3 in CHO
cells attenuated apoptosis induced by wild type BAD or its S113A
mutant, whereas the S137A mutant that is incapable of binding 143-3
in yeast cells retained its apoptosis-inducing activity even when
143-3 was coexpressed. Thus, wild type BAD is presumably
phosphorylated at serine 137 whereas the S137A mutant resembles an
underphosphorylated form and shows minimal interaction with 143-3. In
contrast to our data using both yeast and CHO cells, S137A BAD mutant
could be coprecipitated with 143-3 in FL5.12 cells (9), thus
suggesting the phosphorylation pattern of BAD is probably cell
type-specific, as determined by the levels of different kinases. Our
finding that the S137A BAD mutant could not induce apoptosis more than
wild type BAD further suggests that endogenous 143-3 proteins may be
compartmentalized in CHO cells.
P11 is also known as 42C or calpactin I light chain and belongs to the
S100 family of calcium-binding proteins (18). It was originally
identified as an early response gene after NGF stimulation of a rat
pheochromocytoma (PC12) cell line (18). Of interest, overexpression of
P11 induces neurite outgrowth and enhances PC12 cell survival in the
absence of NGF (19). This protein exists in an unstable soluble form by
itself or complexes with P36 to form a stable and membrane-bound
calpactin I tetramer of (P11)2 (P36)2 (34). In
diverse cell lines, both P11 and P36 were increased after
transformation induced by viral oncogenes (35). The present finding of
P11 as a BAD-interacting protein capable of attenuating the
pro-apoptotic effect of BAD provides a molecular mechanism for the
observed survival function of P11. However, P11 appears to be less
effective than 143-3 in the present transfection assay, probably
because it is a labile protein. Because cellular levels of P11 are
stabilized by P36 or annexin II (34), initially identified as a
substrate for the transforming protein of Rous sarcoma virus (36), it
is of interest to study the effect of coexpressing P11 and P36 on
BAD regulation of apoptosis. PCTAIRE-1, a protein homologous to
cyclin-dependent kinases, was also found to interact with both 143-3
and P11 (37). Although binding domains between specific protein pairs
have not been characterized, it is likely that 143-3 and P11 could
interact with different proteins through similar motifs, and P11 could
have a role in other 143-3-regulated signaling pathways in addition
to apoptosis regulation. Since the expression of P11 is hormonally
regulated and tissue-specific (18), the present observation indicated
that extracellular hormonal signals could modulate BAD-induced
apoptosis through induction of BAD-binding proteins in addition to
regulating the interaction between BAD and 143-3 via BAD
phosphorylation (9). Future studies will reveal the role of P11 and BAD
in hormonally regulated apoptosis models such as ovarian follicles,
from which these cDNAs were isolated.
The use of the S137A BAD mutant with one less hydroxyl group as a bait
allowed us to isolate P11, which showed minimal binding with the
presumably phosphorylated wild type BAD in yeast. However, coexpression
of P11 attenuated the pro-apoptotic activity of both wild type and
S137A BAD in CHO cells. Because immunoblot analysis suggests BAD in CHO
cells existed in both phosphorylated and underphosphorylated forms and
recombinant P11 could interact with wild type BAD in vitro,
P11 could prevent BAD-induced apoptosis by interacting preferentially
with the presumably pro-apoptotic, underphosphorylated form of BAD in
CHO cells. These data suggest the phosphorylation status of BAD is
critical for the regulation of its proapoptotic activity;
phosphorylated BAD interacts with 143-3 whereas underphosphorylated
BAD preferentially interacts with P11 (Fig. 6
). Identification of 143-3 and P11 as
BAD-interacting proteins expands recent findings showing Bcl-2
interaction with other non-Bcl-2 family proteins, such as BAG-1, Nip1,
Nip2, Nip3, Raf-1, R-ras p23, 53BP2, and Ha-Ras p21 (38, 39, 40, 41, 42, 43).

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Figure 6. BAD Plays a Pivotal Role in Mediating Communication
between Different Signal Transduction Pathways and Apoptotic Mechanisms
Controlled by Bcl-2 Family Members
NGF induces P11 whereas diverse extracellular signals may regulate the
accessibility of 143-3 through the regulation of Raf-1 kinase,
protein kinase C isoforms, phosphatidyl inositol-3 kinase, or other
signaling proteins. The hormone-inducible P11 preferentially binds
underphosphorylated forms of BAD whereas the widely expressed 143-3
interacts with BAD phosphorylated at the 143-3-binding sites. Both
P11 and 143-3 attenuate the pro-apoptotic activity of BAD. Survival
factors may induce P11 expression or phosphorylate BAD at 143-3 sites
to dampen BAD-induced cell killing.
|
|
In conclusion, we have demonstrated that the unique Bcl-2 member
BAD interacts with widely expressed 143-3 proteins as well as the
hormone-inducible P11. The observed interactions suggest that
diverse extracellular signals could attenuate the pro-apoptotic
activity of BAD through multiple modulatory pathways, depending on the
phosphorylation status of BAD. Because 143-3 proteins are known to
interact with key enzymes in mitogenic and other signaling pathways,
whereas P11 is capable of substituting the survival and neurite
extension functions of NGF, their interactions with BAD may play a
pivotal role in apoptosis regulation (Fig. 6
). These control mechanisms
may serve as key steps to integrate various hormonal signal
transduction pathways and the apoptosis machinery, thus maintaining
homeostasis.
