of April 12, 2018.This information is current as

ApoptosisRegulator in IL-2 Deprivation-Induced

as a Caspase-9αIdentification of PP1

RebolloFleischer, Ata Ghadiri, Marianne Duhamel and Angelita Frédéric Dessauge, Xavier Cayla, Juan Pablo Albar, Aarne

http://www.jimmunol.org/content/177/4/2441doi: 10.4049/jimmunol.177.4.2441

2006; 177:2441-2451; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/177/4/2441.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Identification of PP1� as a Caspase-9 Regulator in IL-2Deprivation-Induced Apoptosis

Frederic Dessauge,* Xavier Cayla,† Juan Pablo Albar,‡ Aarne Fleischer,* Ata Ghadiri,*Marianne Duhamel,* and Angelita Rebollo1*

One of the mechanisms that regulate cell death is the reversible phosphorylation of proteins. ERK/MAPK phosphorylatescaspase-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/threoninephosphatase type 1� (PP1�) associates with phosphorylated caspase-9. IL-2 deprivation induces PP1� dephosphorylation, whichleads to its activation and, as a consequence, dephosphorylation and activation of caspase-9 and subsequent dissociation of bothmolecules. In cell-free systems supplemented with ATP caspase-9 activation is induced by addition of cytochrome c and we showthat in this process PP1� is indispensable for triggering caspase-9 as well as caspase-3 cleavage and activation. Moreover, PP1�associates with caspase-9 in vitro and in vivo, suggesting that it is the phosphatase responsible for caspase-9 dephosphorylationand activation. Finally, we describe two novel phosphatase-binding sites different from the previously described PP1� consensusmotifs, and we demonstrate that these novel sites mediate the interaction of PP1� with caspase-9. The Journal of Immunology,2006, 177: 2441–2451.

A poptosis is a regulated process important for differenti-ation, control of cell number, and removal of damagedor autoreactive cells (1, 2). Extracellular as well as in-

tracellular signals, including growth factor deprivation, radiation,chemotherapeutic drugs, or cross-linking of receptors, can triggeran apoptotic response, leading to the controlled activation of cys-teine proteases known as caspases (3). Two major pathways ofcaspase activation have been described, one involving the releaseof cytochrome c, among other molecules from mitochondria, andthe other involving cell surface death receptor activation (4).

The mitochondrial apoptotic pathway can be regulated at mul-tiple stages to promote or suppress cell death, the release of cyto-chrome c being a key step (5). Once cytochrome c is released, itassociates with Apaf-1 and procaspase-9 to form the apoptosomecomplex, which facilitates the autoprocessing of initiator caspase-9(6). Subsequently, activated caspase-9 cleaves downstreamcaspases such as caspase-3, -6, and -7. In turn, caspase-3 fullyprocesses caspase-9 via a feedback loop leading to amplification ofthe apoptotic signal and cell degradation (7).

Two recent studies have indicated that caspase-9 can also beactivated by mechanisms involving neither cytochrome c releasenor Apaf-1 activation (8, 9). The first study showed that in re-sponse to endoplasmic reticular stress caspase-12 was able to pro-cess 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 incaspase-mediated apoptosis. First, the phosphorylation status of acaspase 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 phos-phatases (11–14). Finally, some caspases are themselves subjectedto reversible phosphorylation. It has been demonstrated that theERK/MAPK pathway inhibits caspase-9 activation by direct phos-phorylation of a threonine at position 125 (Thr125) (15). This phos-phorylation is sufficient to block caspase-9 processing and subse-quent caspase-3 activation, suggesting that caspase-9 inhibition byphosphorylation promotes cell survival (15). Caspase-9 can also bephosphorylated at serine residues 99, 183, and 195 by protein ki-nase A (PKA),2 inhibiting its activation. However, inhibition ofcaspase-9 activation by PKA does not necessarily require directphosphorylation of caspase-9 (16).

Serine/threonine phosphatases are usually classified as type 1(PP1) or type 2 (PP2), depending on their substrate specificity andsensitivity to inhibitors. PP1 represents a family of holoenzymesgenerated by specific interactions between catalytic subunits and awide variety of regulatory or anchoring proteins (17, 18). The cat-alytic subunit of serine/threonine phosphatase type 1 (PP1c) is a38-kDa protein highly conserved throughout evolution. Four iso-forms of the enzyme, a, b, g1, and g2, encoded by three genes (g1and g2 result from alternative splicing) are differentially expressedin mammals (19). Protein variations among these isoforms are ob-served mainly at the C terminus (20), which plays a regulatory rolein the catalytic activity, as demonstrated by proteolysis and phos-phorylation studies (21). PP1 is a major eukaryotic serine/threo-nine phosphatase that regulates diverse cellular processes such ascell cycle progression, proliferation, protein synthesis, muscle con-traction, carbohydrate metabolism, transcription, cytokinesis, andneuronal signaling (22–25). During the cell cycle, PP1 activity is

*Laboratoire d’Immunologie Cellulaire et Tissulaire, Hopital Pitie-Salpetriere, UniteInstitut National de la Sante, et de la Recherche Medicale, Paris, France; †EquipeHypophyse, Unite Mixte de Recherche 6175, Institut National de la RechercheAgronomique-Centre National de la Recherche, Universite de Tours, Haras-Nation-aux, Physiologie de la Reproduction et des Comportements, Nouzilly, France; and‡Centro Nacional de Biotecnologia, Campus de Cantoblanco, Universidad Autonomade Madrid, Madrid, Spain

