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The Journal of Immunology, 2006, 177: 2441-2451.
Copyright © 2006 by The American Association of Immunologists

Identification of PP1{alpha} as a Caspase-9 Regulator in IL-2 Deprivation-Induced Apoptosis

Frédéric Dessauge*, Xavier Cayla{dagger}, Juan Pablo Albar{ddagger}, Aarne Fleischer*, Ata Ghadiri*, Marianne Duhamel* and Angelita Rebollo1,*

* Laboratoire d’Immunologie Cellulaire et Tissulaire, Hôpital Pitié-Salpêtrière, Unité Institut National de la Santé, et de la Recherche Médicale, Paris, France; {dagger} Equipe Hypophyse, Unité Mixte de Recherche 6175, Institut National de la Recherche Agronomique-Centre National de la Recherche, Université de Tours, Haras-Nationaux, Physiologie de la Reproduction et des Comportements, Nouzilly, France; and {ddagger} Centro Nacional de Biotecnologia, Campus de Cantoblanco, Universidad Autonoma de Madrid, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylates caspase-9 at Thr125, and this phosphorylation is crucial for caspase-9 inhibition. Until now, the phosphatase responsible for Thr125 dephosphorylation has not been described. Here, we demonstrate that in IL-2-proliferating cells, phosphorylated serine/threonine phosphatase type 1{alpha} (PP1{alpha}) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1{alpha} dephosphorylation, which leads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of both molecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we show that in this process PP1{alpha} is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1{alpha} associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylation and activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1{alpha} consensus motifs, and we demonstrate that these novel sites mediate the interaction of PP1{alpha} with caspase-9.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Apoptosis is a regulated process important for differentiation, control of cell number, and removal of damaged or autoreactive cells (1, 2). Extracellular as well as intracellular signals, including growth factor deprivation, radiation, chemotherapeutic drugs, or cross-linking of receptors, can trigger an apoptotic response, leading to the controlled activation of cysteine proteases known as caspases (3). Two major pathways of caspase activation have been described, one involving the release of cytochrome c, among other molecules from mitochondria, and the other involving cell surface death receptor activation (4).

The mitochondrial apoptotic pathway can be regulated at multiple stages to promote or suppress cell death, the release of cytochrome c being a key step (5). Once cytochrome c is released, it associates with Apaf-1 and procaspase-9 to form the apoptosome complex, which facilitates the autoprocessing of initiator caspase-9 (6). Subsequently, activated caspase-9 cleaves downstream caspases such as caspase-3, -6, and -7. In turn, caspase-3 fully processes caspase-9 via a feedback loop leading to amplification of the apoptotic signal and cell degradation (7).

Two recent studies have indicated that caspase-9 can also be activated by mechanisms involving neither cytochrome c release nor Apaf-1 activation (8, 9). The first study showed that in response to endoplasmic reticular stress caspase-12 was able to process and activate caspase-9, whereas the other study proposed a novel pathway of caspase-9 cleavage triggered by viral infection. Phosphorylation and dephosphorylation are crucial steps in caspase-mediated apoptosis. First, the phosphorylation status of a caspase substrate may decide whether the protein is cleaved or not. Second, caspases directly affect protein phosphatases (10). Third, caspases may be, directly or indirectly, controlled by protein phosphatases (11, 12, 13, 14). Finally, some caspases are themselves subjected to reversible phosphorylation. It has been demonstrated that the ERK/MAPK pathway inhibits caspase-9 activation by direct phosphorylation of a threonine at position 125 (Thr125) (15). This phosphorylation is sufficient to block caspase-9 processing and subsequent caspase-3 activation, suggesting that caspase-9 inhibition by phosphorylation promotes cell survival (15). Caspase-9 can also be phosphorylated at serine residues 99, 183, and 195 by protein kinase A (PKA),2 inhibiting its activation. However, inhibition of caspase-9 activation by PKA does not necessarily require direct phosphorylation of caspase-9 (16).

Serine/threonine phosphatases are usually classified as type 1 (PP1) or type 2 (PP2), depending on their substrate specificity and sensitivity to inhibitors. PP1 represents a family of holoenzymes generated by specific interactions between catalytic subunits and a wide variety of regulatory or anchoring proteins (17, 18). The catalytic subunit of serine/threonine phosphatase type 1 (PP1c) is a 38-kDa protein highly conserved throughout evolution. Four isoforms of the enzyme, a, b, g1, and g2, encoded by three genes (g1 and g2 result from alternative splicing) are differentially expressed in mammals (19). Protein variations among these isoforms are observed mainly at the C terminus (20), which plays a regulatory role in the catalytic activity, as demonstrated by proteolysis and phosphorylation studies (21). PP1 is a major eukaryotic serine/threonine phosphatase that regulates diverse cellular processes such as cell cycle progression, proliferation, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, cytokinesis, and neuronal signaling (22, 23, 24, 25). During the cell cycle, PP1 activity is regulated by phosphorylation (26, 27, 28). PP1 play a key role in the mitotic transition by dephosphorylating proteins that are essential in these cellular functions (29, 30, 31, 32, 33, 34). It has been shown that phosphorylation of PP1{alpha} at threonine residue 320 by cyclin-dependent kinases blocks the enzymatic activity of PP1{alpha} (35). In agreement, a constitutive active mutant of PP1{alpha} that is resistant to Cdk phosphorylation at Thr320 prevents cells from entering the S phase of the cell cycle (27).

