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Molecular and Cellular Biology, January 2002, p. 182-195, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.182-195.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Tyrosine Phosphorylation of Protein Kinase C{delta} Is Essential for Its Apoptotic Effect in Response to Etoposide

Michal Blass,1 Ilana Kronfeld,1 Gila Kazimirsky,1 Peter M. Blumberg,2 and Chaya Brodie1*

Gonda (Goldschmied) Medical Diagnosis Research Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel,1 Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208922

Received 28 March 2001/ Returned for modification 23 May 2001/ Accepted 19 September 2001


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ABSTRACT
 
Protein kinase C{delta} (PKC{delta}) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKC{delta} in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G1/S phase of the cell cycle. Overexpression of PKC{delta} increased the apoptotic effect induced by etoposide, whereas the PKC{delta} selective inhibitor rottlerin and the PKC{delta} dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKC{delta} and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKC{delta} were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKC{delta} in the effects of etoposide was examined using cells overexpressing a PKC{delta} mutant in which five tyrosine residues were mutated to phenylalanine (PKC{delta}5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKC{delta}. Likewise, activation of caspase 3 and the cleavage of the PKC{delta}5 mutant were significantly lower in cells overexpressing PKC{delta}5. Using mutants of PKC{delta} altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKC{delta} in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKC{delta} as an important regulator of this effect.


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INTRODUCTION
 
Protein kinase C (PKC) comprises a family of phospholipid-dependent serine-threonine kinases which play important roles in various cellular functions (45, 46, 56). PKC consists of 12 isoforms showing diversity in their structures, cellular distributions, and biological functions (27). Based on their structures and cofactor regulation, the PKC isoforms are divided into the classical PKCs ({alpha}, ß1, ß2, and {gamma}), the novel PKCs ({delta}, {varepsilon}, {eta}, and {theta}), and the atypical PKCs (PKC{zeta} and PKC{iota}/{lambda}). In addition, two other members, PKCµ and PKC{nu}, exhibit unique characteristics (28). All PKC isoforms can be divided into an N-terminal regulatory domain and a C-terminal catalytic domain with serine-threonine kinase activity (26, 44). PKC chimeras have been used to study the role of the regulatory and catalytic domains of different PKC isoforms (2, 57).

Various PKC isoforms have been reported to play important roles in cell apoptosis (10, 17). Thus, PKC{alpha} and PKC{varepsilon} inhibit apoptosis by phosphorylating or increasing the expression of Bcl2, respectively (23, 52). In contrast, PKC{delta}, -{theta}, and -µ have been implicated as proapoptotic kinases, mostly by being targets of caspase 3 (14, 34). Thus, apoptotic stimuli, such as ionizing radiation and etoposide, induced the cleavage of PKC{delta} and the accumulation of the PKC{delta} catalytic fragment, which is constitutively active (21, 34). Overexpression of the catalytic domain of PKC{delta} in HeLa cells induced nuclear fragmentation and cell apoptosis (21). PKC{delta} has also been reported to be essential for the spontaneous apoptosis of neutrophils (48), for the apoptosis of parotid cells in response to etoposide (49), and for the apoptosis of keratinocytes and LaNCap cells in response to phorbol myristate acetate (PMA) (19, 36). Likewise, PKC{theta} has been shown to be cleaved by caspase 3 in vitro and in vivo and the catalytic domain of PKC{theta}-induced apoptosis in U937 cells (11). Similarly, PKC{zeta} can be cleaved at the hinge region by caspases in response to apoptotic stimuli, such as tumor necrosis factor alpha or etoposide (18).

Various studies suggest that PKC{delta} associates with different tyrosine kinases and undergoes tyrosine phosphorylation in response to various stimuli. PKC{delta} has been shown to be tyrosine phosphorylated in response to PMA, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) (5, 13, 37), and ligands for the immunoglobulin E (IgE) receptor (25, 54). In addition, apoptotic stimuli, such as H2O2 (32) and {gamma}-irradiation (58), induce tyrosine phosphorylation of PKC{delta}, and c-Abl is implicated in these latter responses. Depending on the stimulus, tyrosine residues in both the regulatory and catalytic domains may undergo phosphorylation.

