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The Journal of Immunology, 2001, 166: 7345-7352.
Copyright © 2001 by The American Association of Immunologists

Bcl-2 Targets Protein Phosphatase 1{alpha} to Bad1

Verónica Ayllón*, Xavier Cayla{dagger}, Alphonse García{ddagger}, Fernando Roncal*, Raul Fernández*, Juan Pablo Albar*, Carlos Martínez-A.* and Angelita Rebollo2,*

* Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain; {dagger} Laboratoire de Physiologie de la Reproduction, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Paris, France; and {ddagger} Département d’Immunologie, Laboratoire de Signalisation Immunoparasitaire, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1{alpha} is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1{alpha} and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1{alpha} to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1{alpha}. Disruption of Bcl-2/PP1{alpha} association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1{alpha} to Bad.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Bcl-2 family of proteins serves as critical regulators of pathways involved in apoptosis, acting to either inhibit or promote cell death (1). This family is divided into three subfamilies. The first one comprises proteins that are similar to Bcl-2 and are antiapoptotic (Bcl-2-like proteins). The second group includes proteins that are structurally related to Bcl-2 but are proapoptotic (Bax-like proteins). The third group contains proteins that share only the BH3 domain and are proapoptotic (BH3-only proteins). All of these proteins have emerged as key components in the regulation of apoptosis and integrate signals from survival-inducing and death-promoting pathways (2, 3, 4).

The Bcl-2 family proteins homo- and heterodimerize. This balance between specific homo- and heterodimers is thought to be critical for the maintenance of cell survival or for the induction of apoptosis (5, 6, 7). Whereas up- or down-regulation of these proteins may account for the survival of certain cell types in response to extracellular stimuli, it also is possible that survival factors may use protein kinases or phosphatases to alter the ability of apoptotic proteins to promote cell survival or death. Bcl-2 is a positive regulator of cell survival, protecting various cell types from death induced by growth factor deprivation, heat shock, and viral agents (1, 8). Several signal transducing proteins have been reported to directly or indirectly interact with Bcl-2. These include R-Ras, H-Ras, Raf-1, Apaf, and the phosphatase calcineurin (9, 10, 11, 12, 13, 14).

The Bad protein belongs to the third Bcl-2 family subgroup (15, 16). Bad forms heterodimers with both Bcl-2 and Bcl-x, thereby neutralizing their antiapoptotic effects and thus promoting cell death (17, 18, 19). In the presence of IL-3, TNF, nerve growth factor, and GM-CSF, Bad becomes serine phosphorylated (20, 21, 22, 23, 24), resulting in its association with the 14-3-3 protein, which abolishes its interaction with Bcl-2 or Bcl-x (25, 17). In addition, it has been shown recently that association of the 14-3-3 protein to Bad is dependent on Ser155 phosphorylation of Bad (26, 27, 28).

Serine/threonine phosphatases are usually classified as type 1 (PP1)3 or type 2 (PP2), depending on their substrate specificity and sensitivity to inhibitors. PP1 is a major eukaryotic Ser/Thr phosphatase that regulates diverse cellular processes such as cell cycle progression, proliferation, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, cytokinesis, and neuronal signaling (29, 30, 31, 32, 33, 34, 35). PP1 represents a family of holoenzymes generated by specific interactions between catalytic subunits and a wide variety of regulatory or anchoring proteins involved in targeting as well as in controlling phosphatase activity (36, 37).

The catalytic subunit of PP1 (PP1c) is a 38-kDa protein that is highly conserved throughout evolution. Four isoforms of the enzyme, {alpha}, {beta}, {gamma}1, and {gamma}2, encoded by three genes ({gamma}1 and {gamma}2 result from alternative splicing) are differentially expressed in mammals (38). Protein sequence variations among these isoforms have been observed mainly within the carboxyl termini (39). We have shown recently that PP1{alpha} dephosphorylates the proapoptotic molecule Bad, thereby acting as a regulator of apoptosis induced by IL-2 deprivation (40). In the present study, we report that Bcl-2 is a new targeting subunit of PP1{alpha} that controls its association with Bad.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

TS1{alpha}{beta} is a murine T cell line stably transfected with the {alpha}- and {beta}-chains of the IL-2 receptor (41) and can be maintained in the presence of IL-2, IL-4, or IL-9. Cells were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 5% heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 2 mM glutamine, 10 mM HEPES, 0.55 mM arginine, 0.24 mM asparagine, 50 µM 2-ME, and 5 ng/ml rIL-2.