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

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Angelita Rebollo, Laboratoired’Immunologie Cellulaire et Tissulaire, Unite 543, Institut National de la Sante, et dela Recherche Medicale, Hopital Pitie-Salpetriere, Batiment CERVI, 83, Bd del’Hopital, 75013 Paris, France. E-mail address: rebollo@chups.jussieu.fr

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

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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regulated by phosphorylation (26–28). PP1 play a key role in themitotic transition by dephosphorylating proteins that are essentialin these cellular functions (29–34). It has been shown that phos-phorylation of PP1� at threonine residue 320 by cyclin-dependentkinases blocks the enzymatic activity of PP1� (35). In agreement,a constitutive active mutant of PP1� that is resistant to Cdk phos-phorylation at Thr320 prevents cells from entering the S phase ofthe cell cycle (27).

We have previously shown that the mitochondrial apoptoticpathway is implicated in IL-2 deprivation-induced apoptosis (36).We have also shown that IL-2 deprivation-induced apoptosis op-erates by regulating Bad dephosphorylation through the PP1�phosphatase (37). In this article, we demonstrate that PP1� is as-sociates with caspase-9 to induce its dephosphorylation and, as aconsequence, its protease activity. In addition, we have mapped thenovel binding sites of caspase-9 to PP1�.

Materials and MethodsCells and cultures

TS1�� is a murine T cell line stably transfected with the �- and �-chainsof the human IL-2R (38); it can be independently propagated in the pres-ence of IL-2, IL-4, or IL-9; when they are deprived of growth factor, theydie by apoptosis. Cells were cultured in RPMI 1640 (BioWhittaker) sup-plemented with 5% heat-inactivated FCS (Invitrogen Life Technologies), 2mM glutamine, 10 mM HEPES, 0.55 mM arginine, 0.24 mM asparagine,50 �M 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 forWestern blotting according to standard protocols: caspase-3; phospho-Thr320 PP1�; phospho-Thr125 caspase-9 (Cell Signaling); caspase-9 (CellSignaling or Santa Cruz Biotechnology); and PP1 (BD Transduction Lab-oratories). Glutathione agarose was from Santa Cruz Biotechnology. Kitsfor estimation of caspase-9 and caspase-3 activity were purchased fromR&D Systems. Okadaic acid (OA), tautomycin, and cytochrome c werefrom Calbiochem or Sigma-Aldrich. Recombinant caspase-9 and PP1 werefrom Calbiochem. GST-Casp-9 plasmid was a gift from Prof. P. Clarke(University of Dundee, Dundee, U.K.). Peroxidase (PO)-conjugated sec-ondary Abs were from DAKO. Protein A-Sepharose, ECL, and ECL Pluswere purchased from GE Healthcare. Nonidet P-40 was from Roche Di-agnostics. Annexin was from Immunotech.

Isolation of S100 fraction

A total of 20 � 106 cells was IL-2 stimulated or deprived, harvested, andwashed with chilled PBS. Cell pellet was resuspended in 5 volumes ofice-cold buffer A (20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mMMgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 250 mMsucrose) supplemented with protease inhibitors. Cells were disrupted in aDounce homogenizer, the nuclei were centrifuged (1000 � g, 10 min,4°C), and the supernatant further centrifuged (10,000 � g, 15 min, 4°C).The resulting mitochondrial pellet was resuspended in buffer A and storedat �80°C. The supernatant was further centrifuged (100,000 � g, 1 h,4°C),and the resulting cytosolic fraction was washed at �80°C.

Immunoprecipitation and Western blotting

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

Immunodepletion of PP1

Washed protein A-Sepharose (40 �l) was incubated with anti-PP1 Ab in200 �l 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 washingwith incubation buffer. A total of 500 �g of TS1�� S100 lysate was addedto the protein A-Sepharose and incubated at 4°C for 3 h. Protein A-Sepha-rose was recovered by centrifugation, and the supernatant was incubated at4°C for 3 h with a second aliquot of anti-PP1� Ab bound to protein A-Sepharose. The protocol was repeated a total of four times for completePP1� depletion. After centrifugation, the supernatant was recovered andused as PP1-depleted extract.

Caspase assays

Incubations were performed at 30°C in a total volume of 50 �l of reactionbuffer containing 400 �g of S100 TS�� extract, 1 mM ATP, 10 mg/mlcreatine kinase, and 5 mM creatine phosphate. Where required, cytochromec was added to a final concentration of 5 �M. At specific time points, thereaction was mixed with 60 �l of reaction buffer containing caspase-9 orcaspase-3 colorimetric substrate (LEDH or DEVD) coupled to p-nitroala-nine (pNA). After incubation at 37°C for 60 min, release of pNA wasmeasured at 405 nm using a spectrophotometer microtiter plate reader.

In vitro phosphatase assay

Cells (1 � 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 or-thovanadate, and protease inhibitor mixture), and supernatants were im-munoprecipitated with anti-caspase-9 Ab overnight and then incubatedwith protein A-Sepharose (1 h, 4°C). Immunoprecipitates were washedwith phosphatase buffer (50 mM Tris-HCl (pH 7.5), 0.1% 2-ME, 0.1 mMEDTA, 1 mg/ml BSA) and mixed with phosphatase buffer containing phos-phorylase a as substrate and supplemented with 4 mM caffeine (final con-centration). The reaction was incubated at 30°C for 40 min and stopped byadding 100 �l of 20% trichloroacetic acid and 100 �l of 1 mg/ml BSA andthen centrifuged. A total of 185 �l were used to estimate the generation offree phosphate liberated from [32P]phosphorylase a. For phosphorylasephosphatase activity inhibition, OA or tautomycin was added to the reac-tion mixture.

