Interference of BAD (Bcl-xL/Bcl-2-Associated Death Promoter)-Induced Apoptosis in Mammalian Cells by 14–3-3 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 14–3-3, 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 14–3-3 interacting proteins abolished the interaction between BAD and 14–3-3 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 14–3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14–3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14–3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14–3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14–3-3 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 14–3-3 proteins was demonstrated (9). In addition, serine phosphorylation of BAD was shown to be important for 14–3-3 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 14–3-3 protein bind strongly to BAD through a putative 14–3-3 binding site. The highly conserved ubiquitous 14–3-3 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 14–3-3 suppresses apoptosis induced by BAD in Chinese hamster ovary (CHO) cells, indicating that interactions between them may allow coordination of cell death and 14–3-3-regulated intracellular signaling pathways. Using a BAD mutant (S137A BAD) not capable of binding 14–3-3 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Specific Interaction between BAD and 14–3-3 Family of Proteins: Serine to Alanine Mutation in One of the Two Putative 14–3-3 Binding Sites of BAD Abolishes BAD Binding to 14–3-3 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, {eta}, and {theta} forms of 14–3-3 cDNAs. Sequence analysis also showed that one cDNA clone for the ß-form of 14–3-3 encodes a novel splicing variant with a shorter 3'- untranslated region. Studies using yeast cells further indicated comparable interactions between BAD and four 14–3-3 isoforms (ß, {eta}, {theta}, and {epsilon}; Fig. 1AGo; left panel). In the same assay, neither Bcl-2 (Fig. 1AGo; right panel) nor Bax or lamin C (data not shown) interacted with any of the 14–3-3 proteins, suggesting the interaction between BAD and 14–3-3 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 14–3-3 in Yeast Cells

A, Different 14–3-3 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 14–3-3 (ß, {eta}, {theta}, and {epsilon}) 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 14–3-3 fused to the GAL4 activation domain. B, Diagrammatic drawing of BAD sequence showing two putative 14–3-3-binding sites and mutant BAD constructs with point mutations within the 14–3-3-binding motifs or with deletions. C, Interaction between 14–3-3 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 14–3-3; –, Absence of expression of both reporter genes. Ser to Ala mutation in one of the two putative 14–3-3-binding sites of BAD abolished interactions between BAD and 14–3-3. Furthermore, truncation of residues 1–139, but not 1–42, 1–61, or 189–205 of BAD, disrupted its interaction with 14–3-3, whereas BAD 141–205 and BAD 1–188 showed no interaction with Bcl-2.

 
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 14–3-3 binding motifs RxRSxSxP (Fig. 1BGo and Ref.16). To test the structural requirement for interactions between BAD and 14–3-3, BAD mutants with single amino acid changes within the putative 14–3-3 binding motifs or with truncations were generated (Fig. 1BGo and Ref.22). In yeast two-hybrid analysis (Fig. 1CGo), S137A, but not S113A, mutation in the binding motif of BAD abolished interaction of BAD with 14–3-3. Likewise, double mutations in both sites also abolished BAD binding to 14–3-3. 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 1–140, but not 1–42, 1–61, or 189–205, in BAD disrupted its interaction with 14–3-3 (Fig. 1CGo).

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 14–3-3 binding site is essential for interaction of 14–3-3 with different signaling proteins (16). We hypothesized that the S137A BAD mutant not capable of binding 14–3-3 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. 2AGo (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 14–3-3 also showed strong interaction with P11. In the same assay, wild type BAD, but not its S137A and S113A/S137A mutants, interacted with 14–3-3 (Fig. 2AGo; 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 14–3-3 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. 1CGo.

 
Further studies on interactions between P11 and different truncated forms of the S137A BAD in the two-hybrid assay demonstrated that truncation of residues 1–140 or 189–205, but not 1–61, 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. 2BGo).

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. 3AGo (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. 3AGo; right panel). Immunocytochemical analysis (Fig. 3BGo) 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. 3CGo). 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. 3CGo, 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.