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MATERIALS AND METHODS
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Two-Hybrid Screening of BAD-Binding Proteins and
Interactions between BAD and 143-3 or P11
Using a GAL4-activation domain (AD)-tagged ovarian
Matchmaker cDNA library prepared from 27-day-old female
Sprague-Dawley rats primed for 36 h with PMSG (CLONTECH Lab,
Inc., Palo Alto, CA), we isolated multiple clones of BAD cDNAs based on
their ability to interact with human Bcl-2 in an HF7c yeast reporter
strain (20). To identify BAD-interacting proteins, we fused rat BAD
cDNA to the binding domain (BD) of GAL4 in a yeast shuttle vector pGBT9
and screened the same ovarian cDNA library using a two-step procedure.
In the first step, yeast cells were transformed with pGBT9-BAD as bait
and selected on plates deficient for tryptophan. In the second step,
selected cells were further transformed with the library cDNAs, and
clones harboring interacting proteins for BAD were selected in plates
lacking tryptophan, leucine, and histidine. Positive transformants were
then selected for growth in media containing 30 mM
3-aminotriazole and for lac-Z reporter gene expression. Individual
AD-fusion cDNAs in positive yeast cells were retrieved after
transformation of HB101 strain Escherichia coli cells. Among
the positive clones sequenced, different isoforms of 143-3 cDNAs were
identified. Using the same approach, P11 was identified as an
interacting protein for the mutant BAD S137A.
Interactions between BAD and its binding proteins were assessed further
using the pGBT9 GAL4-BD and pGADGH GAL4-AD vectors (21). Specific
binding of different protein pairs in yeast was evaluated based on the
activation of GAL1-HIS3 and GAL4-lacZ reporter genes. A minimum of
three independent transformants with each pair of hybrid cDNAs were
analyzed for the expression of two reporter genes. For GAL1-HIS3
reporter expression, cells were grown in a medium lacking leucine,
tryptophan, and histidine but contained 30 mM
3-aminotriazole to inhibit endogenous histidine production. The
activation of the GAL4-lacZ reporter gene was monitored using a filter
lift assay for ß-galactosidase. Yeast cells patched on leucine(-)
and tryptophan(-) plates were incubated for 36 h at 30 C before
lysis by freezing and thawing and placed on filters presoaked in Z
buffer (Na2HPO4, 10 mM KCl, 1
mM 2-mercaptoethanol, pH 7.0) containing 0.4 mg/ml
5-bromo-4-chloro-3-indolyl-ß-D-galactoside. Appearance of
a blue color indicated ß-galactosidase activity.
Construction of Expression Vectors Encoding BAD Mutants
The serine to alanine BAD mutants were generated using
oligonucleotide-directed, two-step PCR mutagenesis (22), whereas the
truncated BAD mutants were derived using PCR amplification. For yeast
studies, mutant cDNAs were subcloned into the pGBT9 expression vector,
whereas the same cDNAs were subcloned into the pcDNA3 expression vector
(Invitrogen, Inc., San Diego, CA) for mammalian cell studies.
Analysis of Apoptosis in Transfected CHO Cells
Apoptosis was monitored after transfection of different cDNAs as
previously described (3, 23). CHO cells were plated at a density of
2 x 105 cells per well in DMEM/F12 supplemented with
10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine. One day later, cells were transfected using
the lipofectamine procedure (Life Technologies, Gaithersburg, MD) with
the empty pcDNA3 expression vector or the same vector containing
different cDNAs, together with 1/10 equivalent of an indicator plasmid
pCMV-ß-gal to allow the identification of transfected cells.
Inclusion of 10-fold excess expression vectors, as compared with the
pCMV-ß-gal reporter plasmid, ensured that most of the
ß-galactosidase-expressing cells also expressed the protein(s) under
investigation. Cells were incubated with plasmids in a serum-free
medium for 4 h, followed by the addition of FBS to a final
concentration of 5% and further incubation for 14 h. After an
additional culture in fresh medium for 18 h, cells were fixed by
0.25% glutaraldehyde and stained with X-gal [0.4 mg/ml in buffer
containing 150 mM NaCl, 100 mM
Na2HPO4, 1 mM MgCl2,
3.3 mM K4Fe(CN)6. 3H2O,
and 3.3 mM K3Fe(CN)6, pH 7.0] for
6 h at 37 C to detect ß-galactosidase expression. The number of
blue cells were counted by microscopic examination and scored as either
live (flat or spindle-shaped blue cells) or dead (fragmented or
rounded-up blue cells) (3, 23). Data are expressed as the percentage
(mean ± SEM) of blue cells exhibiting signs of
apoptosis based on counting of six independent samples (at least 500
cells per 35-mm dish) from three or more separate experiments.