We have previously shown that the mitochondrial apoptotic pathway is implicated in IL-2 deprivation-induced apoptosis (36). We have also shown that IL-2 deprivation-induced apoptosis operates by regulating Bad dephosphorylation through the PP1{alpha} phosphatase (37). In this article, we demonstrate that PP1{alpha} is associates with caspase-9 to induce its dephosphorylation and, as a consequence, its protease activity. In addition, we have mapped the novel binding sites of caspase-9 to PP1{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and cultures

TS1{alpha}beta is a murine T cell line stably transfected with the {alpha}- and beta-chains of the human IL-2R (38); it can be independently propagated in the presence of IL-2, IL-4, or IL-9; when they are deprived of growth factor, they die by apoptosis. Cells were cultured in RPMI 1640 (BioWhittaker) supplemented with 5% heat-inactivated FCS (Invitrogen Life Technologies), 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 Chiron. The following Abs were used for Western blotting according to standard protocols: caspase-3; phospho-Thr320 PP1{alpha}; phospho-Thr125 caspase-9 (Cell Signaling); caspase-9 (Cell Signaling or Santa Cruz Biotechnology); and PP1 (BD Transduction Laboratories). Glutathione agarose was from Santa Cruz Biotechnology. Kits for estimation of caspase-9 and caspase-3 activity were purchased from R&D Systems. Okadaic acid (OA), tautomycin, and cytochrome c were from Calbiochem or Sigma-Aldrich. Recombinant caspase-9 and PP1 were from Calbiochem. GST-Casp-9 plasmid was a gift from Prof. P. Clarke (University of Dundee, Dundee, U.K.). Peroxidase (PO)-conjugated secondary Abs were from DAKO. Protein A-Sepharose, ECL, and ECL Plus were purchased from GE Healthcare. Nonidet P-40 was from Roche Diagnostics. Annexin was from Immunotech.

Isolation of S100 fraction

A total of 20 x 106 cells was IL-2 stimulated or deprived, harvested, and washed with chilled PBS. Cell pellet was resuspended in 5 volumes of ice-cold buffer A (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 250 mM sucrose) supplemented with protease inhibitors. Cells were disrupted in a Dounce homogenizer, the nuclei were centrifuged (1000 x g, 10 min, 4°C), and the supernatant further centrifuged (10,000 x g, 15 min, 4°C). The resulting mitochondrial pellet was resuspended in buffer A and stored at –80°C. The supernatant was further centrifuged (100,000 x g, 1 h, 4°C),and the resulting cytosolic fraction was washed at –80°C.

Immunoprecipitation and Western blotting

Cells (1 x 107) were IL-2 stimulated or deprived, followed by lysis for 20 min at 4°C in lysis buffer (50 mM Tris (pH 8), 1% Nonidet P-40, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, and protease inhibitor mixture). Lysates (800 µg of protein) were immunoprecipitated with the appropriate Ab for 2 h at 4°C, and protein A-Sepharose was added for 1 h at 4°C. After washing with 1x TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20), immunoprecipitates were separated by SDS-PAGE. Alternatively, cells were lysed in Laemmli sample buffer, and protein extracts were separated by SDS-PAGE, transferred to polyvinylidine difluoride (PVDF), blocked (5% nonfat dry milk in TBST), and incubated with the primary Ab in TBS-0.5% nonfat dry milk. The membrane was washed and incubated with PO-conjugated secondary Ab. Protein detection was performed using the ECL or ECL Plus system.

Immunodepletion of PP1

Washed protein A-Sepharose (40 µl) was incubated with anti-PP1 Ab in 200 µl of incubation buffer (200 mM HEPES-KOH (pH 7.5), 10 mM KCl, 2 mM MgCl2) at 4°C overnight. Unbound Ab was removed by washing with incubation buffer. A total of 500 µg of TS1{alpha}beta S100 lysate was added to the protein A-Sepharose and incubated at 4°C for 3 h. Protein A-Sepharose was recovered by centrifugation, and the supernatant was incubated at 4°C for 3 h with a second aliquot of anti-PP1{alpha} Ab bound to protein A-Sepharose. The protocol was repeated a total of four times for complete PP1{alpha} depletion. After centrifugation, the supernatant was recovered and used as PP1-depleted extract.

Caspase assays

Incubations were performed at 30°C in a total volume of 50 µl of reaction buffer containing 400 µg of S100 TS{alpha}beta extract, 1 mM ATP, 10 mg/ml creatine kinase, and 5 mM creatine phosphate. Where required, cytochrome c was added to a final concentration of 5 µM. At specific time points, the reaction was mixed with 60 µl of reaction buffer containing caspase-9 or caspase-3 colorimetric substrate (LEDH or DEVD) coupled to p-nitroalanine (pNA). After incubation at 37°C for 60 min, release of pNA was measured at 405 nm using a spectrophotometer microtiter plate reader.

In vitro phosphatase assay

Cells (1 x 107) were lysed in lysis buffer (50 mM Tris-HCl (pH 8), 1% Nonidet P-40, 137 mM NaCl, 1 mM CaCl2, 10% glycerol, 1 mM orthovanadate, and protease inhibitor mixture), and supernatants were immunoprecipitated with anti-caspase-9 Ab overnight and then incubated with protein A-Sepharose (1 h, 4°C). Immunoprecipitates were washed with phosphatase buffer (50 mM Tris-HCl (pH 7.5), 0.1% 2-ME, 0.1 mM EDTA, 1 mg/ml BSA) and mixed with phosphatase buffer containing phosphorylase a as substrate and supplemented with 4 mM caffeine (final concentration). The reaction was incubated at 30°C for 40 min and stopped by adding 100 µl of 20% trichloroacetic acid and 100 µl of 1 mg/ml BSA and then centrifuged. A total of 185 µl were used to estimate the generation of free phosphate liberated from [32P]phosphorylase a. For phosphorylase phosphatase activity inhibition, OA or tautomycin was added to the reaction mixture.

Peptide synthesis

Overlapping peptides covering the whole caspase-9 or PP1{alpha} molecule were prepared by automated spot synthesis into an amino-derivatized cellulose membrane as previously described (39, 40). The membrane was blocked, incubated with purified PP1{alpha} or caspase-9 protein and, after several washing steps, incubated with anti-PP1{alpha} or anti-caspase-9 Ab followed by the PO-conjugated secondary Ab. Protein interactions were visualized using the ECL system. Similarly, caspase-9 site 1 or site 2 peptides comprising amino acids MDEADRQLLRRCRVRLVS (site 1) or LDRDKLEHRFRWLRFM (site 2), as well as mutated peptides, were synthesized in an automated multiple peptide synthesizer with solid phase procedure and standard Fmoc chemistry. The purity and composition of the peptides were confirmed by reverse phase HPLC and by amino acid analysis. These peptides were used for protein-protein interaction competition studies.