Lymphokines, Abs, reagents, and plasmids

Human rIL-2 was provided by Roussel Uclaf (Paris, France). The anti-Bad Ab was obtained from Calbiochem (La Jolla, CA), Transduction Laboratories (Lexington, KY), or Santa Cruz Biotechnology (Santa Cruz, CA). The anti-PP1{alpha} Abs and anti-14-3-3 Ab, recognizing all 14-3-3 isoforms, were obtained from Upstate Biotechnology (Lake Placid, NY), Calbiochem, or Transduction Laboratories. The anti-Bcl-2 Ab was obtained from Transduction Laboratories or Calbiochem. The anti-IL-2 Ab was a gift from J. L. Moreau (Institut Pasteur, Paris, France), and anti-histones Ab was obtained from Chemicon International (Temecula, CA). The anti-GST Ab was obtained from Santa Cruz Biotechnology. Peroxidase (PO)-conjugated goat anti-rabbit or -mouse Ab was obtained from Dako (Glostrup, Denmark). Cy3-, Cy2- and Alexa 488-conjugated secondary Abs were obtained from Molecular Probes (Eugene, OR). ECL and ECL-Plus reagents were obtained from Amersham (Little Chalfont, U.K.). Nonidet P-40 was obtained from Boehringer Mannheim (Mannheim, Germany). Glutathione-agarose beads, okadaic acid, and protease inhibitors cocktail were obtained from Sigma (St. Louis, MO). Bcl-2, Bad, and PP1{alpha} cloned into the pLex10 or pGAD vectors were provided by Dr. A. Germani (Hôpital Cochin, Paris, France).

cDNA libraries and the two-hybrid screen

Two cDNA libraries from IL-2-stimulated or -deprived TS1{alpha}{beta} cells from polyadenylated RNA were constructed in fusion with the Gal4 activation domain in pGAD10. Bcl-2 cloned into the pLex10 vector was used as bait to screen the cDNA libraries in the Saccharomyces cerevisiae L40 strain (MATa, trp1, leu2, his3, LYS::lexA-His3, URA::lexA-lacZ) by standard procedures (42).

Sequence analysis

Sequencing of cDNA inserts from positive clones of the two-hybrid screening was performed on both strands with an automatic sequencer (Applied Biosystems, Foster City, CA). Sequences were compared using the FASTA program.

Preparation of GST fusion proteins

PP1{alpha} and Bad were inserted into pGEX-4T1 or pEBG vectors, respectively (Pharmacia Biotech, Uppsala, Sweden and New England Biolabs, Beverly, MA). Fusion proteins were isolated from lysates by affinity chromatography with glutathione-agarose beads. For purity controls, proteins were eluted from agarose beads, separated by SDS-PAGE, and stained with Coomassie blue.

Immunoprecipitation and Western blotting

Cells (1 x 107) were IL-2-stimulated or -deprived, followed by their lysis for 20 min at 4°C in lysis buffer (50 mM Tris-HCl (pH 8), 1% Nonidet P-40, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, and protease inhibitors cocktail). Lysates were immunoprecipitated with the appropriate Ab and protein A-Sepharose was added. After washing, immunoprecipitates were separated by SDS-PAGE. Alternatively, cells were lysed in Laemmli sample buffer, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose, blocked, and incubated with the primary Ab. The membrane was washed and incubated with the PO-conjugated secondary Ab. Proteins detection was performed with the ECL or ECL Plus system.

In vitro phosphatase assay

IL-2-stimulated or -deprived cells (1 x 107) were lysed in lysis buffer as described above. The supernatants were immunoprecipitated with the corresponding Ab followed by incubation with protein A-Sepharose. Immunoprecipitates were washed with phosphatase buffer (50 mM Tris-HCl (pH 7.5), 0.1% 2-ME, 0.1 mM EDTA, and 1 mg/ml BSA) and mixed with [32P]phosphorylase a, diluted in phosphatase buffer supplemented with caffeine. The reaction was incubated (40 min at 30°C), stopped with 200 µl 20% TCA, and centrifuged. A total of 185 µl of the supernatant were used to estimate the generation of free phosphate liberated from [32P]phosphorylase a.

Peptide synthesis

Overlapping dodecapeptides covering the whole Bcl-2 molecule were prepared as described previously (43, 44) by automated spot synthesis (Abimed, Langerfeld, Germany) onto an amino-derivatized cellulose membrane. The membrane was blocked (SuperBlock; Pierce, Rockford, IL), incubated with purified PP1{alpha} and after several washing steps incubated with an anti-PP1{alpha} Ab followed by the PO-conjugated secondary Ab. Protein interactions were visualized with the ECL system.

Bcl-2C or Bcl-2C* peptides comprising amino acid residues 141–151 (wild-type or mutated sequence, respectively) from Bcl-2 were synthesized on an automated multiple peptide synthesizer (AMS 422; Abimed) with the solid-phase procedure and standard F-moc chemistry (45). The purity and composition of the peptides was confirmed by reverse-phase HPLC and by amino acid analysis.