Statistical differences among treatment groups were analyzed using
one-way ANOVA and Scheffe F-test.
Immunoblotting and Immunocytochemical Studies
At 36 h posttransfection, CHO cells were washed with PBS
and lysed in NP-40 lysis buffer (100 mM NaCl, 20
mM Tris, pH 8.0, 1 mM EDTA, and 0.1% NP-40)
supplemented with 1 mM phenylmethylsulfonyl fluoride and 1
µg/ml leupeptin. Protein concentrations were determined by the
Bradford method (Bio-Rad Laboratory, Hercules, CA). Aliquots of cell
lysates were then boiled in Laemmli solubilization buffer before being
electrophoresed on 1315% SDS polyacrylamide gels, transferred onto
Immobilon-P membranes, and blotted for 2 h with primary antibodies
(at 1:5,000 for BAD and at 1:20,000 for ß 143-3; Santa Cruz
Biotechnology, Santa Cruz, CA). After washing with Tris-buffered saline
(TBS)/0.1% Tween 20, membranes were incubated with a goat anti-rabbit
second antibody conjugated to horseradish peroxidase, and signals were
detected using enhanced chemiluminescence (ECL) (Amersham, Arlington
Heights, IL). For immunocytochemistry of BAD, cells were fixed and
quenched with 1% hydrogen peroxide in methanol for 30 min, followed by
three washes in TBS. After incubation with 5% nonimmune goat serum in
TBS, cells were treated with a polyclonal antibody against BAD (at
1:200 in TBS; Santa Cruz Biotechnology) for 30 min, followed by TBS
washing (8 x 3 min) before incubation (30 min) with a goat
anti-rabbit second antibody conjugated to horseradish peroxidase
(Amersham). The signals were developed using the substrate
3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA).
Coprecipitation Experiments
To confirm interactions between BAD and 143-3, CHO cells
transfected with the expression vector encoding BAD or an empty vector
were harvested at 36 h after transfection. After washing, cells
were lysed in binding buffer (50 mM Tris, pH 7.5, 150
mM NaCl, 10 µg/ml trypsin inhibitor, and 0.1% NP-40),
and the supernatant was precleared with 1 µg/ml of nonimmune goat
serum and 10 µl Protein A Sepharose (Pharmacia Biotech, Uppsala,
Sweden). After removal of Protein A Sepharose beads by centrifugation,
cell lysates were incubated with the anti-BAD antibody for 3 h and
Protein A Sepharose (50 µl) for 1 h at 4 C, washed three times
with the binding buffer, and boiled in a 4x Laemmli buffer for 5 min.
Immunoprecipitated proteins were electrophoresed using 13% SDS-PAGE
and analyzed for the presence of 143-3 with an anti-143-3 antibody
(K19 at 1:5,000, Santa Cruz Biotechnology).
In Vitro Protein Interaction Assay
Recombinant hemmagglutinin epitope-tagged P11 (HA-P11) and
GST-BAD were prepared by subcloning rat P11 and BAD cDNAs into
prokaryotic expression vector pTricB and pGEXT-4T-1 (Invitrogen),
respectively. Plasmids were transformed into DH5-á bacteria, and
protein synthesis was induced with 0.5 mM
isopropyl-ß-D-thiogalactoside. The bacteria were lysed in
PBS containing 1% Triton X-100, sonicated, and clarified by
centrifugation before purification of recombinant proteins using
Nickle-NTA-agarose (Qiagen Inc., Chatsworth, CA) or GST-Sepharose
(Pharmacia Biotech) chromatography. In vitro binding assays
were performed by incubating equal amounts of recombinant GST or
GST-BAD immobilized onto glutathione-Sepharose beads, with recombinant
HA-P11 diluted in 0.4 ml Tris buffer (20 mM, pH 8.0)
containing 100 mM NaCl, 0.15% NP-40. The slurry was
incubated at 4 C for 4 h, washed five times with the same buffer,
and resuspended in Laemmli buffer. Proteins retained on the agarose
beads were boiled for 5 min and resolved using 15% SDS-PAGE before
analysis using an anti-HA-peroxidase monoclonal antibody (Boehringer
Mannheim, Indianapolis, IN) or a goat anti-GST polyclonal antibody
(Pharmacia Biotech).
 |
ACKNOWLEDGMENTS
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We thank Dr. Lois K. Miller (University of Georgia, Athens, GA)
for the gift of P35 cDNA. We are also grateful to Dr. Marco Conti for
helpful discussions. The GenBank accession number for rat BAD is
AF003523.
 |
FOOTNOTES
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Address requests for reprints to: A. J. W. Hseuh, Department of Obstetrics/Gynecology, Stanford University School of Medicine, Division of Reproductive Biology, 300 Pasteur Drive, Room A344, Stanford, California 94305-5317.
This study was supported by NIH Grant HD31566 (AJWH).
Received for publication May 12, 1997.
Revision received August 11, 1997.
Accepted for publication August 21, 1997.
 |
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