Protein kinase C ζ  associates with death inducing signaling complex and regulates Fas ligand-induced apoptosis

Cellular Signalling 17 (2005) 1149 – 1157 www.elsevier.com/locate/cellsig

Protein kinase C ~ associates with death inducing signaling complex and regulates Fas ligand-induced apoptosis Ingrid Leroy, Aure´lie de Thonel1, Guy Laurent, Anne Quillet-MaryT INSERM U563/CPTP, CHU Purpan, Baˆt B, Dpt G. DELSOL, Equipe G. LAURENT, CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France Received 8 November 2004; received in revised form 17 December 2004; accepted 17 December 2004 Available online 17 February 2005

Abstract Previous studies have shown that Protein kinase C (PKC) stimulation may interfere with Fas signaling pathway and Fas ligand (FasL)induced apoptosis. In this study, we investigated in Jurkat cells, a FasL-sensitive human T-cell model, whether PKC~ targets apical events of Fas signaling. We describe for the first time that in Jurkat cells, both PKC~ and Prostate apoptosis response-4 (Par-4), one of the major endogenous PKC~ regulators, are components of the death inducing signaling complex (DISC). Using PKC~ overexpressing cells or si-RNA depletion, we demonstrate that PKC~ interferes neither with Fas expression nor Fas clustering in raft microdomains, but negatively regulates FasL-induced apoptosis by interfering with DISC formation and subsequent caspase-8 processing. D 2005 Elsevier Inc. All rights reserved. Keywords: Protein kinase C; Par-4; Fas; Death inducing signaling complex; Signal transduction; Apoptosis

1. Introduction Fas (APO-1/CD95) is a well-characterized member of the tumor necrosis factor (TNF) receptor superfamily [1,2]. Ligation of CD95 with Fas ligand (FasL) or agonistic antibodies triggers apoptosis in a large variety of cell types. Different apoptotic signaling pathways have been described after Fas engagement depending on the cell type. In type I cells, stimulation of CD95 results in the clustering of the CD95 receptor in lipid (raft) microdomains [3], DISC (Death-Inducing Signaling Complex) formation through FADD and caspase-8 recruitment to Fas, and subsequent release of caspase-8 as an active hetero-tetramer containing p18 and p10. These subunits, in turn, trigger the activation of effector caspases such as caspase-3 [4–7]. In type II cells, Abbreviations: PKC~, protein kinase C ~; Par-4, prostate apoptosis response-4; DISC, death inducing signaling complex; FasL, Fas ligand; FADD, Fas-associated death domain protein; si-RNA, small interference RNA. T Corresponding author. Tel.: +33 5 62 74 45 43; fax: +33 5 62 74 45 58. E-mail address: [email protected] (A. Quillet-Mary). 1 Current address: Turku Centre for Biotechnology, Turku, Finland. 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2004.12.013

activation of caspase-8 at DISC level is also involved but CD95 signaling is amplified by the mitochondrial pathway, mediated by Bid cleavage [8]. Nevertheless the relevance of these two cell types in vivo remains controversial [9]. Several proteins have been implicated in the regulation of FasL-induced apoptosis at the DISC level (see Ref. [10] for review). Among them, the inactive caspase-8 homologue, cellular FLICE inhibitory protein (c-FLIP), could interfere with the activation of procaspase-8 after DISC formation [11], even if a pro-apoptotic role of c-FLIP has been proposed [12]. More recently, caspase-10, as caspase-8, has been described to interact with FADD and processed within the DISC although the substrate(s) of these two caspases are different [13–15]. Moreover, other Fas-associated proteins have been characterized, including Fas-associated phosphatase (FAP-1), Fas associated-factor 1 (FAF-1) and receptor interacting protein (RIP), although their physiological role remains controversial [10]. Protein kinase C (PKC) has emerged as a potent negative regulator of apoptosis induced not only by Fas but also by other death receptors, including TNF-related apoptosisinducing ligand (TRAIL) receptor or TNF-a receptor [16,17]. Previous studies have shown that phorbol ester-

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induced PKC stimulation may interfere with Fas oligomerization [18], FADD recruitment [19], or Bid cleavage [20]. Based on phorbol ester specificity, these results suggest that classical or novel PKC isozymes do play an important role in regulating FasL-induced apoptosis. However, although little is known about the function of phorbol insensitive, socalled atypical, PKC isozymes, more recent studies suggest that these enzymes may also contribute to Fas signaling regulation [21,22]. In the present study, we investigated in a Fas sensitive Tcell model whether PKC~ is a DISC component and interferes with FADD recruitment, caspase-8 processing and apoptosis upon Fas activation.

2. Materials and methods

to FasL) was kindly given by Pascal Schneider (Lausanne, Switzerland). Neutralizing anti-Fas (clone ZB4) and secondary monoclonal antibodies were from Beckman/Coulter (Villepinte, France). Anti-Par-4, anti-PKC~ and anti-Fas polyclonal antibodies were purchased from Santa Cruz Biotechnology (TEBU, Le Perray-en-Yvelines, France). Anti-caspase-8 and anti-phospho-PKC~ antibodies were purchased from Cell Signaling Technology (Ozyme, St Quentin en Yvelines, France). Anti-FADD and anti-Flotillin antibodies were purchased from BD Transduction Laboratories (Le Pont de Claix, France). Anti-Bcl-2 antibody was from Dako (Trappes, France) and anti-actin from NeoMarkers (Interchim, Montluc¸on, France). Anti-FLIPL antibody was from Upstate (Euromedex, Souffelweyersheim, France). PKC~ pseudo-substrate was produced by MilleGen Biotechnologies (Toulouse, France). Other products were purchased from Sigma (Saint Quentin-Fallavier, France).