Protein-protein interaction competition

The Bcl-2/PP1{alpha} interaction was competed by using the Bcl-2C peptide, NWGRIVAFFEF (comprising the motif R/KXV/IXF), or Bcl-2C* peptide, NWGRIAAAFEF, in which valine and phenylalanine were substituted for alanine. Lysates from IL-2-stimulated cells were immunoprecipitated with anti-Bcl-2 or anti-Bad Abs, and Protein A-Sepharose was added. The Bcl-2/PP1{alpha} interaction was competed with different concentrations of Bcl-2C or Bcl-2C* peptides (30 min, room temperature). After washing, immunoprecipitates were either assayed for protein phosphatase activity or transferred to nitrocellulose and blotted with the corresponding Ab.

Indirect immunofluorescence

IL-2-stimulated cells were harvested and centrifuged in a cytospin (Heraeus, New York, NY) for 10 min at 600 x g on glass slides. Cells were fixed with methanol for 2 min at -20°C, followed by permeabilization with 50 µg/ml of lysophosphatidylcholine in PBS for 2 min at room temperature. Anti-Bcl-2, anti-Bad, anti-PP1{alpha} or anti-histones, diluted in PBS/1% BSA, were added to the slides under a cover slip and incubated in an humidified chamber (1 h, room temperature). After washing with PBS and PBS/BSA, the secondary Ab was added and incubated for 45 min as above. The slides were washed and mounted with antifading agents and analyzed immediately.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Bad and the serine/threonine phosphatase PP1{alpha} as Bcl-2-interacting proteins in TS1{alpha}{beta} cells

With Bcl-2 as bait in the two-hybrid method to screen a cDNA library from IL-2-stimulated or -deprived cells, we identified two specific clones that showed a 100% match to a partial cDNA encoding the catalytic PP1{alpha} subunit and the proapoptotic molecule Bad. cDNAs encoding Bad and PP1{alpha} full length were cloned in the pGAD and tested for interaction with Bcl-2 by induction of LacZ expression (Fig. 1Go). The Bad and PP1{alpha} interaction also was analyzed in the two-hybrid system. Bad was cloned in pLex and tested for interaction with PP1{alpha} by induction of LacZ expression (Fig. 1Go). Bad or Bcl-2 alone did not restore the enzymatic activity. A Ras/Raf interaction was used as a positive control.



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  		FIGURE 1. Interaction of Bcl-2, Bad, and the catalytic subunit of PP1{alpha} phosphatase in the two-hybrid system. The S. cerevisiae L40 reporter strain was transformed with the indicated plasmids. The interaction between the two hybrid proteins is indicated by the induction of Lac Z expression (blue patches). L40 carrying Ras and Raf were used as the positive control. BD, Fusion with the DNA-binding domain of Lex10. AD, Fusion with the activation domain of Gal4.

 
To validate the results obtained with the yeast two-hybrid system, the interaction of Bcl-2, PP1{alpha}, and Bad was studied in intact cells by coimmunoprecipitation with Bcl-2-, Bad-, and PP1{alpha}-specific Abs. We performed reciprocal coimmunoprecipitation experiments of cytoplasmic proteins under IL-2-stimulation or -deprivation conditions. High PP1{alpha} levels were detected by Western blotting in anti-Bcl-2 immunoprecipitates of IL-2-stimulated cells. The amount decreased throughout the starvation period (Fig. 2GoA). Bad was detected in anti-Bcl-2 immunoprecipitates from IL-2-stimulated cells, decreasing 24 h after IL-2 deprivation (Fig. 2GoA). Reblotting the membrane with an anti-Bcl-2 Ab showed a decrease in Bcl-2 throughout IL-2 deprivation. Immunoprecipitation of cytoplasmic lysates with an irrelevant Ab (anti-IL-2) was not able to detect Bad, PP1{alpha}, or Bcl-2. Bcl-2 was detected in Bad immunoprecipitates from IL-2 stimulated cells, decreasing after lymphokine deprivation (Fig. 2GoB). Reblotting the membrane with anti-Bad Ab showed similar amount of Bad in IL-2-stimulated or -deprived cells. PP1{alpha} was detected in control IL-2-stimulated cells, decreasing after the starvation period, as indicated by reblotting the membrane with anti-PP1{alpha} Ab. Bcl-2 was detected in anti-PP1{alpha} immunoprecipitates of control IL-2-stimulated cells, decreasing after IL-2 deprivation (Fig. 2GoB). Bad levels diminished after deprivation, as shown by reblotting the membrane with an anti-Bad Ab. The membrane also was probed with an anti-PP1{alpha} Ab, which showed a similar amount of PP1{alpha} in IL-2-stimulated or -deprived cells (Fig. 2GoB). These results show that Bcl-2/PP1{alpha}/Bad complex is recovered in TS1{alpha}{beta} cells. Association of Bcl-2 and PP2A in myeloid cells on IL-3 or bryostatin 1 stimulation has been observed (46). However, we did not detect coimmunoprecipitation of PP2A with Bcl-2 (data not shown).