Protein-protein interaction competition

The caspase-9/PP1{alpha} interaction was competed using peptides corresponding to site 1 (MDEADRQLLRRCRVRLVS) and/or site 2 (LDRDKLEHRFRWLRFM). Lysates from IL-2-stimulated cells were immunoprecipitated with anti-caspase-9 or anti-PP1{alpha} Ab, and protein A-Sepharose was added. The caspase-9/PP1{alpha} interaction was competed with 1 mM site 1 and/or site 2 peptides (30 min, room temperature). After a washing, immunoprecipitates were transferred to nitrocellulose and blotted with the corresponding Ab.

Preparation of GST fusion proteins

PP1 was inserted into pGEX-4T1 vector. Expression of recombinant protein was induced in Escherichia coli BLR (DE3) at 30°C for 4 h by addition of 0.2 mM isopropyl beta-D-thiogalactoside. GST-tagged proteins were affinity purified with glutathione-Sepharose 4B before elution in buffer (10 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.1 mM PMSF, and 1 µg/ml protease inhibitor mixture containing 15 mM glutathione). For purity control, proteins were eluted from agarose beads in SDS sample buffer, separated in 10% polyacrylamide gels, and stained with Coomassie blue.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-2 deprivation induces caspase-9 and PP1{alpha} dephosphorylation

We have previously shown that the protein phosphatase PP1{alpha} is activated upon IL-2 deprivation and that this correlates with its dephosphorylation and apoptosis induction. In addition, we have shown that IL-2 deprivation-induced apoptosis operates by regulating Bad phosphorylation through the PP1{alpha} phosphatase (37). We have also described that apoptosis triggered by IL-2 deprivation leads to cytochrome c release, as well as caspase-9 and caspase-3 activation (36). According to our results and given that caspase-9 activation is inhibited by phosphorylation (15), we were interested therefore in analyzing whether PP1{alpha} could be involved in caspase-9 activation. First, we analyzed the level of phosphorylation of caspase-9 and PP1{alpha} in proliferating and apoptotic cells. Phosphorylated PP1{alpha} was detected in control IL-2-stimulated cells, decreasing throughout the starvation period analyzed (Fig. 1A). Reprobing the membrane with an anti-PP1{alpha} Ab showed that there were similar amounts of PP1{alpha} in IL-2-stimulated and -deprived cells (Fig. 1A). Similarly, phosphorylated caspase-9 was detected in control IL-2-proliferating cells, decreasing in deprived cells and being almost undetectable after 12 h of IL-2 deprivation. In parallel to caspase-9 dephosphorylation, IL-2 deprivation induces caspase-9 cleavage (Fig. 1A). Treatment of IL-2-stimulated cells for 6 h with the PP1{alpha} inhibitor OA blocks caspase-9 dephosphorylation. Inhibition of caspase-9 dephosphorylation progressively increased with the OA concentration used (Fig. 1B). Finally, when IL-2-maintained cells are deprived of the lymphokine, they undergo apoptosis (Fig. 1C), given that TS1{alpha}beta is a growth factor-dependent cell line.


Figure 1
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  		FIGURE 1. Effect of IL-2 deprivation on PP1{alpha} and caspase-9 phosphorylation. A, TS1{alpha}beta cells were IL-2 stimulated or deprived for the indicated times and then lysed. Protein extracts were separated by SDS-PAGE, transferred to PVDF, and hybridized with anti-phospho-PP1{alpha}, anti-phopho-caspase-9, anti-caspase-9, and anti-PP1{alpha} Ab, the two latter as internal control of protein loading. Protein bands were detected using the ECL system. Molecular masses (Mw) of the corresponding proteins are shown. Similar results were obtained in three independent experiments. B, IL-2-stimulated cells were treated with different OA concentrations for 6 h and then lysed and processed as above. The blot was proved with anti-phospho-caspase-9 and anti-caspase-9, the latter as internal control. Molecular masses of the proteins is shown. C, Cells were cultured in the presence or absence of IL-2, harvested at different times, and stained with annexin and propidium iodide. Cells were analyzed using fluorescence flow cytometry. SD is shown for n = 3.

 
Identification of caspase-9 as a PP1{alpha}-interacting protein

To address the hypothesis of whether PP1{alpha} associates with caspase-9 and whether PP1{alpha} is directly responsible of caspase-9 dephosphorylation, we analyzed the interaction of PP1{alpha} and caspase-9 in intact cells by immunoprecipitation. Reciprocal coimmunoprecipitations of cytoplasmic proteins was performed under IL-2 stimulation or deprivation conditions (Fig. 2A). High caspase-9 levels were observed in anti-PP1{alpha} immunoprecipitates of IL-2-stimulated cells. The amount of caspase-9 associated with PP1{alpha} decreased upon IL-2 deprivation, with low levels detected after 24 h, in contrast to PP1{alpha} levels that did not diminish (Fig. 2A). In reciprocal experiments, high PP1{alpha} levels were detected in anti-caspase-9 immunoprecipitates from IL-2-stimulated cells, slightly decreasing upon 12 h of IL-2 deprivation and being almost undetectable after 24 h of deprivation (Fig. 2B). Reprobing the membrane with an anti-caspase-9 Ab showed that there were similar levels of caspase-9 in IL-2-stimulated cells but that it decreased upon IL-2 deprivation (Fig. 2B). Similarly, the interaction between PP1{alpha} and caspase-9 was also observed in freshly isolated thymocytes (Fig. 2C). No interaction was observed between p53 and caspase-9. The anti-PP1{alpha} and anti-caspase-9 Abs recognized both phosphorylated and nonphosphorylated PP1{alpha} and caspase-9. This result strongly suggests that PP1{alpha} interacts with caspase-9 in TS1{alpha}beta cells in vivo.