In a recent study (5), we reported that phosphorylation of PKC{delta} on distinct tyrosine residues plays important roles in C6 cell proliferation and in the expression of the astrocytic marker glutamine synthetase. In the present study, we find that tyrosine phosphorylation of PKC{delta} is essential for the cleavage of caspase 3 and PKC{delta} and for the apoptotic effect of PKC{delta} in response to etoposide.


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MATERIALS AND METHODS
 
Materials. An affinity-purified polyclonal anti-PKC{varepsilon} antibody against a polypeptide corresponding to amino acids 726 to 737 of PKC{varepsilon} was purchased from GIBCO-BRL Life Technologies (Gaithersburg, Md.). Monoclonal anti-PKC{delta} antibody directed against the regulatory domain was obtained from Transduction Laboratories (Lexington, Ky.), and polyclonal anti-PKC antibodies were from Santa Cruz (Santa Cruz, Calif.). Etoposide was from Alexis Co. (San Diego, Calif.), and an anti-active caspase 3 antibody was obtained from New England Biolabs (Beverly, Mass.). The caspase inhibitors DEVD-FMK, Z-VAD-FMK, and YVAD were obtained from Calbiochem (La Jolla, Calif.). Leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and sodium vanadate were obtained from Sigma Chemical Co. (St. Louis, Mo.). The Caspase 3 Cellular Activity Assay Kit PLUS was obtained from BIOMOL (Plymouth Meeting, Pa.), and the Cell Death Detection enzyme-linked immunosorbent assay (ELISA) Kit was from Roche Molecular Biochemicals.

Generation of PKC chimeras. The PKC chimeras were generated by exchanging the regulatory and catalytic domains of PKC{alpha} and PKC{delta} as previously described (1). PKC{alpha}/{delta} refers to the chimera with the PKC{alpha} regulatory domain and the PKC{delta} catalytic domain, and PKC{delta}/{alpha} refers to the reciprocal chimera. The PKC cDNAs were subcloned into the metallothionein promoter-driven eukaryotic expression vector (MTH). The vector sequence encodes a C-terminal PKC{varepsilon}-derived 12-amino-acid tag ({varepsilon}MTH) that is added to the expressed proteins (47). The expression of these chimeras and their activities in C6 cells were recently described (5).

Site-directed mutagenesis of PKC{delta}. Mouse PKC{delta} was cloned into the pGEM-T vector (Promega, Madison, Wis.) as described previously (5). This plasmid served as our "master" vector for the site-directed mutagenesis, using the Transformer Site-Directed Mutagenesis Kit from Clontech (Palo Alto, Calif.). Conversion of tyrosine residues at sites 52, 64, 155, 187, and 565 into phenylalanine was performed as previously described (5). PKC{delta} and the PKC{delta} mutants were subcloned into the metallothionein promoter-driven eukaryotic expression vector ({varepsilon}MTH). A PKC{delta} K376R dominant mutant was generated as previously described (36).

Construction of PKC{delta}-GFP fusion protein. cDNAs encoding the murine PKC{delta} and the PKC{delta}5 mutant were fused into the N-terminal enhanced green fluorescent protein (GFP) vector pEGFP-N1 (Clontech Laboratories). The original pEGFP-N1 vector was modified by the insertion of an MluI site into the plasmid polylinker. The restriction site was created by ligating a phosphorylated linker containing the MluI site into pEGFP-N1 digested with SmaI. The construct was verified by sequencing. The clones containing GFP-PKC{delta} or GFP-PKC{delta}5 were constructed by the excision of PKC{delta} or PKC{delta}5 from MTH-PKC plasmids by digestion with XhoI and MluI. The inserts were then ligated into the modified GFP vector by using the same restriction sites. DNA sequencing of the GFP-PKC constructs confirmed the intended reading frame.

C6 glial cultures and cell transfection. C6 cells (105 cells/ml) were seeded on tissue culture dishes (10-cm diameter) and were grown in medium consisting of Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (50 U/ml), and streptomycin (0.05 mg/ml). The cells were transfected either with the empty vectors or with the PKC{delta} and PKC{delta}5 expression vectors by using Lipofectamine (Gibco-BRL Life Technologies) as previously described (6). Experiments were routinely carried out on a clone of the transfected cells, but all the results were confirmed on one pool and two additional individual clones.