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  		FIGURE 2. Association of Bcl-2, Bad, and PP1{alpha} in vitro and in vivo. A, Cytoplasmic lysates from IL-2-stimulated (5 ng/ml) or -deprived cells were immunoprecipitated with an anti-Bcl-2 Ab, transferred to nitrocellulose, and immunoblotted with an anti-PP1{alpha}, an anti-Bad, and an anti-Bcl-2, the latter as an internal control. Proteins were detected by the ECL system. The molecular weights of the proteins are shown. Similar results were obtained in three independent experiments. I, immunoprecipitation with an irrelevant Ab (anti-IL-2). B, Cytoplasmic lysates from IL-2-stimulated or -deprived cells were immunoprecipitated with an anti-PP1{alpha} Ab, transferred to nitrocellulose, and immunoblotted with an anti-Bad, an anti-Bcl-2, and an anti-PP1{alpha} Ab. Similarly, Bad was immunoprecipitated from cytoplasmic lysates of IL-2-stimulated or -deprived cells and blotted with an anti-Bcl-2, an anti-PP1{alpha}, and an anti-Bad Ab. Proteins were detected as above. Similar results were obtained in three independent experiments. C, GST-PP1{alpha} or GST-Bad fusion proteins were isolated by affinity chromatography with glutathione-agarose beads and incubated with cytoplasmic lysates from IL-2-stimulated or -deprived cells. Eluted proteins were transferred to nitrocellulose and blotted with an anti-Bcl-2, an anti-Bad, an anti-PP1{alpha}, and an anti-GST Ab. Proteins were detected with the ECL system. The molecular weights of the corresponding proteins are indicated.

 
The Bcl-2/Bad/PP1{alpha} interaction was studied in in vitro binding experiments. Bad or PP1{alpha} was produced as GST fusion proteins and purified on glutathione-agarose beads. Cytoplasmic lysates from IL-2-stimulated or -deprived cells were incubated with GST-Bad and GST-PP1{alpha}. Thereafter, proteins were resolved by SDS-PAGE and blots developed with anti-Bcl-2, anti-PP1{alpha}, anti-Bad, and anti-GST Abs. Bcl-2 and PP1{alpha} from IL-2-stimulated cells interact with GST-Bad (Fig. 2GoC). The level of interaction decreases progressively throughout the starvation period. Bcl-2 and Bad from IL-2-stimulated cells interacted with the GST-PP1{alpha} fusion protein. A weak interaction was observed with cytoplasmic extracts from 24 h IL-2-deprived cells (Fig. 2GoC). For internal controls, blots were reprobed with an anti-GST Ab. We did not detect interaction with GST alone (data not shown). These results confirm that we recovered Bcl-2/PP1{alpha}/Bad interaction in vitro as well as in vivo. Finally, colocalization of Bcl-2, PP1{alpha} and Bad was studied in intact cells by confocal microscopy. IL-2-stimulated cells were attached to cover slips, fixed, permeabilized, and, after blocking of nonspecific binding, incubated with anti-Bcl-2, anti-Bad, anti-PP1{alpha}, and anti-histones Abs, followed by Cy2-, Alexa 488- or Cy3-conjugated secondary Abs. Double labeling showed areas of overlapping fluorescence, in addition to areas of green and red fluorescence (Fig. 3Go). As a control, the distinct localization in the cell of Bcl-2 and histones is shown.



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  		FIGURE 3. Colocalization of Bcl-2/PP1{alpha}, Bcl-2/Bad and Bad/PP1{alpha} in IL-2-stimulated cells. Cells were attached to a glass slide, fixed, permeabilized with lysophosphatidylcholine and stained with anti-Bcl-2 and anti-PP1{alpha}, anti-Bcl-2 and anti-Bad, anti-Bad and anti-PP1{alpha} and anti-Bcl-2 and anti-histones. Cells were washed and stained with a Cy2-, Alexa 488-, or Cy3-conjugated secondary Ab, and after several washing steps, samples were mounted with antifading agents and analyzed. Similar results were obtained in two independent experiments.

 
In the presence of IL-3, Bad becomes serine phosphorylated, which results in its association with the 14-3-3 protein, thereby abolishing its interaction with Bcl-x (23). We have shown that IL-2 also induces serine phosphorylation of Bad (40). Fig. 4Go shows that serine phosphorylation of Bad in response to IL-2 does not result in its binding to the 14-3-3 protein. This protein was detected in total extracts from control IL-2-stimulated cells (Fig. 4Go, lane T); however, it was not detected in either PP1{alpha} or in Bcl-2 or Bad immunoprecipitates of IL-2-stimulated or -deprived cells. As internal control, the interaction of Raf and the 14-3-3 protein in Raf immunoprecipitates from control IL-2-stimulated cells is shown (Fig. 4Go, lane C).