2.1. Cell lines 2.3. Confocal microscopy Jurkat lymphoid cell line was obtained from the ATCC (Rockville, MD, USA). Jurkat cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 1 mM sodium pyruvate and 10 mM Hepes buffer. Cells were maintained at 37 8C in a fully humidified 5% CO2 incubator. All media and reagents were provided by Invitrogen (Cergy Pontoise, France). For PKC~ overexpression, stable transfection of Jurkat cells was performed by electroporation with a p2Rc/CMV vector containing cDNA of PKC~ from Xenopus (kind gift from J. Moscat, Madrid, Spain). Clone selection was done using 2 mg/ml geneticin. PKC~ gene suppression was done using GeneSuppressor kit provided by Imgenex (Clinisciences, Montrouge, France) according to manufacturer recommendations [23,24]. GeneSuppressor is a plasmid-based system allowing stable siRNA generation after cell transfection. Briefly, human PKC~ oligonucleotide inserts were designed [25]: PKC~ forward: 5V TCG AGATCT TCATCA CCA GCG TGG AGA GTA CTG TCC ACG CTG GTG ATG AAG ATT TTT T 3V; PKC~ reverse: 5V CTA GAA AAA ATC TTC ATC ACC AGC GTG GAC AGT ACT CTC CAC GCT GGT GAT GAA GAT C 3. After primer annealing, inserts were cloned in the pSuppressor plasmid containing Neomycin resistant gene for selection in mammalian cells. Before transfection, the presence of the correct insert was analyzed by sequencing. Jurkat cells were stably transfected by Lipofectaminek 2000 (Invitrogen, Cergy Pontoise, France) with PKC~ si-RNA construction plasmid. Selection was done with 2 mg/ml geneticin. All experiments were done on exponentially growing cells. 2.2. Reagents Recombinant FasL was produced by transfected Neuro2A [26] (a kind gift from A. Fontana, Lausanne, Switzerland). Recombinant FasL–Fc (human Fc fragment coupled

Cells were incubated with or without FasL 0.5 ng/ml, washed in cold PBS and allowed to attach on a polylysin coated glass slide for 7 min at 4 8C. Cells were then fixed for 10 min in 4% paraformaldehyde (w/v) in PBS, permeabilized for 10 min with 0.1% saponin in PBS–BSA (PBS containing 3% bovine serum albumin (w/v) and 1 mM Hepes). Cells were washed and further incubated for 1 h with primary antibodies (10 Ag/ml for anti-Fas antibody and 2 Ag/ml for all other antibodies). After washing with PBS– BSA, cells were stained for 45 min with appropriate fluorochrome-conjugated secondary antibodies (5 Ag/ml). After a final wash in PBS–BSA, cells were mounted in a Dako mounting medium (Dako, Trappes, France). Slides were examined with a Carl Zeis LSM 510 confocal microscope (Carl Zeis, Oberkochen, Germany). Control staining were performed with secondary antibodies alone. Images are single slices and are representative of all the population. 2.4. Western blot analysis Cells were washed with cold PBS and lysed in lysis buffer (30 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP40, 10% glycerol, 1 mM Na3 VO4, 10 mM NaF, 1 mM PMSF, 10 mM h-glycerophosphate, 10 Ag/ml leupeptin and 10 Ag/ml aprotinin) for 30 min on ice, followed by centrifugation at 12,000 g for 10 min at 4 8C. Protein concentration in the supernatants was determined as previously described [27]. For each lysate, 30 Ag total protein was boiled for 5 min at 95 8C in the presence of 5% h-mercaptoethanol. Proteins were separated on 12.5% SDSPAGE and transferred electrophoretically onto nitrocellulose membranes (Hybond-C extra; Amersham Life Science, Cergy-Pontoise, France). Nonspecific binding sites were blocked in PBS containing 0.1% Tween-20 and 10% non-fat milk. Membranes were incubated overnight at 4 8C with

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specific primary antibody diluted at an appropriate concentration in PBS containing 0.1% Tween-20 and 1% nonfat milk. Membranes were then washed three times at room temperature and bound immunoglobulins were detected with an anti-isotype monoclonal antibody coupled to horseradish peroxidase. The signal was visualized by enhanced chemiluminescence (Amersham Life Science, Cergy-Pontoise, France). 2.5. Isolation of membrane microdomains Rafts were isolated from cells as previously described [28]. Briefly, after treatment with or without FasL 15 min, 1108 cells were pelleted by centrifugation, resuspended