Figure 2
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  		FIGURE 2. Interaction of PP1{alpha} and caspase-9 (Casp-9). A, Cytoplasmic lysates from IL-2-stimulated or -deprived cells were immunoprecipitated (IP) with anti-PP1{alpha} Ab, transferred to PVDF, and immunoblotted with anti-caspase-9 and anti-PP1{alpha}, the latter as internal control. B, Similarly, caspase-9 was immunoprecipitated from cytoplasmic lysates of IL-2-stimulated or -deprived cells and immunoblotted with anti-PP1{alpha} or anti-caspase-9, the latter as internal control of protein loading. Molecular masses (Mw) of the proteins are shown. Similar results were obtained in three independent experiments. C, Caspase-9 was immunoprecipitated from cytoplasmic lysates of freshly isolated thymocytes and immunoblotted with anti-PP1{alpha}, anti-caspase-9, and anti-p53, the two latter as internal control. Similar results were obtained in two independent experiments. WB, Western blot.

 
The PP1{alpha}/caspase-9 interaction was also observed in vitro by GST pull down experiments. PP1{alpha} was produced as a GST fusion protein and purified on glutathione-agarose beads. Buffer or cytoplasmic lysates from IL-2-stimulated or -deprived cells were incubated with GST-PP1{alpha}; and after several washing steps, proteins were separated by SDS-PAGE, and the blot was developed with an anti-caspase-9 Ab. Caspase-9 from IL-2-stimulated or -deprived cells interacts with the GST-PP1{alpha} fusion protein (Fig. 3), whereas no interaction was observed in the absence of TS1{alpha}beta extracts (Fig. 3, lane E). In reciprocal experiments, GST-caspase-9 was purified on glutathione-agarose beads; after a washing, proteins were resolved by SDS-PAGE, and the blot was developed as previously described here (Fig. 3). PP1{alpha} from IL-2-stimulated or -deprived cells interacts with GST-caspase-9 fusion protein. No interaction was observed in the absence of TS1{alpha}beta extracts (Fig. 3, lane E). These results confirm the interaction previously demonstrated by coimmunoprecipitation experiments in T cells and in thymocytes.


Figure 3
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  		FIGURE 3. In vitro interaction of PP1{alpha} and caspase-9. GST-PP1 (A) or GST-caspase-9 (B) fusion proteins were induced with isopropyl beta-D-thiogalactoside for 4 h, and proteins were isolated by affinity chromatography with glutathione-agarose beads and incubated with or without (lane E) cytoplasmic lysates from IL-2-stimulated or -deprived cells. After a washing, eluted proteins were separated by SDS-PAGE, transferred to PVDF, and blotted with anti-PP1{alpha} or anti-caspase-9 Ab. Molecular masses (Mw) of the corresponding proteins are indicated. Similar results were obtained in three independent experiments.

 
PP1{alpha} is required for caspase-9 activity

Cell-free systems that reproduce in vitro the regulation of apoptosis have been proved to be useful for dissecting the biochemical mechanisms controlling caspase activation. To investigate the signaling pathways that control caspase-9 activation, we used a cell-free system derived from TS1{alpha}beta cells supplemented with an ATP-regenerating system in which caspase activation is induced by the addition of cytochrome c. Caspase-9 and caspase-3 activations were assayed by cleavage of the colorimetric tetrapeptide substrate LEHD-pNA and DEVD-pNA, respectively. In cytosolic extracts of IL-2-stimulated TS1{alpha}beta, caspase-9 activation was detected after 5 min of incubation with cytochrome c, progressively increasing during the incubation period analyzed (Fig. 4A). Caspase-9 was not activated upon PP1{alpha} depletion from the cytosol (Fig. 4A) compared with control lysates without cytochrome c addition. As internal control, we also used lamin-depleted extracts, showing that in the absence of lamin, caspase-9 is activated by the presence of cytochrome c and PP1{alpha}. Similarly, caspase-3 activation was also detected upon 5 min of incubation of cytosolic TS1{alpha}beta extracts with cytochrome c, increasing during the incubation period analyzed (Fig. 4B). Depletion of PP1{alpha} from the cytosol inhibits around 50% the caspase-3 activity (Fig. 4B). No significant caspase-3 activity was detected in control cytosolic extracts without cytochrome c addition. As internal control, the experiment was also done in lamin-depleted extracts. Fig. 4C shows the internal control of PP1{alpha} depletion upon four sequential immunoprecipitations with the specific anti-PP1{alpha} Ab. After PP1{alpha} depletion, caspase-9 is still present in the supernatant.


Figure 4
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  		FIGURE 4. PP1{alpha} depletion blocks caspase activation. Caspase-9 (A) and caspase-3 (B) activation in TS1{alpha}beta cytosolic extracts was assayed by measuring cleavage of LEHD-pNA or DEVD-pNA, respectively. Extracts incubated with or without cytochrome c (Cyt c) were used as a control. PP1{alpha} or lamin depletion was achieved by four sequential anti-PP1{alpha} immunoprecipitations from cytosolic extracts. SD is shown for n = 3. C, PP1{alpha} depletion was confirmed by Western blot (WB) with specific anti-PP1{alpha} Ab. Western blot was probed with anti-PP1{alpha} or anti-caspase-9. D, Caspase-9 cleavage in cytosolic extracts was induced by incubation with cytochrome c in the presence of PP1{alpha}. Samples were immunoblotted with anti-caspase-9 Ab. Caspase-9 cleavage was inhibited by PP1{alpha} depletion from the cytosol. Extracts incubated without cytochrome c were used as a control. Molecular masses (Mw) of the proteins are shown. E, Caspase-3 cleavage in TS1{alpha}beta cytosolic extracts was induced by incubation with cytochrome c in the presence of PP1{alpha}. Samples were immunoblotted with anti-caspase-3 Ab. Caspase-3 cleavage was partially blocked by PP1{alpha} depletion from cytosolic extracts. Extracts incubated without cytochrome c were used as a control. Molecular masses of the proteins are shown.