Peptide synthesis

Overlapping peptides covering the whole caspase-9 or PP1� moleculewere prepared by automated spot synthesis into an amino-derivatized cel-lulose membrane as previously described (39, 40). The membrane wasblocked, incubated with purified PP1� or caspase-9 protein and, after sev-eral washing steps, incubated with anti-PP1� or anti-caspase-9 Ab fol-lowed by the PO-conjugated secondary Ab. Protein interactions were vi-sualized using the ECL system. Similarly, caspase-9 site 1 or site 2peptides comprising amino acids MDEADRQLLRRCRVRLVS (site 1) orLDRDKLEHRFRWLRFM (site 2), as well as mutated peptides, were syn-thesized in an automated multiple peptide synthesizer with solid phaseprocedure and standard Fmoc chemistry. The purity and composition of thepeptides were confirmed by reverse phase HPLC and by amino acid anal-ysis. These peptides were used for protein-protein interaction competitionstudies.

Protein-protein interaction competition

The caspase-9/PP1� interaction was competed using peptides correspond-ing to site 1 (MDEADRQLLRRCRVRLVS) and/or site 2 (LDRDKLEHRFRWLRFM). Lysates from IL-2-stimulated cells were immunoprecipitatedwith anti-caspase-9 or anti-PP1� Ab, and protein A-Sepharose was added.The caspase-9/PP1� interaction was competed with 1 mM site 1 and/or site2 peptides (30 min, room temperature). After a washing, immunoprecipitateswere transferred to nitrocellulose and blotted with the corresponding Ab.

Preparation of GST fusion proteins

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

ResultsIL-2 deprivation induces caspase-9 and PP1�dephosphorylation

We have previously shown that the protein phosphatase PP1� isactivated upon IL-2 deprivation and that this correlates with its

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dephosphorylation and apoptosis induction. In addition, we haveshown that IL-2 deprivation-induced apoptosis operates by regu-lating Bad phosphorylation through the PP1� phosphatase (37).We have also described that apoptosis triggered by IL-2 depriva-tion leads to cytochrome c release, as well as caspase-9 andcaspase-3 activation (36). According to our results and given thatcaspase-9 activation is inhibited by phosphorylation (15), we wereinterested therefore in analyzing whether PP1� could be involvedin caspase-9 activation. First, we analyzed the level of phosphor-ylation of caspase-9 and PP1� in proliferating and apoptotic cells.Phosphorylated PP1� was detected in control IL-2-stimulatedcells, decreasing throughout the starvation period analyzed (Fig.1A). Reprobing the membrane with an anti-PP1� Ab showed thatthere were similar amounts of PP1� in IL-2-stimulated and -de-prived cells (Fig. 1A). Similarly, phosphorylated caspase-9 wasdetected in control IL-2-proliferating cells, decreasing in deprivedcells and being almost undetectable after 12 h of IL-2 deprivation.In parallel to caspase-9 dephosphorylation, IL-2 deprivation in-duces caspase-9 cleavage (Fig. 1A). Treatment of IL-2-stimulatedcells for 6 h with the PP1� inhibitor OA blocks caspase-9 dephos-phorylation. Inhibition of caspase-9 dephosphorylation progres-sively increased with the OA concentration used (Fig. 1B). Finally,when IL-2-maintained cells are deprived of the lymphokine, theyundergo apoptosis (Fig. 1C), given that TS1�� is a growth factor-dependent cell line.

Identification of caspase-9 as a PP1�-interacting protein

To address the hypothesis of whether PP1� associates withcaspase-9 and whether PP1� is directly responsible of caspase-9dephosphorylation, we analyzed the interaction of PP1� andcaspase-9 in intact cells by immunoprecipitation. Reciprocal co-immunoprecipitations of cytoplasmic proteins was performed un-der IL-2 stimulation or deprivation conditions (Fig. 2A). Highcaspase-9 levels were observed in anti-PP1� immunoprecipitatesof IL-2-stimulated cells. The amount of caspase-9 associated withPP1� decreased upon IL-2 deprivation, with low levels detectedafter 24 h, in contrast to PP1� levels that did not diminish (Fig.2A). In reciprocal experiments, high PP1� levels were detected inanti-caspase-9 immunoprecipitates from IL-2-stimulated cells,slightly decreasing upon 12 h of IL-2 deprivation and being almostundetectable after 24 h of deprivation (Fig. 2B). Reprobing themembrane with an anti-caspase-9 Ab showed that there were sim-ilar levels of caspase-9 in IL-2-stimulated cells but that it de-creased upon IL-2 deprivation (Fig. 2B). Similarly, the interactionbetween PP1� and caspase-9 was also observed in freshly isolatedthymocytes (Fig. 2C). No interaction was observed between p53and caspase-9. The anti-PP1� and anti-caspase-9 Abs recognizedboth phosphorylated and nonphosphorylated PP1� and caspase-9.This result strongly suggests that PP1� interacts with caspase-9 inTS1�� cells in vivo.