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in 1 ml of ice-cold MBS-buffered saline (150 nM NaCl, 25 mM 2-(N-morphilino)-ethane-sulfonic-acid, pH 6.5) containing 1% Triton X-100. After 30 min on ice and homogeneization, ice-cold MBS (1.5 ml) was added and 2 ml of this suspension were mixed with 2 ml of 80% sucrose (w/v) in MBS. This mixture was subsequently loaded under a linear gradient consisting of 8 ml 5–40% (w/v) sucrose in MBS. All solutions contained appropriate protease inhibitors. Gradients were centrifuged in a Beckman SW 41 swinging-rotor at 39,000 rpm for 20 h at 4 8C. Twelve fractions of 1 ml each were collected (from top to bottom). Flotillin was used as a marker of rafts [29]. For Western blot analysis fractions were pooled 3 by 3 (fractions 1, 2 and 3 became fraction I; 4, 5 and 6, II; 7, 8 and 9, III; 10, 11 and 12, IV), and 60 Al of fractions I, II, corresponding to Rafts fraction, III and IV were boiled for 5 min at 95 8C in the presence of 5% h-mercaptoethanol and loaded on the gel. 2.6. DISC formation analysis Exponentially growing cells (2107 cells/ml) were incubated with 0.5 or 0.3 Ag/ml FasL–Fc for the indicated time. For basal level, cells were incubated 10 min at 4 8C with FasL–Fc. Cold PBS (5 ml) was then added to stop the reaction and cells were centrifuged at 100 g for 5 min at 4 8C and lysed in lysis buffer (0.2% NP40, 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM Na3 VO4, 10% glycerol and protease inhibitor cocktail tablets (Complete Mini, Roche Diagnostics GmbH, Mannheim, Germany)), before protein G-sepharose was added. Immunoprecipitates were washed 3 times in lysis buffer without proteases inhibitors before Western blot analysis. Negative control was done without FasL–Fc (CT lane).

Fig. 1. PKC~ is a new DISC component. (A) Confocal analysis. Jurkat cells were treated or not with FasL 0.5 ng/ml for 5 min, and then fixed and permeabilized. Cells were stained with monoclonal anti-Fas antibody (clone ZB4, in green) and with polyclonal anti-PKC~ antibody (in blue). Cells were analyzed by confocal microscopy. Results are representative of all cells in the population analyzed in 3 independent experiments. (B) Raft isolation. Jurkat cells were treated or not with FasL (2.5 ng/ml) and pooled Triton soluble fractions I, II and III were isolated as described in Materials and methods. Raft microdomains fraction was characterized by Western blot analysis using anti-Flotillin and anti-Bcl-2 antibodies. (C) Raft analysis. Jurkat cells were treated or not with FasL (2.5 ng/ml) and fractions containing raft microdomains were isolated. Proteins localized in raft microdomains were analyzed by Western blot using appropriate antibodies. (D) DISC analysis. Jurkat cells were treated for the indicated times with 0.3 Ag/ml of FasL–Fc. Proteins associated to Fas were directly immunoprecipitated and submitted to Western blot analysis using appropriate antibodies. CT line represents the same experiment without FasL–Fc. For B, and C, results are representative of 3 experiments. (E) Effect of PKC~ pseudosubstrate on FasL-induced apoptosis. Jurkat cells were pretreated or not with PKC~ pseudosubstrate (PS) for 1 h prior exposure to FasL (0.5 ng/ml) for the indicated times; (5) FasL alone; ( ) PS 50 nM alone; (n) PS 50 nM + FasL; ( ) PS 100 nM alone; (n) PS 100 nM + FasL. Percentage of apoptotic cells was evaluated by DAPI staining. Results are the meanFSD of 3 experiments *, Pb0.05.

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2.7. Cytochemical staining After FasL treatment, changes in nuclear chromatin were evaluated by DAPI staining as previously described [21]. Apoptotic cells were scored and expressed as the number of cells exhibiting a morphology typical of apoptosis per 200 cells counted. 2.8. Flow cytometry analysis

of PKC~ and Fas whereas FADD and caspase-8 were not detected. However, upon Fas activation, both PKC~ and Fas accumulated into raft microdomains in which FADD and caspase-8 were also detected (Fig. 1C). These results showed that after FasL exposure PKC~ co-localized with DISC components into raft microdomains and could associate with DISC proteins. In order to confirm this hypothesis, we examined the presence of PKC~ in Fas receptor complex in Jurkat cells

After a wash in PBS containing 1% FCS, 5105 cells were incubated at 4 8C for 30 min in presence of anti-Fas (clone ZB4) monoclonal antibody (from 0.0625 Ag/ml to 2 Ag/ml) or irrelevant mouse IgG (2 Ag/ml). After 2 washes in PBS containing 1% FCS, cells were incubated with a Phycoerythrinconjugated goat anti-mouse IgG antibody for 30 min at 4 8C. Fluorescence intensities were evaluated using flow cytometry (Facscan, Becton Dickinson, Pont-de Claix, France). 2.9. Statistics Quantitative experiments were analyzed using Student’s t test. All P values resulted from the use of 2-sided tests.

3. Results 3.1. PKC~ is a functional DISC component We first investigated whether PKC~ co-localized with Fas using confocal microscopy in both unstimulated and FasL stimulated Jurkat cells. As shown in Fig. 1A, in control cells, PKC~ was mainly distributed in the cytoplasm but was also present at the plasma membrane. Interestingly, double staining reveals that membrane-associated PKC~ colocalized with Fas (Fig. 1A). However, in Fas-activated cells, PKC~ membrane distribution displayed a patchy pattern and a part of the enzyme co-localized with clustered Fas receptor (Fig. 1A, see arrows). Previous studies [3,30] have shown that, upon activation, a significant fraction of Fas, FADD and caspase-8 were relocalized to raft microdomains. However a recent report demonstrates, in mouse T cells that, upon Fas ligation, FADD and caspase-8 form caps at the plasma membrane but not in lipid rafts [31]. Nevertheless, we hypothesized that PKC~ was preferentially translocated to detergent-soluble extracts. Rafts microdomains of Jurkat cells were isolated as described in Materials and Methods. The purity of the preparation was tested using anti-Flotillin (only present in Raft fractions) [29] and anti-Bcl-2 (only present in heavy fractions) [32] antibodies. As shown in Fig. 1B, raft microdomains were only detected into pooled Tritton soluble fraction II in untreated and FasL-treated cells. For further analysis of raft proteins content, only fraction II was submitted to Western blot analysis. As shown in Fig. 1C, in control Jurkat cells, raft microdomains contained a fraction