 
The effect of PP1{alpha} depletion on caspase-9 processing was analyzed in cytosolic extracts. Depletion of PP1{alpha} from cytosol inhibits procaspase-9 processing (Fig. 4D), whereas cleavage was observed in cytosolic extracts incubated for 15 min with cytochrome c. No caspase-9 processing was observed in the absence of cytochrome c (Fig. 4D). Similarly, the effect of PP1{alpha} depletion on caspase-3 processing was also analyzed. Depletion of PP1{alpha} from cytosol partially inhibits procaspase-3 processing (Fig. 4E). Caspase-3 processing was observed in cytosolic extracts incubated with cytochrome c for 15 min. The processing was measured by the appearance of p19 and p17 fragments. No caspase-3 cleavage was found in the absence of cytochrome c (Fig. 4E). These results suggest a requirement of PP1{alpha} activity for cytochrome c-induced activation of caspase-9 and subsequent caspase-3 activation.

Because PP1{alpha} plays a critical role in caspase-9 activation, we determined whether PP1{alpha} is the phosphatase specifically involved in caspase-9 activation. An in vitro cell-free system reaction was made using cytosolic extracts from IL-2-stimulated cells, supplemented with cytochrome c and ATP. The reaction was incubated with different OA concentrations for 30 min, and caspase-9 activation was measured. Treatment with 1 mM completely prevents caspase-9 activation (Fig. 5), as detected by cleavage of the colorimetric substrate LEDH-pNA.


Figure 5
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  		FIGURE 5. Effect of OA on caspase-9 activity. TS1{alpha}beta extracts were incubated with cytochrome c, ATP, and different OA concentrations for 30 min. Caspase-9 activity was assayed by measuring cleavage of the colorimetric substrate LEDH-pNA. SD is shown for n = 3.

 
PP1{alpha} associates with and controls caspase-9 phosphorylation

To confirm that PP1{alpha} associated to caspase-9 is an active phosphatase, increasing OA concentrations were added to caspase-9 immunoprecipitates, after which phosphatase activity was assayed using phosphorylase a as substrate. Addition of 10–8 M OA to caspase-9 immunoprecipitates results in ~30% inhibition of phosphatase activity, which is almost undetectable after addition of 10–6 M OA (Fig. 6A). Similarly, addition of 10–9 M tautomycin (a specific concentration for PP1{alpha} inhibition) to caspase-9 immunoprecipitates completely inhibits phosphatase activity (data not shown). The selective effect of OA and tautomycin, together with the fact that PP1{alpha} interacts with caspase-9, suggests that PP1{alpha} is an active phosphatase in caspase-9 immunoprecipitates.


Figure 6
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  		FIGURE 6. Estimation of serine/threonine phosphatase and caspase-9 activity. A, Different OA concentrations were added to caspase-9 immunoprecipitates from IL-2-stimulated cells. The activity was estimated using [32P]phosphorylase a as substrate. Phosphatase activity is represented as the percentage of maximal activity in untreated immunoprecipitates. Similar results were obtained in four independent experiments. B, Phosphatase activity was estimated in caspase-9 immunoprecipitates from IL-2-stimuated or -deprived cells using [32P]phosphorylase a as substrate. Phosphatase activity is represented as fold increase compared with control IL-2-stimulated cells. SD is shown for n = 4. Caspase-9 activity was estimated in extracts from IL-2-stimulated or deprived cells using the colorimetric substrate LEHD-pNA. SD is shown for n = 4. C, Cytoplasmic lysates from IL-2-stimulated or -deprived cells were immunoprecipitated (IP) with anti-caspase-9 Ab, transferred to PVDF, and immunoblotted with anti-phospho-PP1{alpha} Ab. D, Cytoplasmic lysates from IL-2-stimulated or 6-h-deprived cells were immunoprecipitated with anti-caspase-9 Ab, transferred to PVDF, and blotted with anti-phospho-PP1{alpha}, anti-phospho-caspase-9, and caspase-9, the latter as internal control of protein loading. Molecular masses (Mw) of the proteins are shown. Similar results were obtained in three independent experiments. E, Cytoplasmic lysates from IL-2-stimulated cells were immunoprecipitated with anti-caspase-9 Ab. Different concentrations of exogenous recombinant active PP1{alpha} were added in the presence of phosphatase buffer. The enzymatic reaction (1 h, 30°C) was separated by SDS-PAGE, transferred to PVDF, and blotted with anti-phospho-caspase-9 and caspase-9. Similar results were obtained in three independent experiments. WB, Western blot.

 
Given that phosphorylation regulates caspase-9 activity and that the caspase-9/PP1{alpha} association can be detected by immunoprecipitation, we explored the possibility that the phosphorylation status of caspase-9 might be controlled by PP1{alpha}. Fig. 6B shows phosphatase activity in caspase-9 immunoprecipitates during IL-2 deprivation. The phosphatase activity in the immunoprecipitates was measured using [32P] phosphorylase a as substrate. Phosphatase activity was detected in caspase-9 immunoprecipitates of IL-2-stimulated cells, progressively increasing upon IL-2 deprivation, reaching a maximum 4–6 h after IL-2 deprivation and decreasing further as the starvation period progressed (Fig. 6B). Caspase-9 activity was also analyzed in extracts from IL-2-stimulated or -deprived cells. The enzymatic activity progressively increases upon IL-2 deprivation, reaching the plateau after 6 h of deprivation (Fig. 6B) and decreasing throughout the starvation period analyzed. This result suggests that IL-2 deprivation induces an increase of caspase-9-associated PP1{alpha} phosphatase activity, followed by caspase-9 activation. We confirmed that the PP1{alpha} associated with caspase-9 is an active phosphatase by adding increasing OA and tautomycin concentrations (data not shown). Fig. 6C shows PP1{alpha} dephosphorylation upon activation by IL-2 deprivation.