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  		FIGURE 4. Effect of IL-2 on the association of the 14-3-3 protein. Total extracts (lane T) or cytoplasmic lysates from IL-2-stimulated or -deprived cells were immunoprecipitated with anti-Bcl-2, anti-Bad, or anti-PP1{alpha} Abs, transferred to nitrocellulose, and blotted with an anti 14-3-3 Ab. Cytoplasmic lysates from IL-2-stimulated cells were precipitated with an anti-14-3-3 Ab and (lane C) and used as a positive control of association. The molecular weights of the protein is shown. Similar results were obtained in two independent experiments.

 
Bcl-2 is a new targeting subunit of PP1{alpha}

We have shown previously that Bad is an in vitro and in vivo substrate for PP1{alpha} phosphatase (40). Given that Bcl-2 also is associated to the PP1{alpha}/Bad complex, we hypothesized that Bcl-2 may be a targeting subunit of PP1{alpha}. To examine this, we performed sequential anti-Bcl-2 immunoprecipitations of cytoplasmic extracts from IL-2-stimulated cells and estimated phosphatase activity. Fig. 5GoA shows Bcl-2 depletion on anti-Bcl-2 immunoprecipitations. The level of Bcl-2 detected after the fourth immunoprecipitation was strongly decreased compared with the level observed after the first anti-Bcl-2 immunoprecipitation. Phosphatase activity estimated in supernatants of the above anti-Bcl-2 immunoprecipitates was not modified (data not shown). The supernatant from the fourth anti-Bcl-2 immunoprecipitation was immunoprecipitated with an anti-Bad Ab (5th), and phosphatase activity was estimated (Fig. 5GoB). Traces of phosphatase activity were detected, compared with the high level of enzymatic activity observed in control anti-Bad immunoprecipitates of IL-2-stimulated cells. Given that in the absence of Bcl-2 we did not detect phosphatase activity, we explored the possibility that Bcl-2 may control the targeting of PP1{alpha} phosphatase to Bad. Anti-Bad immunoprecipitations were performed in control cytoplasmic extracts from IL-2-stimulated cells or in cytoplasmic extracts from the fourth anti-Bcl-2 immunoprecipitation (5th). PP1{alpha} was detected in anti-Bad immunoprecipitates of control cells. On the contrary, PP1{alpha} was not observed in anti-Bad immunoprecipitates depleted of Bcl-2 (Fig. 5GoB). Reblotting the membrane with an anti-Bad Ab showed the presence of Bad. In reciprocal experiments, Bad was detected in anti-PP1{alpha} immunoprecipitates of control cells but was not observed in anti-PP1{alpha} immunoprecipitates depleted of Bcl-2 (Fig. 5GoB). Reprobing the membrane with anti-PP1{alpha} Ab evidenced PP1{alpha}. As internal control, the proapoptotic molecule Bak was not observed in the complex and was only detected in total extracts. These results suggest that Bcl-2 controls the targeting of PP1{alpha} to Bad.



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  		FIGURE 5. Estimation of serine/threonine phosphatase activity after Bcl-2 depletion. A, Bcl-2 was depleted from cytoplasmic extracts of IL-2-stimulated cells by sequential anti-Bcl-2 immunoprecipitations. Cytoplasmic lysates were immunoprecipitated with anti-Bcl-2 Ab (1st). The supernatant was immunoprecipitated three more times (2nd to 4th). Anti-Bcl-2 immunoprecipitates were transferred to nitrocellulose and blotted with an anti-Bcl-2 Ab. Proteins were detected by the ECL system. The molecular weights of protein is shown. Similar results were obtained in three independent experiments. B, Phosphatase activity was estimated in Bad immunoprecipitates from control IL-2-stimulated cells or in Bad immunoprecipitates depleted of Bcl-2 (see A). Phosphatase activity is represented as percentage of the maximal activity detected in control anti-Bad immunoprecipitates. SD is shown (n = 3). The effect of Bcl-2 depletion on Bad/PP1{alpha} interaction also was analyzed. Cytoplasmic lysates from control IL-2-stimulated cells or Bcl-2-depleted cytoplasmic lysates were immunoprecipitated with an anti-Bad Ab, transferred to nitrocellulose, and immunoblotted with anti-PP1{alpha}, anti Bak, and anti-Bad Abs. Total extracts (T) were used as a positive control for Bak expression. Similarly, PP1{alpha} was immunoprecipitated from control and cytoplasmic lysates depleted of Bcl-2 and blotted with an anti-Bad, an anti-Bak, or an anti-PP1{alpha} Ab. Total lysates (T) were used as a positive control for Bak expression. Proteins were detected with the ECL system. Molecular weights of the proteins are shown. Similar results were obtained in three independent experiments.