Fig. 2. B10 and B6 clones characterization. (A) PKC~ expression level and activity. PKC~ expression level and PKC~ activity were analyzed in B10 (n) and B6 (n) cells by Western blot using, respectively, anti-PKC~ and anti-phospho-PKC~ antibodies. Results are the meanFSD of 3 experiments. (B) Cell surface Fas expression. Fas expression at the cell surface of Jurkat ( ), B10 (n) and B6 (E) cells was determined by flow cytometry analysis using anti-Fas (clone ZB4) antibody (a. u.: arbitrary units). Results are representative of 3 experiments. (C) Basal protein expression level. In Jurkat, B10 and B6 cells, expression levels of Fas, FADD, caspase-8, caspase-3 and FLIP were analyzed by Western blot analysis. Actin was used as loading control. Results are representative of 3 experiments.

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stimulated or not with FasL–Fc fusion protein. This system allows to directly immunoprecipitate every protein interacting with the DISC. As expected, DISC analysis revealed that, upon activation, Fas associated with FADD and caspase-8 (Fig. 1D). c-FLIP was also recruited to the DISC but did not interfere with caspase-8 activation as it is recruited after caspase-8 cleavage (Fig. 1D). Furthermore, despite a slight binding of PKC~ to beads, we observed that not only PKC~ associated with Fas before stimulation, but also, upon stimulation, the enzyme accumulated into the DISC (Fig. 1D). Altogether, these results indicate that PKC~ is a component of the DISC and suggest that this enzyme functions as a regulator of Fas signaling. In order to confirm this hypothesis, we investigated whether PKC~ inhibition by its specific pseudo-substrate peptide inhibitor (PS-PKC~) could influence FasL-induced apoptosis. As shown in Fig. 1E, PS-PKC~ was found to facilitate apoptosis induced by FasL in a dose-dependent manner. 3.2. PKC~ expression level influences Fas signaling Based on these findings, we hypothesized that in Jurkat cells PKC~ expression level might influence FasL-induced apoptosis. To confirm this hypothesis, we realized two sets of transfection experiments. At first Jurkat cell line was stably transfected with dominant positive xenopus PKC~. Clones expressing different levels of PKC~ were obtained and among them the clone Jurkat B10 was chosen for

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PKC~-overexpression. In parallel, Jurkat cell line was stably transfected with a plasmid coding for PKC~ si-RNA. Among obtained clones, clone B6 was chosen for PKC~inhibition. Compared to Jurkat wild-type cells, B10 cells display a 40% increase in PKC~ activity and B6 cells a 25% decrease in PKC~ activity, as measured by Western blot analysis with an anti-phospho PKC~ as previously reported [33] (Fig. 2A). Importantly, Fas expression was found to be similar in Jurkat, B10, and B6 cells (Fig. 2B,C), and DISC components as well as caspase-3 and FLIP expression level were similar in the three cell lines (Fig. 2C). Confocal studies revealed that, as for Jurkat wild-type cells, FasL treatment induced Fas clustering in B10 as well as B6 cells. Nevertheless PKC~ localization is different in untreated B10 and B6 cells (Fig. 3A,C). In fact, in PKC~ overexpressing B10 cells PKC~ was present in the cytoplasm and the cytoplasmic membrane where it colocalized with Fas. On the opposite in untreated B6 cells PKC~ was only detected in the cytoplasm. After FasL treatment, in both cell lines as well as in Jurkat cells, a subset of PKC~, forming patches, was co-localized with Fas clusters at the cytoplasmic membrane (Fig. 3A,C, see arrows). Moreover as in Jurkat cells, FasL exposure led to delocalization of DISC components and PKC~ into lipid rafts in B10 and B6 cells (data not shown). Nevertheless, DISC analysis showed that PKC~ expression level seriously interfered with DISC formation. In both clones, PKC~ could associate to the DISC but the kinetic of

Fig. 3. Effect of PKC~ level on DISC formation. (A and C) Confocal analysis. B10 and B6 cells were treated or not with FasL 0.5 ng/ml for 15 min, and then fixed and permeabilized. Cells were stained with monoclonal anti-Fas antibody (clone ZB4, in green) and with polyclonal anti-PKC~ antibody (in blue). Cells were analyzed by confocal microscopy. Results are representative of all cells in the population analyzed in 3 independent experiments. (B and D) DISC analysis. B10 and B6 cells were treated for the indicated times with 0.3 Ag/ml of FasL–Fc. Proteins associated to Fas were directly immunoprecipitated and submitted to Western blot analysis using anti-FADD, anti-PKC~ and anti-caspase-8 antibodies. CT line represents the same experiment without FasL–Fc. Results are representative of 3 experiments.