In the view of the fact that PP1 phosphatase and caspase-9 activities are detected in anti-caspase-9 immunoprecipitates and that dephosphorylation induces caspase-9 as well as PP1{alpha} activation, we analyzed the status of PP1{alpha} and caspase-9 phosphorylation in caspase-9 immunoprecipitates from 6 h IL-2-stimulated or -deprived cells. Caspase-9 and PP1{alpha} are phosphorylated in IL-2-proliferating cells (Fig. 6D). The level of caspase-9 phosphorylation strongly decreases upon 6 h of IL-2 deprivation. Similarly, the level of phosphorylated PP1{alpha} strongly decreases upon 6 h of IL-2 deprivation compared with the control. Total caspase-9 expression was not affected during the deprivation period analyzed (Fig. 6D).

To definitively confirm that PP1{alpha} is able to dephosphorylate caspase-9, we performed an in vitro phosphatase enzymatic assay in caspase-9 immunoprecipitates from IL-2-stimulated cells supplemented with recombinant active PP1{alpha}. Phosphorylated caspase-9 was detected in control immunoprecipitates and the level of phosphorylation decreased upon addition of increasing amounts of exogenous recombinant active PP1{alpha} (Fig. 6E), being almost undetectable upon addition of 2.5 U of recombinant PP1{alpha}. The level of caspase-9 was not modified throughout the enzymatic assay (Fig. 6E). Taken together, our results strongly suggest that PP1{alpha} is the phosphatase involved in caspase-9 dephosphorylation and, as a consequence, its activation.

Determination of the binding site of caspase-9 to PP1{alpha}

To determine the residues of caspase-9 that interact with PP1{alpha}, we generated overlapping peptides (12 aa) from caspase-9, which were immobilized onto a cellulose membrane. The membrane was incubated with purified recombinant PP1{alpha} protein, followed by anti-PP1{alpha} and then a PO-conjugated secondary Ab. Fig. 7A shows the entire mouse caspase-9 amino acid sequence as 222 overlapping peptides each of 12 aa with a 2-aa shift. The sites of interaction of PP1{alpha} with caspase-9 are boxed (sites 1 and 2). Nonlabeled spots reflect background staining, because they could also be detected without primary Ab incubation. Fig. 7B shows the sequence of interacting spots.


Figure 7
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  		FIGURE 7. PP1{alpha}-binding assay on cellulose-bound caspase-9 peptides. A, The sequence of caspase-9 was developed as series of overlapping dodecapeptides. Membrane with caspase-9 peptides was incubated with purified PP1{alpha} followed by anti-PP1{alpha} Ab. After a washing, membrane was incubated with PO-conjugated secondary Ab. Spots were detected with the ECL system. Caspase-9 peptides that interact with PP1{alpha} are boxed. B, Caspase-9 amino acid sequence showing site 1 and site 2 of caspase-9 interaction with PP1{alpha}. Molecular masses (Mw) of the proteins are shown. IP, Immunoprecipitaton; WB, Western blot.

 
Next, we performed competitive protein-protein interactions to confirm that the interaction between caspase-9 and PP1{alpha} is mediated by the newly identified sites 1 and 2 (Casp9 S1 and Casp9 S2). Lysates from IL-2-stimulated cells were immunoprecipitated with the anti-caspase-9 Ab. The caspase-9/PP1{alpha} interaction was competed using 1 mM peptide corresponding to site 1, site 2 (Casp9 S1 and Casp9 S2), or both. PP1{alpha} was detected in control anti-caspase-9 immunoprecipitates (Fig. 8). The amount of PP1{alpha} associated with caspase-9 decreased after competition with Casp9 S1 and Casp9 S2 peptides, becoming undetectable upon competition with both peptides (Fig. 8). An irrelevant peptide (AQAQAQAHAHALALAL) was not able to disrupt the caspase-9/PP1{alpha} interaction. Similar level of caspase-9 was observed in control and peptide-treated anti-caspase-9 immunoprecipitates. In reciprocal experiments, caspase-9 was detected in control PP1{alpha} immunoprecipitates. The amount of caspase-9 associated with PP1{alpha} decreased after competition with peptides corresponding to Casp9 S1 or Casp9 S2, becoming almost undetectable upon competition with both peptides (Fig. 8). The caspase-9/PP1{alpha} interaction was not modified by an irrelevant peptide. Similar levels of PP1{alpha} were observed in control and peptide-treated anti-PP1{alpha} immunoprecipitates.


Figure 8
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  		FIGURE 8. Effect of site 1 and site 2 peptides on the PP1{alpha}/caspase-9 interaction. Cytoplasmic lysates from control cells were immunoprecipitated with anti-caspase-9 or anti-PP1{alpha} Ab. The interaction caspase-9/PP1{alpha} was competed with or without 1 mM of site 1 and/or site 2 (Casp9 S1 and/or Casp9 S2) peptide for 30 min at room temperature. Immunoprecipitates were blotted with anti-PP1{alpha} or anti-caspase-9 Ab. Proteins were detected using the ECL system. Competition with an irrelevant peptide was used as a negative control. Molecular masses (Mw) of the proteins are shown. IP, Immunoprecipitaton; WB, Western blot. Similar results were obtained in three independent experiments.

 
In reciprocal experiments, the sequence of PP1{alpha} was generated as overlapping peptides immobilized onto a cellulose membrane. The membrane was incubated with purified caspase-9, followed by anti-caspase-9 Ab. Fig. 9A shows the PP1{alpha} amino acid sequence as overlapping peptides of 12 aa with a 2-aa shift. The three sites of interaction of caspase-9 with PP1{alpha} are boxed. Fig. 9B shows the sequence of interacting spots. Nonmarked spots reflect the background, because they were also detected when the membrane was probe without primary Ab incubation.