The PP1�/caspase-9 interaction was also observed in vitro byGST pull down experiments. PP1� was produced as a GST fusionprotein and purified on glutathione-agarose beads. Buffer or cyto-plasmic lysates from IL-2-stimulated or -deprived cells were in-cubated with GST-PP1�; and after several washing steps, proteinswere separated by SDS-PAGE, and the blot was developed with ananti-caspase-9 Ab. Caspase-9 from IL-2-stimulated or -deprivedcells interacts with the GST-PP1� fusion protein (Fig. 3), whereasno interaction was observed in the absence of TS1�� extracts (Fig.3, lane E). In reciprocal experiments, GST-caspase-9 was purifiedon glutathione-agarose beads; after a washing, proteins wereresolved by SDS-PAGE, and the blot was developed as previouslydescribed here (Fig. 3). PP1� from IL-2-stimulated or -deprivedcells interacts with GST-caspase-9 fusion protein. No interaction

was observed in the absence of TS1�� extracts (Fig. 3, lane E).These results confirm the interaction previously demonstrated bycoimmunoprecipitation experiments in T cells and in thymocytes.

FIGURE 1. Effect of IL-2 deprivation on PP1� and caspase-9 phosphor-ylation. A, TS1�� cells were IL-2 stimulated or deprived for the indicatedtimes and then lysed. Protein extracts were separated by SDS-PAGE, trans-ferred to PVDF, and hybridized with anti-phospho-PP1�, anti-phopho-caspase-9, anti-caspase-9, and anti-PP1� Ab, the two latter as internalcontrol of protein loading. Protein bands were detected using the ECL system.Molecular masses (Mw) of the corresponding proteins are shown. Similarresults were obtained in three independent experiments. B, IL-2-stimulatedcells were treated with different OA concentrations for 6 h and then lysed andprocessed as above. The blot was proved with anti-phospho-caspase-9 andanti-caspase-9, the latter as internal control. Molecular masses of the proteinsis shown. C, Cells were cultured in the presence or absence of IL-2, harvestedat different times, and stained with annexin and propidium iodide. Cells wereanalyzed using fluorescence flow cytometry. SD is shown for n � 3.

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PP1� is required for caspase-9 activity

Cell-free systems that reproduce in vitro the regulation of apopto-sis have been proved to be useful for dissecting the biochemicalmechanisms controlling caspase activation. To investigate the sig-naling pathways that control caspase-9 activation, we used a cell-free system derived from TS1�� cells supplemented with an ATP-regenerating system in which caspase activation is induced by theaddition of cytochrome c. Caspase-9 and caspase-3 activationswere assayed by cleavage of the colorimetric tetrapeptide substrateLEHD-pNA and DEVD-pNA, respectively. In cytosolic extractsof IL-2-stimulated TS1��, caspase-9 activation was detected after5 min of incubation with cytochrome c, progressively increasingduring the incubation period analyzed (Fig. 4A). Caspase-9 wasnot activated upon PP1� depletion from the cytosol (Fig. 4A) com-pared with control lysates without cytochrome c addition. As in-ternal control, we also used lamin-depleted extracts, showing thatin the absence of lamin, caspase-9 is activated by the presence ofcytochrome c and PP1�. Similarly, caspase-3 activation was alsodetected upon 5 min of incubation of cytosolic TS1�� extractswith cytochrome c, increasing during the incubation period ana-lyzed (Fig. 4B). Depletion of PP1� from the cytosol inhibitsaround 50% the caspase-3 activity (Fig. 4B). No significantcaspase-3 activity was detected in control cytosolic extracts with-out cytochrome c addition. As internal control, the experiment was

also done in lamin-depleted extracts. Fig. 4C shows the internalcontrol of PP1� depletion upon four sequential immunoprecipita-tions with the specific anti-PP1� Ab. After PP1� depletion,caspase-9 is still present in the supernatant.

The effect of PP1� depletion on caspase-9 processing was an-alyzed in cytosolic extracts. Depletion of PP1� from cytosol in-hibits procaspase-9 processing (Fig. 4D), whereas cleavage wasobserved in cytosolic extracts incubated for 15 min with cyto-chrome c. No caspase-9 processing was observed in the absence ofcytochrome c (Fig. 4D). Similarly, the effect of PP1� depletion oncaspase-3 processing was also analyzed. Depletion of PP1� fromcytosol partially inhibits procaspase-3 processing (Fig. 4E).Caspase-3 processing was observed in cytosolic extracts incubatedwith cytochrome c for 15 min. The processing was measured bythe appearance of p19 and p17 fragments. No caspase-3 cleavagewas found in the absence of cytochrome c (Fig. 4E). These resultssuggest a requirement of PP1� activity for cytochrome c-inducedactivation of caspase-9 and subsequent caspase-3 activation.

Because PP1� plays a critical role in caspase-9 activation, wedetermined whether PP1� is the phosphatase specifically involvedin caspase-9 activation. An in vitro cell-free system reaction wasmade using cytosolic extracts from IL-2-stimulated cells, supple-mented with cytochrome c and ATP. The reaction was incubatedwith different OA concentrations for 30 min, and caspase-9 acti-vation was measured. Treatment with 1 mM completely preventscaspase-9 activation (Fig. 5), as detected by cleavage of the col-orimetric substrate LEDH-pNA.