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inhibited in B10 cells (Fig. 4). On the opposite, caspase-8 and caspase-3 cleavage and apoptosis were facilitated in B6 cells compared to parental Jurkat cells (Fig. 4). Altogether, these results showed that PKC~ expression level and activity influence both DISC formation, caspase activation and apoptosis, suggesting that PKC~ acts primarily at the DISC level by interfering not only with FADD recruitment but also with caspase-8 processing and release. 3.3. Par-4 is a new DISC member Par-4 is an endogenous PKC~ negative regulator [34]. Therefore, we hypothesized that Par-4 could also contribute to apical events of Fas signaling regulation. As shown in Fig. 5A, in untreated Jukat cells, a significant fraction of

Fig. 4. Effect of PKC~ level on FasL-induced apoptosis. (A) Caspase processing. Jurkat, B10 and B6 cells were treated by FasL (0.5 ng/ml) for the indicated times. Caspase-8 and caspase-3 processing was analyzed by Western blot using appropriate antibodies. (B) FasL-induced apoptosis. Jurkat (5), B10 (n) and B6 (n) cells were treated by FasL (0.5 ng/ml) for the indicated times. Percentage of apoptotic cells was evaluated by DAPI staining. Results are the meanFSD of 3 experiments *, Pb0.05.

its association and its relative level were quite different. In fact a great amount of PKC~ was detected not only after stimulation but also at the basal state in B10 cells (Fig. 3B). On the contrary, in the absence of stimulation, no PKC~ was present in the DISC in B6 cells according to the results obtained by confocal microscopy. In these cells, after 15 min of FasL–Fc exposure a pool of PKC~ was recruited to Fas receptor (Fig. 3D). Besides, PKC~ overexpression correlated with reduced FADD recruitment and altered caspase-8 processing (Fig. 3B). Thus, whereas the kinetics of caspase-8 association was not affected, p43/44 subunits accumulated from 15 to 75 min in B10 cells (Fig. 3B) but began to be released from the DISC at 45 min in B6 cells (Fig. 3D). Finally, similar level of c-FLIP was recruited to the DISC after caspase-8 cleavage in B10 and B6 cells as in Jurkat cells (data not shown), suggesting that c-FLIP did not play a major role in FasL-induced apoptosis regulation by PKC~. On whole cell extracts Western blot analysis showed that caspase-8 and caspase-3 cleavage as well as apoptosis were

Fig. 5. Par-4 is a new DISC member. (A) Confocal analysis. Jurkat cells were treated or not with FasL 0.5 ng/ml for 15 min, and then fixed and permeabilized. Cells were stained with monoclonal anti-Fas antibody (clone ZB4, in green) and with polyclonal anti-Par-4 antibody (in blue). Cells were analyzed by confocal microscopy. Results are representative of all cells in the population analyzed in 3 independent experiments. (B) Raft analysis. Jurkat cells were treated or not with FasL (2.5 ng/ml) and fractions containing raft microdomains were isolated as described in Materials and methods. The presence of Par-4 in raft microdomains was analyzed by Western blot using appropriate antibody. (C) DISC analysis. Jurkat cells were treated for the indicated times with 0.5 Ag/ml of FasL–Fc. Proteins associated to Fas were directly immunoprecipitated and submitted to Western blot analysis using appropriate antibodies. CT line represents the same experiments without FasL–Fc. (D) Par-4 processing. Jurkat cells were treated by FasL (0.5 ng/ml) for the indicated times. Par-4 processing was analyzed by Western blot using appropriate antibody. For B, C and D results are representative of 3 experiments.

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Par-4 was detected on the plasma membrane and colocalized with Fas. Fas activation resulted in changes in Par-4 membrane distribution with a patchy pattern and colocalization with Fas clusters (Fig. 5A, see arrows) as well as in Par-4 accumulation into raft microdomains (Fig. 5B). Moreover, DISC analysis showed that Par-4 associated with Fas before stimulation, and upon stimulation, we detected a slight accumulation of Par-4 in the DISC. Interestingly, the amount of Par-4 associated with the DISC decreased from 60 min (Fig. 5C). This phenomenon was related to Par-4 cleavage product detection 60–90 min after Fas activation (Fig. 5D). However no Par-4 cleavage products were detected within the DISC. Altogether, these results indicated that: i) Par-4 is constitutively associated with Fas; ii) Par-4 is processed after Fas stimulation according to complex sequential events consisting in the redistribution at the cell surface where it co-aggregates with Fas into raft microdomains, followed by progressive dissociation from the DISC. It is interesting to note that terminal Par-4 processing was concomitant to FADD dissociation and to full activation of caspase-8 and caspase-3, but Par-4 sequence reveals no potential sites for caspase cleavage suggesting that Par-4 degradation could be mediated by other proteases. However, the exact role of Par4 into the DISC remains to be determined.

4. Discussion Our study shows for the first time that, upon Fas activation, at least a fraction of PKC~ interacts with DISC components and confers significant protection against FasLinduced apoptosis in Jurkat cells. Previous studies have described that PKC~ distribution is mainly cytoplasmic, and that the enzyme accumulates into lysosome-targeted endosomal compartment [35]. However, contradictory results have been reported regarding the subcellular redistribution of PKC~ upon activation. Indeed, in most circumstances, including mitogenic stimulation or treatment with differentiating agents or genotoxic stresses, the enzyme is redistributed in other subcellular compartments such as nucleus, or even cleaved through a caspasedependent mechanism [36,37]. However PKC~ translocates to the plasma membrane in cells stressed by UV irradiation or ischemia [38–40]. In our study, in Jurkat cells, PKC~ is mainly distributed in the cytoplasm but is also present at the plasma membrane in which the enzyme co-localizes with Fas. After FasL stimulation, a part of PKC~ associates with Fas clusters in raft microdomains. However, we cannot exclude that a subset of PKC~ might be translocated from cytoplasm to raft micromains. The mechanism by which PKC~ is redistributed in Fas-activated cells was not investigated in our study. However, one can reasonably speculate that activation is a pre-requisite for enzyme translocation. If so, how Fas signaling interferes with PKC~ activity remains to be established. In this perspective,