Figure 9
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  		FIGURE 9. Caspase-9-binding assay on cellulose-bound PP1{alpha} peptides. A, Sequence of PP1{alpha} was developed as series of overlapping dodecapeptides. Membrane with the spots was incubated with purified caspase-9 followed by anti-caspase-9 Ab. After washing, membrane was incubated with PO-conjugated secondary Ab. Spots were developed using ECL. PP1{alpha} peptides that interact with caspase-9 are boxed. B, The PP1{alpha} amino acid sequence showing the PP1{alpha}-binding sites 1, 2, and 3 are shown.

 
On the basis of the three-dimensional structure of caspase-9, we analyzed whether the sequences corresponding to Casp9 S1 and Casp9 S2 (sites 1 and 2) were accessible for interaction with PP1{alpha}. As shown in Fig. 10, A and B, both sites are exposed (yellow spots: A, site 1; B, site 2), strongly suggesting that the interaction is physically possible. Similarly, on the basis of the three-dimensional structure of PP1, sequences corresponding to sites 1, 2, and 3 of the PP1{alpha} are also exposed (yellow) and accessible for interaction with caspase-9 (Fig. 10C). Although sites 1 and 2 of PP1{alpha} are separated in the linear sequence, they are predicted to be close to each other in the crystal structure. The catalytic site of PP1{alpha} is localized on the opposite site of the molecule.


Figure 10
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  		FIGURE 10. Three-dimensional modeling of caspase-9 and PP1{alpha}. A, Caspase-9 sequence from residues 1 to 112 comprising binding site 1 (yellow spots); B, caspase-9 three-dimensional structure from residues 139 to the C-terminal of the protein showing binding site 2 (yellow spots). C, Full length PP1{alpha} structure showing binding sites 1, 2, and 3 (yellow color) and the catalytic site.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Phosphorylation and dephosphorylation affects the function of proteins in almost every conceivable way. It can induce increasing or decreasing enzyme activities, mark a protein for destruction, allow a protein to move from one cellular compartment to another, or enable a protein to interact with or dissociate from other proteins. Kinases and phosphatases can be localized on the same protein, but at different docking sites (41).

Using reciprocal coimmunoprecipitation experiments, we have shown that caspase-9 directly interacts with PP1{alpha} and that this interaction can be blocked by peptides corresponding to the binding sites of caspase-9 to PP1{alpha}. These findings were confirmed by in vitro binding of cellular extracts to purified fusion proteins containing caspase-9 or PP1{alpha}. Under both conditions, we detected a PP1{alpha}/caspase-9 interaction, which could also be recovered in freshly isolated thymocytes. Moreover, we corroborated these results by binding assays using PP1{alpha} or caspase-9 peptides bound to membranes.

The complex PP1{alpha}/caspase-9 detected in control IL-2-stimulated cells shows weak PP1 enzymatic activity, whereas PP1 activity in caspase-9 immunoprecipitates increased along the starvation period, reaching a maximum level 4–6 h after IL-2 deprivation, which corresponds to the point of no return in the apoptotic process. This enzymatic activity in caspase-9 immunoprecipitates was inhibited by OA concentrations that block PP1{alpha} activity. Upon PP1{alpha} activation, we observed a subsequent activation of caspase-9, with a shift in the kinetics of activation with respect to PP1{alpha}, reaching a maximum level upon 6 h of IL-2 deprivation. The caspase-9/PP1{alpha} interaction is observed both in proliferating and 6-h apoptotic cells, the only difference being the phosphorylation status of both proteins. Upon 6 h of IL-2 deprivation, the level of PP1{alpha} and caspase-9 phosphorylation strongly decreases, probably because once PP1{alpha} dephosphorylates caspase-9, PP1{alpha} is dispensable for the complex. It has been described that phosphorylation and dephosphorylation events can affect the formation of complexes. For example, the dynamic phosphorylation and dephosphorylation of Bcl-2 cause conformational changes within the protein and may serve as survival sensor during stress stimuli (42). We do not rule out the possibility that PP1{alpha}/caspase-9 may be a dynamic association and that once PP1{alpha} dephosphorylates caspase-9, the phosphatase loses its affinity for caspase-9. Another hypothesis may be that dephosphorylated caspase-9 changes its conformation, inducing the release of PP1{alpha}, rendering PP1{alpha} available for a new interaction and able to perform further dephosphorylation.

Our results show that PP1{alpha} associates with and dephosphorylates caspase-9 and that the PP1{alpha}/caspase-9 interaction provides a mechanism for controlling caspase-9 phosphorylation and, as a consequence, its activation that correlates with apoptosis induction. In addition, PP1{alpha} may itself be activated by dephosphorylation, suggesting the implication of another phosphatase capable of dephosphorylating and activating PP1{alpha} with protein phosphatase 2A being a likely candidate. In addition, it has been shown that activation of PP1{alpha} plays an important role in Fas-induced apoptosis by stimulating mitochondrial release of cytochrome c and caspase activation in HL-60 and Jurkat cells (43). Moreover, a ceramide-activated protein phosphatase, which is a member of protein phosphatase 2A, is involved in receptor-mediated induction of apoptosis in several cell lines (44), suggesting that protein phosphatase activation may be a common feature of cells undergoing apoptosis.

The finding that PP1 activates RB (45, 46, 47) raises the question as to whether PP1 itself is modulated during the cell cycle. In synchronized MG63 cells, cytoplasmic PP1 activity is maximal in G0-G1, decreasing in the remaining phases of the cell cycle (35). Given that the total amounts of PP1 do not change, posttranslational modifications appear responsible for these activity changes. Purified PP1 is phosphorylated and inactivated in a time-dependent manner, and the phosphorylated residue has been mapped to threonine 320 (35). It seems that inhibitory phosphorylation of PP1 may be required to permit progression into the S phase of the cell cycle (48). A direct correlation between Ras and PP1/protein phosphatase 2A activation has been demonstrated in different studies (49, 50). We have shown that Ras activation leads to apoptotic cell death upon IL-2 deprivation. We also have shown that Ras inhibition prevents cell death through down-modulation of PP1{alpha} activity (50). In summary, several pieces of evidence involve PP1{alpha} in the induction of apoptosis: by dephosphorylation of the proapoptotic Bcl-2 family member Bad (37) and caspase-9 (this article); by stimulating mitochondrial release of cytochrome c; and by dephosphorylation of the retinoblastoma protein that probably becomes a substrate for caspases (51). The dephosphorylation of these proteins by PP1{alpha} may be critical for the initiation of apoptosis. We do not exclude the possibility that cooperation of many proteins and signal pathways, rather than activation of a single one, may be involved in the induction of apoptosis.