 
Determination of the binding site of Bcl-2 to PP1{alpha}

Sequence comparison reveals similarity between different PP1 targeting subunits described and identifies a conserved motif (R/KXV/IXF) that is important for the interaction with PP1 (47, 48). Given that Bcl-2 interacts with PP1{alpha}, we explored the possibility that Bcl-2 may contain the R/KXV/IXF consensus motif. Interestingly, the Bcl-2 sequence revealed the presence of this motif (Fig. 6GoC). To analyze whether this sequence of Bcl-2 was involved in binding to PP1{alpha}, we generated overlapping dodecapeptides from the Bcl-2 protein, which were immobilized onto a cellulose membrane. The membrane was incubated with purified PP1{alpha} protein, followed by anti-PP1{alpha} and then a PO-conjugated secondary Ab. Fig. 6GoA shows the entire Bcl-2 amino acid sequence as 113 overlapping peptides each of 12 aa, with a 2 aa shift. The sites of interaction of PP1{alpha} with Bcl-2 are boxed. Other potential binding sites of Bcl-2 to PP1{alpha} are underlined. Nonlabeled spots reflect the background because they were detected on membrane incubation with the secondary Ab. Fig. 6GoB shows the sequence of interacting spots containing the described motif. The BH1 domain within the Bcl-2 sequence is boxed (Fig. 6GoC). The RIVAF motif is found within in the BH1 domain of Bcl-2. Interestingly, Fig. 6GoD shows the sequence alignment of prosurvival and proapoptotic molecules in the vicinity of RIVAF motif. It is interesting to notice that antiapoptotic molecules share the consensus motif of PP1 targeting subunits, whereas proapoptotic molecules do not share this motif.



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  		FIGURE 6. PP1{alpha} binding assay on cellulose-bound Bcl-2 peptides. A, The sequence of Bcl-2 was developed as series of overlapping dodecapeptides. Membrane with Bcl-2 peptides was incubated with purified PP1{alpha}, followed by an anti-PP1{alpha} Ab. After washing, membrane was incubated with PO-conjugated secondary Ab. Spots were detected with the ECL system. Bcl-2 peptides that interact with PP1{alpha} are boxed. Other potential binding sites of Bcl-2 to PP1{alpha} are underlined. B, Peptide sequence stretches corresponding to the binding site of Bcl-2 to PP1{alpha}. The RIVAF motif is in bold. C, The Bcl-2 amino acid sequence showing the RIVAF motif within the boxed BH1 domain. D, Sequence alignment of prosurvival and proapoptotic molecules in the vicinity of RIVAF motif. This motif is not observed in Bcl-xS because this sequence is deleted by alternative splicing.

 
We performed protein-protein interaction competition experiments to confirm that Bcl-2 interacts with PP1{alpha} through the RIVAF motif. Lysates from IL-2-stimulated cells were immunoprecipitated with an anti-Bcl-2 Ab. The Bcl-2/PP1{alpha} interaction was competed with different concentrations of the Bcl-2C peptide (NWGRIVAFFEF). PP1{alpha} was detected in anti-Bcl-2 immunoprecipitates from control cells, which gradually decreased after competition with increasing concentrations of Bcl-2C peptide (Fig. 7GoA). Bad also was detected in anti-Bcl-2 immunoprecipitates from control cells and progressively decreased after competition with 0.5 or 1 mM Bcl-2C peptide, respectively (Fig. 7GoA). Reprobing the membrane with anti-Bcl-2 Ab showed similar level of Bcl-2 in control or peptide-treated anti-Bcl-2 immunoprecipitates.



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  		FIGURE 7. Effect of Bcl-2C and Bcl-2C* peptide on the Bcl-2/PP1{alpha}/Bad interaction . A, Cytoplasmic lysates from control IL-2-stimulated cells were immunoprecipitated with anti-Bcl-2 or anti-Bad Abs. The interaction Bcl-2/PP1{alpha} was competed with or without different concentrations of the Bcl-2C (NWGRIVAFFEF) or the Bcl-2C* (NWGRIAAAFEF) peptide for 30 min at room temperature. Immunoprecipitates were blotted with anti-Bad, anti-PP1{alpha}, and anti-Bcl-2 Abs. Proteins were detected by using the ECL system. The molecular weights of the proteins are shown. Similar results were obtained in two independent experiments. B, Cytoplasmic lysates from IL-2-stimulated cells were treated as in A and phosphatase activity was estimated. Phosphatase activity is represented as the percentage of the maximal activity in nontreated immunoprecipitates. SD is shown (n = 3). {square}, Control; , 0.5 mM peptide; {blacksquare}, 1 mM peptide. C, GST-fusion protein was isolated by affinity chromatography with glutathione-agarose beads and incubated with cytoplasmic lysates from control IL-2-stimulated cells or Bcl-2-depeleted extracts. Eluted proteins were transferred to nitrocellulose and blotted with an anti-PP1{alpha} and anti-GST Abs. Proteins were detected with the ECL system. Molecular weights of the proteins are shown.