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it should be of interest to evaluate the role of phosphatidic acid or phosphatidyl-choline (PC)-derived diacylglycerol. Indeed, these lipid messengers are produced upon Fas activation [41,42] and have been documented to be potent endogenous stimulators of PKC~ [43,44]. The present study shows that PKC~ influences FADD recruitment to the DISC as well as caspase-8 processing. This result suggests that altered FADD association to Fas is the one of the major mechanism by which PKC~ interferes with Fas signaling. In a myeloid leukemic cell line, we described that PKC~ may directly interact with FADD both in vitro and in vivo [21]. It is therefore possible that PKC~/ FADD interaction results in reduced capacity of FADD to interact with Fas. In a previous study, it has been reported that phorbol ester-mediated PKC stimulation resulted also in marked reduction in FADD recruitment to TRAIL or Fas receptors [16,19]. This observation, together with ours, suggests a common mechanism of PKC isozymes in targeting apical events of death receptor signaling. However, the role of phosphorylation events in this process is unclear. In the case of phorbol ester-stimulated cells, the inhibition of FADD recruitment to TRAIL receptor seemed to be independent of phosphorylation [16]. In our study, we did not investigate whether FADD is phosphorylated by PKC~ in the DISC upon Fas stimulation. However, we have previously shown that PKC~ can phosphorylate FADD in vitro [21]. Moreover, the predictive PKC~ phosphorylation site corresponds to Ser 41 [45] located into FADD Death Effector Domain. This raises the possibility that, upon Fas stimulation, PKC~ recruitment to the DISC facilitates FADD phosphorylation by PKC~ in cells, resulting in FADD conformational changes that alter its association to Fas and could explain subsequent alteration of caspase-8 processing and release from the DISC. This hypothesis should be investigated but unfortunately available anti-phosphoFADD antibody raised against Ser 194 does not correspond to the PKC~ phopshorylation site. Our study shows that PKC~ protects cells against FasLinduced apoptosis. To what extent altered DISC formation contributes to Fas signaling inhibition remains to be investigated since PKC~ could also act by targeting downstream Fas signaling regulators. Indeed, previous studies have largely documented that ERK/MAPK stimulation suppresses Fas-mediated apoptosis downstream DISC formation in different cellular systems, including peripheral T cells [46–48] whereas ERK pathway is considered as an important target for PKC~ [43]. Therefore, PKC~ could act at different levels of Fas signaling by coordinating inhibitory signals resulting in the abrogation of FasLinduced apoptosis. The role of PKC~ in inhibiting apoptosis has been also described in cells treated with anti-cancer agents, oxidative stress and TNF [43,49–51]. These findings suggest a general protective function for this kinase. Because Par-4 gene product is one the major endogenous regulator of PKC~, we hypothesized that Par-4 could also be involved in apical events of Fas signaling. In many

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cell types, Par-4 is primarily localized in the cytoplasm whereas nuclear distribution is also described in human epithelial cancer cells [52–54]. Surprisingly, we found that, in Jurkat cells, Par-4 is mainly detected in the plasma membrane. Moreover, a significant fraction of Par-4 is contained in raft microdomains and associates with Fas. These findings suggest that Par-4 distribution is different in T cells, compared to other cell types. Moreover, upon Fas stimulation, Par-4 co-aggregates with Fas. The fact that Par-4 associates with the DISC suggests a functional role for this protein. However, it remains uncertain whether Par-4 operates by counteracting the inhibitory role of PKC~ in DISC formation. Indeed, several studies have documented that Par-4 displays an intrinsic pro-apoptotic function in which inhibition of NF-nB transcriptional activity plays an essential role [55,56]. So it would be interesting to test if Par-4 inhibition in Jurkat cells influence NF-nB activity. Therefore series of experiments were performed on Jurkat cells using either Par-4 antisense oligonucleotides or Par-4 si-RNA in transient or stable transfections. Unfortunately, no decrease of Par-4 protein was observed, maybe due to a high level and/or stability of this protein in this cell line. Moreover, Par-4 sensitizes lymphoid cells to apoptosis induced by chemotherapeutic agents by down-regulating Bcl-2, promoting disruption of mitochondrial membrane and caspase activation, as well as by interfering with the anti-apoptotic function of IAP [57]. In another study, Rangnekar and co-workers showed that, at least in pancreatic tumor cells, Par-4 facilitates Fas cell membrane trafficking resulting in increased sensitivity to FasL [58]. Interestingly, we found that Par-4 is cleaved during FasLinduced apoptosis. The mechanism of Par-4 proteolysis as well as the significance of this event remains unknown. It should be investigated whether Par-4 proteolysis results in the loss of Par-4 pro-apoptotic function or, conversely, generates more active fragments containing highly functional death-inducing domain as recently described [54]. Nevertheless this last hypothesis seems unlikely since we did not observe Par-4 cleavage fragments within the DISC. Whatever the mechanism by which Par-4 regulates FasLinduced apoptosis, our findings may have important implications in tumor immune escape. Par-4 expression could be reduced in tumor cells in relation with genetic events, such as deletion of chromosome 12, or with aberrant gene regulation due to oncogenic products such as activated ras, raf or src [55,59,60].