For overexpression of the GFP-PKC{delta} fusion proteins, C6 cells were seeded onto 40-mm round glass coverslips at a density of 5 x 104 cells/coverslip. Twenty-four hours later, cells were transfected with the different GFP-PKC{delta} constructs using Lipofectamine Plus reagent according to the manufacturer’s instructions. All experiments were performed 48 h posttransfection.

Measurements of cell apoptosis. Cell apoptosis was measured using propidium iodide (PI) staining and analysis by flow cytometry and by ELISA (Cell Death Detection ELISA Kit) using anti-histone antibodies. Cells (1 x 106/ml) were plated in six-well plates and treated with the indicated treatments for 24 h. Detached cells and trypsinized adherent cells were pooled, fixed in 70% ethanol for 1 h on ice, washed with phosphate-buffered saline (PBS), and treated for 15 min with RNase (50 µM) at room temperature. Cells were then stained with PI (5 µg/ml) and analyzed on a Becton-Dickinson cell sorter.

For anti-histone ELISA (Cell Death Detection ELISA kit), extracts of cells containing histone-associated DNA fragments were incubated in 96-well plates coated with anti-histone antibodies for 2 h. Plates were then washed and incubated with anti-DNA antibodies conjugated to peroxidase for an additional 2 h. Substrate solution was added and absorbance was measured at 405 nm.

Cell viability was also quantitatively assessed by the measurement of lactate dehydrogenase (LDH) in the medium.

Measurement of caspase 3 activity. Caspase 3 activity was measured using the caspase 3 colorimetric assay kit obtained from BIOMOL by using Ac-DEVD-pNA as a substrate according to the manufacturer’s recommendations.

Nuclear and cytosolic fractionation. Nuclear proteins were prepared according to the method described by Haglund and Rothblum (24). Cells (1 x 106 to 5 x 106/ml) were washed once with 1 ml of PBS and once with 1 ml of lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM PMSF, 10 µg of leupeptin/ml, pH 7.9). Cells were lysed by suspending the cell pellet in 20 µl of lysis buffer containing 0.1% Nonidet P-40 (NP-40) for 10 min on ice. To isolate nuclei, the lysates were microcentrifuged for 5 min at 12,000 x g, and the nuclear pellet was washed with lysis buffer without NP-40. Nuclear proteins were obtained by resuspending the nuclear pellet in 20 µl of extraction buffer (420 mM NaCl, 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol, pH 7.9) for 10 min at 4°C. The nuclear suspension was microcentrifuged, the pellet was discarded, and the supernatant was diluted in dilution buffer (50 mM KCl, 20 mM HEPES, 0.2 mM EDTA, and 20% glycerol, pH 7.9). Lack of contamination of the nuclear fraction by the plasma membrane was confirmed using the plasma membrane marker Na-K ATPase.

Preparation of cell homogenates. Cells were washed and resuspended in serum-free medium. The plates were placed on ice, scraped with a rubber policeman, and centrifuged at 1,400 rpm for 10 min. The supernatants were aspirated, and the cell pellets were resuspended in 100 µl of lysis buffer (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5% Na deoxycholate, 2% NP-40, 0.2% sodium dodecyl sulfate [SDS], 1 mM PMSF, 50 µg of aprotinin/ml, 50 µM leupeptin, 0.5 mM Na3VO4) on ice for 15 min. The cell lysates were centrifuged for 15 min at 14,000 rpm in an Eppendorf microcentrifuge, supernatants were removed, and 2x sample buffer was added.

Immunoblot analysis. Lysates (40 µg of protein) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) and were transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS and subsequently stained with the primary antibody. Specific reactive bands were detected using a goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, Calif.), and the immunoreactive bands were visualized by the ECL Western blotting detection kit (Amersham, Arlington Heights, Ill.).