 
In reciprocal experiments, lysates from IL-2-stimulated cells were immunoprecipitated with an anti-Bad Ab and the interaction Bcl-2/PP1{alpha} was competed with different concentrations of Bcl-2C peptide, showing similar results to those observed in anti-Bcl-2 immunoprecipitations (Fig. 7GoA). The specificity of Bcl-2/PP1{alpha} interaction through the RIVAF motif was analyzed by competition experiments with the mutated Bcl-2C* peptide (NWGRIAAAFEF) in which the critical amino acid valine (V) and phenylalanine (F) were mutated to alanine (A). In contrast to the results obtained with Bcl-2C peptide, the mutated peptide did not interfere with the recruitment of PP1{alpha} and Bad to Bcl-2.

To conclusively confirm that Bcl-2 was a targeting subunit of PP1{alpha}, we performed phosphatase activity assays in anti-Bcl-2 immunoprecipitates of IL-2-stimulated cells in which the interaction Bcl-2/PP1{alpha} was competed by Bcl-2C peptide (Fig. 7GoB). Phosphatase activity was detected in anti-Bcl-2 or anti-Bad immunoprecipitates from control IL-2-stimulated cells. Enzymatic activity moderately decreased in anti-Bcl-2 or anti-Bad immunoprecipitates in which Bcl-2/PP1{alpha} interaction was competed with 0.5 mM Bcl-2C peptide and strongly decreased in anti-Bcl-2 or anti-Bad immunoprecipitates competed with 1 mM of Bcl-2C peptide (Fig. 7B). Phosphatase activity in anti-Bcl-2 immunoprecipitates of IL-2 stimulated cells was not affected by addition of increasing concentrations of Bcl-2C* peptide (Fig. 7GoB). Finally, we performed GST-pull down experiments with GST-Bad fusion protein. Cytoplasmic lysates from control IL-2-stimulated cells or Bcl-2 depleted lysates were incubated with GST-Bad. Proteins were resolved by SDS-PAGE and blots developed with an anti-PP1{alpha} or an anti-GST (Fig. 7GoC). The level of interaction Bad/PP1{alpha} strongly decreased in Bcl-2-depleted extracts compared with the interaction observed in control lysates. For internal control, the blot was reproved with an anti-GST Ab. Taken together, our results strongly suggest that Bcl-2 targets PP1{alpha} to Bad.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the search for Bcl-2-interacting proteins with the yeast two-hybrid approach, we show that Bcl-2 interacts with Bad and the serine/threonine phosphatase PP1{alpha}. The formation of the Bcl-2/PP1{alpha}/Bad trimolecular complex was confirmed by in vitro binding experiments and by coimmunoprecipitation. Increasing evidence suggest that some phosphatases do no find their physiological substrates by simple diffusion and that they are frequently directed to their substrates by interaction with targeting subunits (49). The best example for the targeting subunit concept is the serine/threonine phosphatase PP1, one of the major phosphatases of eukaryotic cells (50). PP1 controls diverse cellular processes that are regulated by the PP1 catalytic subunit complex with different targeting subunits that may confer in vivo substrate specificity (29, 31, 34, 35).

Different mammalian PP1 targeting subunits have been characterized, including GM, which targets PP1 to both glycogen and reticulum, the GL subunit, which targets PP1 to liver glycogen, the M110 subunit, which targets PP1 to the skeletal muscle (51), the p53-binding protein (52), and the retinoblastoma gene product (53). We have described recently that PP1{alpha} interacts with and controls Bad dephosphorylation in vitro and in vivo (40). Our results now show that PP1{alpha} is targeted to Bad by Bcl-2. Depletion of Bcl-2 from cytoplasmic lysates of IL-2-stimulated cells strongly decreases Bad-associated phosphatase activity by avoiding the association of PP1{alpha} and Bad, despite the fact that both molecules are present in Bcl-2-depleted extracts. Similar results were observed by using GST-pull down approach.

The proportion of PP1{alpha} directed to Bad is determined by the amount of targeting subunit Bcl-2 synthesized. In keeping with this, a low level of Bcl-2/Bad/PP1{alpha} association, confirmed by coimmunoprecipitation and GST pull down assays, was observed in IL-2-deprived cells because Bcl-2 was the limiting molecule in the formation of the trimolecular complex. Western blotting of total extracts of IL-2-deprived cells also confirmed the down-regulation of Bcl-2 expression but not of Bad and PP1{alpha}. Interestingly, although the level of Bcl-2/PP1{alpha}/Bad association detected in IL-2-deprived cells was lower than in IL-2-stimulated cells, the phosphatase activity, as well as the level of apoptosis, was higher (data not shown). This result suggests a correlation between apoptosis and PP1{alpha} phosphatase activity (40) and, probably, a direct or indirect role of Bcl-2 in controlling PP1{alpha} phosphatase activity, in addition to its role as a PP1{alpha} targeting subunit.