 
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. 3DGo, 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 14–3-3 and P11 in CHO Cells
To test the ability of 14–3-3 to modulate BAD-induced apoptosis, CHO cells were cotransfected with vectors encoding BAD and the ß-isoform of 14–3-3 (Fig. 4AGo). The ability of wild type or S113A BAD to induce apoptosis was reduced after cotransfection with an equivalent amount of the 14–3-3 expression vector (P < 0.01). In contrast, apoptosis induced by the S137A BAD mutant was not affected by 14–3-3 coexpression, consistent with its inability to bind 14–3-3. Also, transfection with the 14–3-3 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. 4AGo, 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 14–3-3, suggesting that the observed attenuation of BAD action by 14–3-3 is not due to changes in BAD expression. In addition, Western blot analysis using a ß-isoform-specific 14–3-3 antibody indicated that cotransfection with the 14–3-3 expression vector increased the level of this protein in transfected cells (Fig. 4AGo, bottom panel). Immunoprecipitation analysis further confirmed direct interaction between BAD and 14–3-3 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 14–3-3 proteins (Fig. 4BGo).



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Figure 4. Suppression of BAD-Induced Apoptosis after Coexpression with 14–3-3

A, Overexpression of 14–3-3 attenuates apoptosis induced by wild type or S113A BAD but not cell death induced by S137A BAD. CHO cells were cotransfected with 14–3-3 (ß-isoform) and/or expression vectors encoding wild type BAD and its mutants. Estimation of apoptosis was determined as described in Fig. 3Go legend. Lower panel shows Western blot analysis of BAD and ß-isoform 14–3-3 in cells expressing BAD or its mutants with or without 14–3-3. B, Coprecipitation of wild type BAD and 14–3-3 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-14–3-3 (K19; Santa Cruz Biotechnology). Specific band for 14–3-3 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.

 
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. 5AGo). 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. 5AGo, 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. 5BGo). 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. 3Go 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).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have identified 14–3-3 family proteins and P11 as binding proteins for the pro-apoptotic Bcl-2 family member BAD; 14–3-3 interacts with wild type BAD that is presumably phosphorylated at the consensus 14–3-3 binding sites whereas P11 interacts preferentially with underphosphorylated BAD. In CHO cells, BAD induces apoptosis, whereas overexpression of 14–3-3 or P11 dampens cell death induced by BAD. These results suggest that 14–3-3 and P11 may interact with BAD to constrain its pro-apoptotic activity. Depending on its phosphorylation status at the 14–3-3 binding sites, BAD induction of apoptosis may be attenuated through preferential interaction with the widely expressed 14–3-3 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 14–3-3 and P11 as binding proteins for BAD and the observation that 14–3-3 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 14–3-3 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 14–3-3 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 14–3-3 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 14–3-3 interferes with BAD-induced apoptosis remain to be defined, it is possible that 14–3-3 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 14–3-3 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 14–3-3 could suppress BAD-induced apoptosis. Because 14–3-3 proteins bind and modulate the activity of Raf-1 (30, 31, 32, 33), interactions between 14–3-3 and BAD may allow coordinated regulation of cell cycle progression and apoptosis.

Phosphorylation of the second serine in the 14–3-3 consensus-binding site (RxRSxSxP) is essential for interaction between 14–3-3 and its binding proteins (14, 16). Among the two potential 14–3-3-binding sites in BAD (Fig. 1BGo), 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 14–3-3 in CHO cells attenuated apoptosis induced by wild type BAD or its S113A mutant, whereas the S137A mutant that is incapable of binding 14–3-3 in yeast cells retained its apoptosis-inducing activity even when 14–3-3 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 14–3-3. In contrast to our data using both yeast and CHO cells, S137A BAD mutant could be coprecipitated with 14–3-3 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 14–3-3 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 14–3-3 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 14–3-3 and P11 (37). Although binding domains between specific protein pairs have not been characterized, it is likely that 14–3-3 and P11 could interact with different proteins through similar motifs, and P11 could have a role in other 14–3-3-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 14–3-3 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 14–3-3 whereas underphosphorylated BAD preferentially interacts with P11 (Fig. 6Go). Identification of 14–3-3 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 14–3-3 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 14–3-3 interacts with BAD phosphorylated at the 14–3-3-binding sites. Both P11 and 14–3-3 attenuate the pro-apoptotic activity of BAD. Survival factors may induce P11 expression or phosphorylate BAD at 14–3-3 sites to dampen BAD-induced cell killing.

 
In conclusion, we have demonstrated that the unique Bcl-2 member BAD interacts with widely expressed 14–3-3 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 14–3-3 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. 6Go). These control mechanisms may serve as key steps to integrate various hormonal signal transduction pathways and the apoptosis machinery, thus maintaining homeostasis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two-Hybrid Screening of BAD-Binding Proteins and Interactions between BAD and 14–3-3 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 14–3-3 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 13–15% 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 14–3-3; 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 14–3-3, 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 14–3-3 with an anti-14–3-3 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
 
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
 
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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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