5. Conclusion Our study shows that in Jurkat cell line, PKC~ and Par-4 associate with the DISC. However the exact role of Par-4 remains unknown. Nevertheless, we propose that PKC~ regulates FasL-induced apoptosis by inhibiting FADD recruitment and subsequent capase-8 processing. Therefore

PKC~ inhibition may represent an interesting approach in order to sensitize malignant cells to immune effectors.

Acknowlegments We thank Dr P. Schneider (Epalinges, Switzerland) for kindly providing FasL–Fc and Dr J. Moscat (Madrid, Spain) for Xenopus PKC~ plasmid. This work was supported by the Ligue Contre le Cancer (comite´ Haute Garonne). I.L. is a recipient of a grant from the Ministe`re de l’Education Nationale, de l’Enseignement Supe´rieur, et de la Recherche.

References [1] S. Nagata, P. Golstein, Science 267 (1995) 1449. [2] U. Gaur, B.B. Aggarwal, Biochem. Pharmacol. 66 (2003) 1403. [3] D. Scheel-Toellner, K. Wang, R. Singh, S. Majeed, K. Raza, S.J. Curnow, M. Salmon, J.M. Lord, Biochem. Biophys. Res. Commun. 297 (2002) 876. [4] A. Algeciras-Schimnich, L. Shen, B.C. Barnhart, A.E. Murmann, J.K. Burkhardt, M.E. Peter, Mol. Cell. Biol. 22 (2002) 207. [5] M.P. Boldin, T.M. Goncharov, Y.V. Goltsev, D. Wallach, Cell 85 (1996) 803. [6] F.C. Kischkel, S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P.H. Krammer, M.E. Peter, EMBO J. 14 (1995) 5579. [7] J.P. Medema, C. Scaffidi, F.C. Kischkel, A. Shevchenko, M. Mann, P.H. Krammer, M.E. Peter, EMBO J. 16 (1997) 2794. [8] B.C. Barnhart, E.C. Alappat, M.E. Peter, Semin. Immunol. 15 (2003) 185. [9] D.C. Huang, J. Tschopp, A. Strasser, Cell Death Differ. 7 (2000) 754. [10] M.E. Peter, P.H. Krammer, Cell Death Differ. 10 (2003) 26. [11] M. Irmler, M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner, J.L. Bodmer, M. Schroter, K. Burns, C. Mattmann, D. Rimoldi, L.E. French, J. Tschopp, Nature 388 (1997) 190. [12] D.W. Chang, Z. Xing, Y. Pan, A. Algeciras-Schimnich, B.C. Barnhart, S. Yaish-Ohad, M.E. Peter, X. Yang, EMBO J. 21 (2002) 3704. [13] F.C. Kischkel, D.A. Lawrence, A. Tinel, H. LeBlanc, A. Virmani, P. Schow, A. Gazdar, J. Blenis, D. Arnott, A. Ashkenazi, J. Biol. Chem. 276 (2001) 46639. [14] J. Wang, H.J. Chun, W. Wong, D.M. Spencer, M.J. Lenardo, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13884. [15] M.R. Sprick, E. Rieser, H. Stahl, A. Grosse-Wilde, M.A. Weigand, H. Walczak, EMBO J. 21 (2002) 4520. [16] N. Harper, M.A. Hughes, S.N. Farrow, G.M. Cohen, M. MacFarlane, J. Biol. Chem. 278 (2003) 44338. [17] S. Nishida, S. Yoshioka, S. Kinoshita-Kimoto, M. Kotani, M. Tsubaki, Y. Fujii, T.T. Tomura, K. Irimajiri, Life Sci. 74 (2003) 781. [18] M.C. Ruiz-Ruiz, M. Izquierdo, G. de Murcia, A. Lopez-Rivas, Eur. J. Immunol. 27 (1997) 1442. [19] M. Gomez-Angelats, J.A. Cidlowski, J. Biol. Chem. 276 (2001) 44944. [20] C. Scaffidi, I. Schmitz, J. Zha, S.J. Korsmeyer, P.H. Krammer, M.E. Peter, J. Biol. Chem. 274 (1999) 22532. [21] A. de Thonel, A. Bettaieb, C. Jean, G. Laurent, A. Quillet-Mary, Blood 98 (2001) 3770. [22] J.A. Hinshaw, C.M. Mueller, D.W. Scott, M.S. Williams, Eur. J. Immunol. 33 (2003) 2490. [23] C.P. Paul, P.D. Good, I. Winer, D.R. Engelke, Nat. Biotechnol. 20 (2002) 505. [24] S.M. Elbashir, J. Harborth, K. Weber, T. Tuschl, Methods 26 (2002) 199. [25] S. Rozen, H. Skaletsky, Methods Mol. Biol. 132 (2000) 365.