PP1� associates with and controls caspase-9 phosphorylation

To confirm that PP1� associated to caspase-9 is an active phos-phatase, increasing OA concentrations were added to caspase-9immunoprecipitates, after which phosphatase activity was assayedusing phosphorylase a as substrate. Addition of 10�8 M OA to

FIGURE 2. Interaction of PP1� and caspase-9 (Casp-9). A, Cytoplas-mic lysates from IL-2-stimulated or -deprived cells were immunoprecipi-tated (IP) with anti-PP1� Ab, transferred to PVDF, and immunoblottedwith anti-caspase-9 and anti-PP1�, the latter as internal control. B, Simi-larly, caspase-9 was immunoprecipitated from cytoplasmic lysates of IL-2-stimulated or -deprived cells and immunoblotted with anti-PP1� or anti-caspase-9, the latter as internal control of protein loading. Molecularmasses (Mw) of the proteins are shown. Similar results were obtained inthree independent experiments. C, Caspase-9 was immunoprecipitatedfrom cytoplasmic lysates of freshly isolated thymocytes and immunoblot-ted with anti-PP1�, anti-caspase-9, and anti-p53, the two latter as internalcontrol. Similar results were obtained in two independent experiments.WB, Western blot.

FIGURE 3. In vitro interaction of PP1� and caspase-9. GST-PP1 (A) orGST-caspase-9 (B) fusion proteins were induced with isopropyl �-D-thio-galactoside for 4 h, and proteins were isolated by affinity chromatographywith glutathione-agarose beads and incubated with or without (lane E)cytoplasmic lysates from IL-2-stimulated or -deprived cells. After a wash-ing, eluted proteins were separated by SDS-PAGE, transferred to PVDF,and blotted with anti-PP1� or anti-caspase-9 Ab. Molecular masses (Mw)of the corresponding proteins are indicated. Similar results were obtainedin three independent experiments.

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

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caspase-9 immunoprecipitates results in �30% inhibition of phos-phatase activity, which is almost undetectable after addition of10�6 M OA (Fig. 6A). Similarly, addition of 10�9 M tautomycin(a specific concentration for PP1� inhibition) to caspase-9 immu-noprecipitates completely inhibits phosphatase activity (data notshown). The selective effect of OA and tautomycin, together withthe fact that PP1� interacts with caspase-9, suggests that PP1� isan active phosphatase in caspase-9 immunoprecipitates.

Given that phosphorylation regulates caspase-9 activity and thatthe caspase-9/PP1� association can be detected by immunopre-cipitation, we explored the possibility that the phosphorylation sta-tus of caspase-9 might be controlled by PP1�. Fig. 6B shows phos-phatase activity in caspase-9 immunoprecipitates during IL-2deprivation. The phosphatase activity in the immunoprecipitateswas measured using [32P] phosphorylase a as substrate. Phospha-tase 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 decreasingfurther as the starvation period progressed (Fig. 6B). Caspase-9activity was also analyzed in extracts from IL-2-stimulated or -de-prived cells. The enzymatic activity progressively increases uponIL-2 deprivation, reaching the plateau after 6 h of deprivation (Fig.6B) and decreasing throughout the starvation period analyzed. Thisresult suggests that IL-2 deprivation induces an increase ofcaspase-9-associated PP1� phosphatase activity, followed bycaspase-9 activation. We confirmed that the PP1� associated withcaspase-9 is an active phosphatase by adding increasing OA andtautomycin concentrations (data not shown). Fig. 6C shows PP1�dephosphorylation upon activation by IL-2 deprivation.

In the view of the fact that PP1 phosphatase and caspase-9 ac-tivities are detected in anti-caspase-9 immunoprecipitates and thatdephosphorylation induces caspase-9 as well as PP1� activation,we analyzed the status of PP1� and caspase-9 phosphorylation incaspase-9 immunoprecipitates from 6 h IL-2-stimulated or -de-prived cells. Caspase-9 and PP1� are phosphorylated in IL-2-pro-liferating cells (Fig. 6D). The level of caspase-9 phosphorylationstrongly decreases upon 6 h of IL-2 deprivation. Similarly, thelevel of phosphorylated PP1� strongly decreases upon 6 h of IL-2deprivation compared with the control. Total caspase-9 expressionwas not affected during the deprivation period analyzed (Fig. 6D).

To definitively confirm that PP1� is able to dephosphorylatecaspase-9, we performed an in vitro phosphatase enzymatic assayin caspase-9 immunoprecipitates from IL-2-stimulated cells sup-plemented with recombinant active PP1�. Phosphorylatedcaspase-9 was detected in control immunoprecipitates and the

level of phosphorylation decreased upon addition of increasingamounts of exogenous recombinant active PP1� (Fig. 6E), beingalmost undetectable upon addition of 2.5 U of recombinant PP1�.The level of caspase-9 was not modified throughout the enzymaticassay (Fig. 6E). Taken together, our results strongly suggest thatPP1� is the phosphatase involved in caspase-9 dephosphorylationand, as a consequence, its activation.

Determination of the binding site of caspase-9 to PP1�

To determine the residues of caspase-9 that interact with PP1�, wegenerated overlapping peptides (12 aa) from caspase-9, whichwere immobilized onto a cellulose membrane. The membrane wasincubated with purified recombinant PP1� protein, followed byanti-PP1� and then a PO-conjugated secondary Ab. Fig. 7A showsthe entire mouse caspase-9 amino acid sequence as 222 overlap-ping peptides each of 12 aa with a 2-aa shift. The sites of inter-action of PP1� with caspase-9 are boxed (sites 1 and 2). Nonla-beled spots reflect background staining, because they could also bedetected without primary Ab incubation. Fig. 7B shows the se-quence of interacting spots.