It has been published that serine phosphorylation of Bad in response to IL-3 results in binding to the 14-3-3 protein, thereby avoiding its heterodimerization with Bcl-x (17). We have shown that Ser112 and Ser136 phosphorylation of Bad also is induced by IL-2, and its dephosphorylation correlates with the appearance of apoptosis (40). Our results indicate that neither phosphorylated nor dephosphorylated Bad is associated to 14-3-3 protein, showing that Bad can interacts with Bcl-2 in IL-2-stimulated or -deprived cells because it is not sequestered by the 14-3-3 protein. Our findings are in agreement with recent publications showing that association of Bad to 14-3-3 is dependent on Ser155 phosphorylation of Bad (26, 27, 28). The lack of 14-3-3 association to Bad in IL-2-stimulated cells is not surprising because this lymphokine does not induce Ser155 phosphorylation of Bad (data not shown).

The finding that Bcl-2/PP1{alpha}/Bad form a trimolecular complex implies that recognition sites for binding are different. This result also suggests that the Bcl-2 binding site to PP1{alpha} is distinct from the catalytic site of PP1{alpha}. Our data are consistent with the observation that PP1 attached to microcystin-Sepharose affinity columns maintains an intact regulatory subunit-binding site (54). Because Bcl-2 targets PP1{alpha} to Bad, this suggests that the function of the two proteins may be coordinated. The targeting subunit Bcl-2 provides proximity of enzyme (PP1{alpha}) and substrate (Bad). We do not exclude the possibility that, in addition to Ras, Bcl-2 may have a direct or indirect role in the control of PP1{alpha} phosphatase activity. In addition, we cannot rule out the possibility that the Bcl-2/Bad interaction is stabilized by PP1{alpha}.

It has been shown that most, if not all, targeting subunits have a PP1-binding motif. Bcl-2 contains this conserved PP1-binding motif as described in other PP1 targeting subunits (47, 48). Of interest, Bcl-xL and Bcl-w, other antiapoptotic molecules included in the Bcl-2-like family, also contain the PP1-binding motif, suggesting that they also may function as targeting subunits of PP1{alpha}. On the contrary, the proapoptotic molecules Bak and Bax do not share this motif. In agreement, it has been shown that mutation of phenylalanine amino acid on the RIVAF motif abolishes binding of targeting subunits to PP1{alpha} (48). Interestingly, the proapoptotic molecule Bcl-xS, generated by alternative splicing of Bcl-xL retains only a short fragment of the BH1 domain and lacks the RIVAF motif. IL-4-stimulated TS1{alpha}{beta} cells do not express Bcl-2, but instead express Bcl-x (8), leading to the speculation that Bcl-x may replace Bcl-2 as targeting subunit of PP1{alpha} in IL-4-stimulated cells. The finding that Bcl-2C peptide disrupted the interaction between Bcl-2 and PP1{alpha} implies that at least this motif is critical for the interaction with PP1{alpha}, although we do not exclude that Bcl-2 may have other sites of interaction with PP1{alpha}. This is in agreement with recent results showing that some regulatory proteins such as GM and M110 have multiple sites of interaction with PP1 (55). This result also suggests the specificity of the Bcl-2/PP1{alpha} interaction, which also was confirmed by colocalization and coimmunoprecipitation. It also has been speculated that hormonal or growth factor regulation of PP1 may involve a control on the number and identity of interaction sites between PP1 and regulatory subunits (47, 48). Our results and previous studies from other groups have revealed that mutation of valine and phenylalanine to alanine within the recognition motif affects the ability of PP1{alpha} to bind Bcl-2, as well as the interaction of PP1{alpha} with Bad.

Our studies demonstrate that Bcl-2/PP1{alpha} interaction requires the RIVAF motif. This motif respects the consensus motif previously shown to mediate interaction of most of targeting subunits with the PP1 catalytic subunit. The mechanism by which regulatory subunits modulate the substrate specificity of PP1 is unknown, although we can speculate that regulatory subunits either alter the conformation of the PP1 catalytic subunit or simply target PP1 to its substrate. Both mechanisms could operate in vivo, depending on the targeting subunit. Our findings also show the importance of the short peptide RIVAF in mediating specific protein-protein interactions. In addition, the mutation of this motif could disrupt the interaction of many targeting subunits with PP1. In summary, our results show for the first time that PP1{alpha} is targeted to Bad by Bcl-2.


   Acknowledgments
 
We thank Dr. F. Romero (Seville, Spain), Dr. J. L. Moreau (Paris, France), and Dr. A. Germani (Paris, France) for gifts of reagents used in this study and Dr. D. R. Jones for critical reading of the manuscript.


   Footnotes
 
1 This work was partially supported by Association pour la Recherche sur le Cancer Grants 9294 and 9449 and grants from the Dirección General de Investigación y Ciencia. The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council and Pharmacia (Piscataway, NJ) and Upjohn. Back

2 Address correspondence and reprint requests to Dr. Angelita Rebollo, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Universidad Autonoma de Madrid, 28049 Madrid, Spain. E-mail address: arebollo{at}cnb.uam.es Back

3 Abbreviations used in this paper: PP, protein phosphatase; PO, peroxidase. Back

Received for publication December 15, 2000. Accepted for publication April 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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