I. Leroy et al. / Cellular Signalling 17 (2005) 1149–1157 [26] M. Shimizu, A. Fontana, Y. Takeda, H. Yagita, T. Yoshimoto, A. Matsuzawa, J. Immunol. 162 (1999) 7350. [27] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [28] M.P. Lisanti, Z.L. Tang, P.E. Scherer, M. Sargiacomo, Methods Enzymol. 250 (1995) 655. [29] P.E. Bickel, P.E. Scherer, J.E. Schnitzer, P. Oh, M.P. Lisanti, H.F. Lodish, J. Biol. Chem. 272 (1997) 13793. [30] A.O. Hueber, A.M. Bernard, Z. Herincs, A. Couzinet, H.T. He, EMBO Rep. 3 (2002) 190. [31] L.A. O’Reilly, U. Divisekera, K. Newton, K. Scalzo, T. Kataoka, H. Puthalakath, M. Ito, D.C. Huang, A. Strasser, Cell Death Differ. 11 (2004) 724. [32] S. Grazide, N. Maestre, R.J. Veldman, C. Bezombes, S. Maddens, T. Levade, G. Laurent, J.P. Jaffrezou, FASEB J. 16 (2002) 1685. [33] A.C. Newton, Methods Enzymol. 345 (2002) 499. [34] M.T. Diaz-Meco, M.M. Municio, S. Frutos, P. Sanchez, J. Lozano, L. Sanz, J. Moscat, Cell 86 (1996) 777. [35] P. Sanchez, G. De Carcer, I.V. Sandoval, J. Moscat, M.T. Diaz-Meco, Mol. Cell. Biol. 18 (1998) 3069. [36] Y. Mizukami, S. Kobayashi, F. Uberall, K. Hellbert, N. Kobayashi, K. Yoshida, J. Biol. Chem. 275 (2000) 19921. [37] L. Smith, Z. Wang, J.B. Smith, Biochem. J. 375 (2003) 663. [38] C. Huang, J. Li, N. Chen, W. Ma, G.T. Bowden, Z. Dong, Mol. Carcinog. 27 (2000) 65. [39] K. Kurkinen, R. Busto, G. Goldsteins, J. Koistinaho, M.A. PerezPinzon, Neurochem. Res. 26 (2001) 1139. [40] B.J. Padanilam, Kidney Int. 59 (2001) 1789. [41] M.G. Cifone, P. Roncaioli, R. De Maria, G. Camarda, A. Santoni, G. Ruberti, R. Testi, EMBO J. 14 (1995) 5859. [42] J.H. Kim, Y.D. Yoon, I. Shin, J.S. Han, IUBMB Life 48 (1999) 445. [43] V.M. Mas, H. Hernandez, I. Plo, C. Bezombes, N. Maestre, A. QuilletMary, R. Filomenko, C. Demur, J.P. Jaffrezou, G. Laurent, Blood 101 (2003) 1543. [44] M. Lorenzo, T. Teruel, R. Hernandez, A.G. Kayali, N. Webster, J. Exp. Cell. Res. 278 (2002) 146.

1157

[45] J.C. Obenauer, L.C. Cantley, M.B. Yaffe, Nucleic Acids Res. 31 (2003) 3635. [46] T.H. Holmstrom, I. Schmitz, T.S. Soderstrom, M. Poukkula, V.L. Johnson, S.C. Chow, P.H. Krammer, J.E. Eriksson, EMBO J. 19 (2000) 5418. [47] S.E. Tran, T.H. Holmstrom, M. Ahonen, V.M. Kahari, J.E. Eriksson, J. Biol. Chem. 276 (2001) 16484. [48] N. Engedal, H.K. Blomhoff, J. Biol. Chem. 278 (2003) 10934. [49] R. Filomenko, F. Poirson-Bichat, C. Billerey, J.P. Belon, C. Garrido, E. Solary, A. Bettaieb, Cancer Res. 62 (2002) 1815. [50] S.J. Kim, S.G. Hwang, I.C. Kim, J.S. Chun, J. Biol. Chem. 278 (2003) 42448. [51] C. Bezombes, A. de Thonel, A. Apostolou, T. Louat, J.P. Jaffrezou, G. Laurent, A. Quillet-Mary, Mol. Pharmacol. 62 (2002) 1446. [52] E.R. Boghaert, S.F. Sells, A.J. Walid, P. Malone, N.M. Williams, M.H. Weinstein, R. Strange, V.M. Rangnekar, Cell Growth Differ. 8 (1997) 881. [53] Q. Guo, W. Fu, J. Xie, H. Luo, S.F. Sells, J.W. Geddes, V. Bondada, V.M. Rangnekar, M.P. Mattson, Nat. Med. 4 (1998) 957. [54] N. El-Guendy, Y. Zhao, S. Gurumurthy, R. Burikhanov, V.M. Rangnekar, Mol. Cell. Biol. 23 (2003) 5516. [55] A. Nalca, S.G. Qiu, N. El-Guendy, S. Krishnan, V.M. Rangnekar, J. Biol. Chem. 274 (1999) 29976. [56] I. Garcia-Cao, M.J. Lafuente, L.M. Criado, M.T. Diaz-Meco, M. Serrano, J. Moscat, EMBO Rep. 4 (2003) 307. [57] S. Boehrer, K.U. Chow, F. Beske, N. Kukoc-Zivojnov, E. Puccetti, M. Ruthardt, C. Baum, V.M. Rangnekar, D. Hoelzer, P.S. Mitrou, E. Weidmann, Cancer Res. 62 (2002) 1768. [58] M. Chakraborty, S.G. Qiu, K.M. Vasudevan, V.M. Rangnekar, Cancer Res. 61 (2001) 7255. [59] S.G. Qiu, S. Krishnan, N. el-Guendy, V.M. Rangnekar, Oncogene 18 (1999) 7115. [60] M. Barradas, A. Monjas, M.T. Diaz-Meco, M. Serrano, J. Moscat, EMBO J. 18 (1999) 6362.