Next, we performed competitive protein-protein interactions toconfirm that the interaction between caspase-9 and PP1� is medi-ated by the newly identified sites 1 and 2 (Casp9 S1 and Casp9 S2).Lysates from IL-2-stimulated cells were immunoprecipitated withthe anti-caspase-9 Ab. The caspase-9/PP1� interaction was com-peted using 1 mM peptide corresponding to site 1, site 2 (Casp9 S1and Casp9 S2), or both. PP1� was detected in control anti-caspase-9 immunoprecipitates (Fig. 8). The amount of PP1� as-sociated with caspase-9 decreased after competition with Casp9 S1and Casp9 S2 peptides, becoming undetectable upon competitionwith both peptides (Fig. 8). An irrelevant peptide (AQAQAQA-HAHALALAL) was not able to disrupt the caspase-9/PP1�interaction. Similar level of caspase-9 was observed in control andpeptide-treated anti-caspase-9 immunoprecipitates. In reciprocalexperiments, caspase-9 was detected in control PP1� immunopre-cipitates. The amount of caspase-9 associated with PP1� decreasedafter competition with peptides corresponding to Casp9 S1 orCasp9 S2, becoming almost undetectable upon competitionwith both peptides (Fig. 8). The caspase-9/PP1� interaction wasnot modified by an irrelevant peptide. Similar levels of PP1�were observed in control and peptide-treated anti-PP1�immunoprecipitates.

In reciprocal experiments, the sequence of PP1� was generatedas overlapping peptides immobilized onto a cellulose membrane.The membrane was incubated with purified caspase-9, followed byanti-caspase-9 Ab. Fig. 9A shows the PP1� amino acid sequenceas overlapping peptides of 12 aa with a 2-aa shift. The three sitesof interaction of caspase-9 with PP1� are boxed. Fig. 9B shows thesequence of interacting spots. Nonmarked spots reflect the back-ground, because they were also detected when the membrane wasprobe without primary Ab incubation.

On the basis of the three-dimensional structure of caspase-9, weanalyzed whether the sequences corresponding to Casp9 S1 andCasp9 S2 (sites 1 and 2) were accessible for interaction with PP1�.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 isphysically possible. Similarly, on the basis of the three-dimen-sional structure of PP1, sequences corresponding to sites 1, 2, and3 of the PP1� are also exposed (yellow) and accessible for inter-action with caspase-9 (Fig. 10C). Although sites 1 and 2 of PP1�are separated in the linear sequence, they are predicted to be closeto each other in the crystal structure. The catalytic site of PP1� islocalized on the opposite site of the molecule.

FIGURE 5. Effect of OA on caspase-9 activity. TS1�� extracts wereincubated with cytochrome c, ATP, and different OA concentrations for 30min. Caspase-9 activity was assayed by measuring cleavage of the color-imetric substrate LEDH-pNA. SD is shown for n � 3.

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DiscussionPhosphorylation and dephosphorylation affects the function of pro-teins in almost every conceivable way. It can induce increasing ordecreasing enzyme activities, mark a protein for destruction, allowa protein to move from one cellular compartment to another, orenable a protein to interact with or dissociate from other proteins.Kinases and phosphatases can be localized on the same protein, butat different docking sites (41).

Using reciprocal coimmunoprecipitation experiments, we haveshown that caspase-9 directly interacts with PP1� and that thisinteraction can be blocked by peptides corresponding to the bind-ing sites of caspase-9 to PP1�. These findings were confirmed byin vitro binding of cellular extracts to purified fusion proteins con-taining caspase-9 or PP1�. Under both conditions, we detected aPP1�/caspase-9 interaction, which could also be recovered infreshly isolated thymocytes. Moreover, we corroborated these re-sults by binding assays using PP1� or caspase-9 peptides bound tomembranes.

The complex PP1�/caspase-9 detected in control IL-2-stim-ulated cells shows weak PP1 enzymatic activity, whereas PP1activity in caspase-9 immunoprecipitates increased along the

FIGURE 6. Estimation of serine/threonine phosphatase and caspase-9activity. A, Different OA concentrations were added to caspase-9 immu-noprecipitates from IL-2-stimulated cells. The activity was estimated using[32P]phosphorylase a as substrate. Phosphatase activity is represented asthe percentage of maximal activity in untreated immunoprecipitates. Sim-ilar results were obtained in four independent experiments. B, Phosphataseactivity was estimated in caspase-9 immunoprecipitates from IL-2-stimuated or -deprived cells using [32P]phosphorylase a as substrate. Phos-phatase activity is represented as fold increase compared with control IL-2-stimulated cells. SD is shown for n � 4. Caspase-9 activity wasestimated in extracts from IL-2-stimulated or deprived cells using the col-orimetric substrate LEHD-pNA. SD is shown for n � 4. C, Cytoplasmiclysates from IL-2-stimulated or -deprived cells were immunoprecipitated

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

(IP) with anti-caspase-9 Ab, transferred to PVDF, and immunoblotted withanti-phospho-PP1� Ab. D, Cytoplasmic lysates from IL-2-stimulated or6-h-deprived cells were immunoprecipitated with anti-caspase-9 Ab, trans-ferred to PVDF, and blotted with anti-phospho-PP1�, anti-phospho-caspase-9, and caspase-9, the latter as internal control of proteinloading. Molecular masses (Mw) of the proteins are shown. Similar resultswere obtained in three independent experiments. E, Cytoplasmic lysatesfrom IL-2-stimulated cells were immunoprecipitated with anti-caspase-9Ab. Different concentrations of exogenous recombinant active PP1� wereadded in the presence of phosphatase buffer. The enzymatic reaction (1 h,30°C) was separated by SDS-PAGE, transferred to PVDF, and blotted withanti-phospho-caspase-9 and caspase-9. Similar results were obtained inthree independent experiments. WB, Western blot.