It has been shown that many of the PP1{alpha}-targeting subunits have a PP1-binding motif, but caspase-9 does not contain these conserved PP1-binding motifs (RxVxF and/or FxxRxR, respectively) (52, 53, 54, 55). We describe here two novel sites of interaction between PP1{alpha} and caspase-9 that have not been previously described. The finding that site 1 and site 2 (Casp9 S1 and Casp9 S2) peptides disrupt the interaction between PP1{alpha} and caspase-9 implies that these motifs are critical for the interaction, although we do not exclude that caspase-9 may have additional sites of interaction with PP1{alpha}. This is in agreement with results showing that some PP1{alpha}-targeting proteins have multiple sites of interaction with PP1 (56). We can conclude that caspase-9 is a PP1{alpha} substrate, and we entertain the possibility that it might also be a PP1{alpha}-regulatory subunit. Our results also underline the importance of Casp9 S1 and Casp9 S2 peptides in mediating specific protein interactions. Both sequences are highly conserved in human, mouse, and rat caspase-9. The importance of short peptide sequences in mediating crucial protein-protein interactions and in determining subcellular localization of proteins has become apparent in recent years. The ability of proteins to recognize short sequences with a high degree of specificity provides a mechanism for generation of specific signaling responses.

Upon growth factor stimulation, caspase-9 is phosphorylated at threonine 125 by ERK/MAPK (15), thus inhibiting its proapoptotic function. Other sites of phosphorylation by PKA have been described in caspase-9, notably at serines 99, 183, and 195, but inhibition of caspase-9 activation by PKA does not appear to require direct phosphorylation of caspase-9. It is obvious that phosphorylation of caspase-9 requires a phosphatase activity to reverse the reaction; accordingly upon full activation of PP1{alpha}, we were not able to detect caspase-9 phosphorylation. In addition, the PP1{alpha}/caspase-9 association strongly decreases after IL-2 starvation. This is probably due to the fact that once caspase-9 is dephosphorylated, the affinity of PP1{alpha} for caspase-9 diminishes, or that cleaved caspase-9 has a different conformational structure that is less appropriate for PP1{alpha} binding. The site of procaspase-9 cleavage does not block interaction with PP1{alpha}, because it is far from sites 1 and 2 (linear sequence). Furthermore, we are able to detect an interaction between murine caspase-9 and human PP1{alpha}, and also between murine PP1{alpha} and human caspase-9, suggesting that the binding site is conserved between human and mouse proteins. In in vitro binding experiments, phosphorylation is not mandatory for association between caspase-9 and PP1{alpha}, because we are able to detect association in GST pull downs, as well as in peptide spot binding experiments. In a cell-free system where PP1{alpha} has been depleted, we are able to completely inhibit caspase-9 activity, whereas only 50% of caspase-3 activity was blocked. This likely reflects the control of caspase-3 activation by other molecules, in addition to caspase-9.

Although full length caspase-9 has not been crystallized yet, the structures of the individual caspase recruitment domain 7 and catalytic domain have been solved, but structural data for the 40 aa between these domains is lacking. The crystal structure was determined at 3.5 Å resolution for human caspase-9 lacking the first 138 residues (57). The caspase recruitment domain domain was probably removed during preparation of recombinant protein due to an E. coli protease. According to our findings, this structure shows site 2 to be exposed on the surface. Similarly, the crystal structure from aa 1–112 of caspase-9 has also been solved and (58) similarly shows a surface-exposed site 1. PP1c is folded into a single elliptical domain consisting of a central beta sandwich of two mixed beta sheets surrounded on one side of seven {alpha} helices and on the other by a subdomain consisting of three {alpha} helices and three beta sheets at the top of the beta sandwich, creating a catalytic channel (54). The catalytic site of PP1 contains a binuclear metal site consisting of Mn2+ and Fe2+ (54). PP1{alpha} structure shows, according to our results, accessible sites 1, 2 and 3, which correlates with the possibility of association to caspase-9.

To be qualified as a tumor suppressor, a protein must inhibit oncogenic transformation and tumorigenicity, permit cell death, and should have undergone loss-of-function mutations in malignant tumors. PP1{alpha} meets at least one of these criteria, because its activity is able to trigger exit from the cell cycle and suppress cellular transformation by Ras and other oncogenes that disrupt cell cycle regulation (59, 60) Taken together, our results help to understand the mechanisms that regulate PP1{alpha} phosphatase-mediated caspase-9 dephosphorylation that is required for its executor function. The future challenge will be to understand to what extent the functions of PP1 in cell cycle and apoptosis are integrated and/or regulated differentially.


   Acknowledgments
 
We thank Gordon Langsley for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Angelita Rebollo, Laboratoire d’Immunologie Cellulaire et Tissulaire, Unité 543, Institut National de la Santé, et de la Recherche Médicale, Hôpital Pitié-Salpêtrière, Bâtiment CERVI, 83, Bd de l’Hôpital, 75013 Paris, France. E-mail address: rebollo{at}chups.jussieu.fr Back

2 Abbreviations used in this paper: PKA, protein kinase A; PP1. serine/threonine phosphatase type 1; PP1c, catalytic subunit of PP1; PVDF, polyvinylidine difluoride; OA, okadaic acid; PO, peroxidase; pNA, p-nitroalanine. Back

Received for publication March 3, 2006. Accepted for publication May 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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