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starvation period, reaching a maximum level 4 – 6 h after IL-2deprivation, which corresponds to the point of no return in theapoptotic process. This enzymatic activity in caspase-9 immu-noprecipitates was inhibited by OA concentrations that blockPP1� activity. Upon PP1� activation, we observed a subsequentactivation of caspase-9, with a shift in the kinetics of activationwith respect to PP1�, reaching a maximum level upon 6 h ofIL-2 deprivation. The caspase-9/PP1� interaction is observedboth in proliferating and 6-h apoptotic cells, the only differencebeing the phosphorylation status of both proteins. Upon 6 h ofIL-2 deprivation, the level of PP1� and caspase-9 phosphory-lation strongly decreases, probably because once PP1� dephos-phorylates caspase-9, PP1� is dispensable for the complex. Ithas been described that phosphorylation and dephosphorylationevents can affect the formation of complexes. For example, thedynamic phosphorylation and dephosphorylation of Bcl-2 causeconformational changes within the protein and may serve assurvival sensor during stress stimuli (42). We do not rule out thepossibility that PP1�/caspase-9 may be a dynamic associationand that once PP1� dephosphorylates caspase-9, the phospha-tase loses its affinity for caspase-9. Another hypothesis may bethat dephosphorylated caspase-9 changes its conformation, in-ducing the release of PP1�, rendering PP1� available for a newinteraction and able to perform further dephosphorylation.

Our results show that PP1� associates with and dephosphory-lates caspase-9 and that the PP1�/caspase-9 interaction provides amechanism for controlling caspase-9 phosphorylation and, as aconsequence, its activation that correlates with apoptosis induc-tion. In addition, PP1� may itself be activated by dephosphoryla-tion, suggesting the implication of another phosphatase capable ofdephosphorylating and activating PP1� with protein phosphatase2A being a likely candidate. In addition, it has been shown thatactivation of PP1� plays an important role in Fas-induced apopto-sis by stimulating mitochondrial release of cytochrome c andcaspase activation in HL-60 and Jurkat cells (43). Moreover, aceramide-activated protein phosphatase, which is a member of pro-

tein phosphatase 2A, is involved in receptor-mediated induction ofapoptosis in several cell lines (44), suggesting that protein phos-phatase activation may be a common feature of cells undergoingapoptosis.

The finding that PP1 activates RB (45– 47) raises the questionas to whether PP1 itself is modulated during the cell cycle. Insynchronized MG63 cells, cytoplasmic PP1 activity is maximalin G0-G1, decreasing in the remaining phases of the cell cycle(35). Given that the total amounts of PP1 do not change, post-translational modifications appear responsible for these activitychanges. Purified PP1 is phosphorylated and inactivated in atime-dependent manner, and the phosphorylated residue hasbeen mapped to threonine 320 (35). It seems that inhibitoryphosphorylation of PP1 may be required to permit progressioninto the S phase of the cell cycle (48). A direct correlationbetween Ras and PP1/protein phosphatase 2A activation hasbeen demonstrated in different studies (49, 50). We have shownthat Ras activation leads to apoptotic cell death upon IL-2 de-privation. We also have shown that Ras inhibition prevents celldeath through down-modulation of PP1� activity (50). In sum-mary, several pieces of evidence involve PP1� in the inductionof apoptosis: by dephosphorylation of the proapoptotic Bcl-2family member Bad (37) and caspase-9 (this article); by stim-ulating mitochondrial release of cytochrome c; and by dephos-phorylation of the retinoblastoma protein that probably be-comes a substrate for caspases (51). The dephosphorylation ofthese proteins by PP1� may be critical for the initiation ofapoptosis. We do not exclude the possibility that cooperation ofmany proteins and signal pathways, rather than activation of asingle one, may be involved in the induction of apoptosis.

It has been shown that many of the PP1�-targeting subunitshave a PP1-binding motif, but caspase-9 does not contain theseconserved PP1-binding motifs (RxVxF and/or FxxRxR, respec-tively) (52–55). We describe here two novel sites of interactionbetween PP1� and caspase-9 that have not been previously de-scribed. The finding that site 1 and site 2 (Casp9 S1 and Casp9S2) peptides disrupt the interaction between PP1� and

FIGURE 8. Effect of site 1 and site 2 peptides on the PP1�/caspase-9interaction. Cytoplasmic lysates from control cells were immunoprecipi-tated with anti-caspase-9 or anti-PP1� Ab. The interaction caspase-9/PP1�was competed with or without 1 mM of site 1 and/or site 2 (Casp9 S1and/or Casp9 S2) peptide for 30 min at room temperature. Immunopre-cipitates were blotted with anti-PP1� or anti-caspase-9 Ab. Proteins weredetected using the ECL system. Competition with an irrelevant peptide wasused as a negative control. Molecular masses (Mw) of the proteins areshown. IP, Immunoprecipitaton; WB, Western blot. Similar results wereobtained in three independent experiments.

FIGURE 9. Caspase-9-binding assay on cellulose-bound PP1� pep-tides. A, Sequence of PP1� was developed as series of overlapping dode-capeptides. Membrane with the spots was incubated with purifiedcaspase-9 followed by anti-caspase-9 Ab. After washing, membrane wasincubated with PO-conjugated secondary Ab. Spots were developed usingECL. PP1� peptides that interact with caspase-9 are boxed. B, The PP1�amino acid sequence showing the PP1�-binding sites 1, 2, and 3 are shown.

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caspase-9 implies that these motifs are critical for the interac-tion, although we do not exclude that caspase-9 may have ad-ditional sites of interaction with PP1�. This is in agreementwith results showing that some PP1�-targeting proteins havemultiple sites of interaction with PP1 (56). We can concludethat caspase-9 is a PP1� substrate, and we entertain the possi-bility that it might also be a PP1�-regulatory subunit. Our re-sults also underline the importance of Casp9 S1 and Casp9 S2peptides in mediating specific protein interactions. Both se-quences are highly conserved in human, mouse, and ratcaspase-9. The importance of short peptide sequences in medi-ating crucial protein-protein interactions and in determiningsubcellular localization of proteins has become apparent in re-cent years. The ability of proteins to recognize short sequenceswith a high degree of specificity provides a mechanism for gen-eration of specific signaling responses.

Upon growth factor stimulation, caspase-9 is phosphorylatedat threonine 125 by ERK/MAPK (15), thus inhibiting its pro-apoptotic function. Other sites of phosphorylation by PKA havebeen described in caspase-9, notably at serines 99, 183, and195, but inhibition of caspase-9 activation by PKA does notappear to require direct phosphorylation of caspase-9. It is ob-vious that phosphorylation of caspase-9 requires a phosphataseactivity to reverse the reaction; accordingly upon full activationof PP1�, we were not able to detect caspase-9 phosphorylation.In addition, the PP1�/caspase-9 association strongly decreasesafter IL-2 starvation. This is probably due to the fact that oncecaspase-9 is dephosphorylated, the affinity of PP1� forcaspase-9 diminishes, or that cleaved caspase-9 has a differentconformational structure that is less appropriate for PP1� bind-ing. The site of procaspase-9 cleavage does not block interac-tion with PP1�, because it is far from sites 1 and 2 (linearsequence). Furthermore, we are able to detect an interactionbetween murine caspase-9 and human PP1�, and also betweenmurine PP1� and human caspase-9, suggesting that the bindingsite is conserved between human and mouse proteins. In in vitrobinding experiments, phosphorylation is not mandatory for as-sociation between caspase-9 and PP1�, because we are able todetect association in GST pull downs, as well as in peptide spotbinding experiments. In a cell-free system where PP1� has beendepleted, we are able to completely inhibit caspase-9 activity,whereas only 50% of caspase-3 activity was blocked. Thislikely reflects the control of caspase-3 activation by other mol-ecules, in addition to caspase-9.

Although full length caspase-9 has not been crystallized yet, thestructures of the individual caspase recruitment domain 7 and cat-alytic domain have been solved, but structural data for the 40 aabetween these domains is lacking. The crystal structure was de-termined at 3.5 Å resolution for human caspase-9 lacking the first138 residues (57). The caspase recruitment domain domain wasprobably removed during preparation of recombinant protein dueto an E. coli protease. According to our findings, this structureshows site 2 to be exposed on the surface. Similarly, the crystalstructure from aa 1–112 of caspase-9 has also been solved and (58)similarly shows a surface-exposed site 1. PP1c is folded into asingle elliptical domain consisting of a central � sandwich of twomixed � sheets surrounded on one side of seven � helices and onthe other by a subdomain consisting of three � helices and three �sheets at the top of the � sandwich, creating a catalytic channel(54). The catalytic site of PP1 contains a binuclear metal site con-sisting of Mn2� and Fe2� (54). PP1� structure shows, accordingto our results, accessible sites 1, 2 and 3, which correlates with thepossibility of association to caspase-9.

FIGURE 10. Three-dimensional modeling of caspase-9 and PP1�. A,Caspase-9 sequence from residues 1 to 112 comprising binding site 1 (yel-low spots); B, caspase-9 three-dimensional structure from residues 139 tothe C-terminal of the protein showing binding site 2 (yellow spots). C, Fulllength PP1� structure showing binding sites 1, 2, and 3 (yellow color) andthe catalytic site.

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To be qualified as a tumor suppressor, a protein must inhibitoncogenic transformation and tumorigenicity, permit cell death,and should have undergone loss-of-function mutations in malig-nant tumors. PP1� meets at least one of these criteria, because itsactivity is able to trigger exit from the cell cycle and suppresscellular transformation by Ras and other oncogenes that disruptcell cycle regulation (59, 60) Taken together, our results help tounderstand the mechanisms that regulate PP1� phosphatase-me-diated caspase-9 dephosphorylation that is required for its executorfunction. The future challenge will be to understand to what extentthe functions of PP1 in cell cycle and apoptosis are integratedand/or regulated differentially.

AcknowledgmentsWe thank Gordon Langsley for critical reading of the manuscript.

DisclosuresThe authors have no financial conflict of interest.

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