Immunoprecipitation. Immunoprecipitation was performed as previously described (5). Briefly, C6 cells overexpressing PKC{delta} or PKC{delta}5 were serum starved overnight and treated for different periods of time with PMA (10 nM) or PDGF (100 ng/ml). The samples were preabsorbed with 25 µl of protein A/G-Sepharose (50%) for 10 min, and immunoprecipitation was performed using 4 µg of antibody/ml for 1 h at 4°C and then incubated with 30 µl of protein A/G-Sepharose for an additional hour. Following washes, the pellets were resuspended in 25 µl of SDS sample buffer and boiled for 5 min. The entire supernatants were subjected to Western blotting. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were washed and visualized by the enhanced chemiluminescence (ECL) system.

Immunofluorescence staining. Cells were grown on glass coverslips. Following etoposide treatment (3 to 24 h), cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min. Subsequently, cells were washed in PBS, and after blocking with staining buffer (2% bovine serum albumin [BSA] and 0.1% Triton X-100 in PBS) for 30 min at room temperature, cells were incubated with a mouse (regulatory domain) or a rabbit (catalytic domain) anti-PKC antibody. Following washes in PBS, cells were incubated with an anti-rabbit antibody or with Alexa FluorTM 546 goat anti-mouse IgG for an additional 60 min and were mounted in FluoroGuard antifade reagent. Cells were viewed and photographed using confocal microscopy with x63 magnification at excitation wavelengths of 543 and 488 nm.

For the translocation of GFP-PKC{delta} and GFP-PKC{delta}5, cells were treated for 6 and 24 h with etoposide, were fixed in 4% PFA for 10 min, and were mounted in FluoroGuard antifade reagent.

Statistical analysis. The results are presented as the mean values ± standard error (SE). Data were analyzed using analysis of variance (ANOVA) and a paired Student’s t test to determine the level of significance between the different groups.


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RESULTS
 
Etoposide induces apoptosis and cell cycle arrest in C6 glioma cells. Treatment of the cells with etoposide arrested the cells in the G1/S phase of the cell cycle and induced cell apoptosis (Fig. 1A). Using PI staining and fluorescence-activated cell sorter (FACS) analysis, we found that approximately 22% ± 3.9% of the cells underwent apoptosis in response to etoposide after 24 h of treatment, whereas approximately 63% ± 7.1% of the cells were apoptotic after 48 h. Similar results were obtained using anti-histone ELISA (Fig. 1B) and using measurements of LDH levels in the cell supernatants (data not shown).



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  		FIG. 1. Etoposide induces apoptosis in C6 glioma cells. C6 cells were treated with etoposide (50 µM) for 24 or 48 h. Cell apoptosis was determined using PI staining and FACS analysis (A) or anti-histone ELISA (B). The results are from one representative experiment out of six similar experiments. (A) The optical densities of etoposide-treated cells (48 h) were designated 100% (total apoptosis), and all other values are presented as percent of this total. (B) The results represent the means ± SE of triplicate measurements in each of three experiments. (C) The morphology of the cells (48 h) was monitored under a phase contrast light microscope. The results are representative of four similar experiments.

Treatment of the cells with etoposide also resulted in lower cell number and in the appearance of rounded and detached cells, which are characteristic of apoptotic cells (Fig. 1C).

Role of PKC{delta} in apoptotic effect of etoposide. Recent studies suggested that PKC{delta} plays a role in the apoptotic effect of etoposide in salivary gland acinar cells (49). To explore the role of PKC{delta} in the effect of etoposide on C6 cells, we utilized rottlerin, which has been reported to be a PKC{delta} selective inhibitor (22). Treatment of C6 cells with rottlerin (5 µM) did not affect the basal level of cell apoptosis; however, rottlerin inhibited the apoptotic effect of etoposide (Fig. 2A), reducing cell apoptosis by approximately 50%. Higher concentrations of rottlerin itself induced morphologic changes in the cells and some cell apoptosis and therefore could not be used.



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  		FIG. 2. Effects of rottlerin, PKC{delta} DN, and PKC{delta} overexpression on the apoptosis of C6 cells induced by etoposide. C6 cells were treated with etoposide (50 µM) in the absence and presence of rottlerin (5 µM) for 48 h (A) or cells overexpressing control vector (CV), PKC{delta} DN (B), or PKC{delta} (C) were treated with etoposide. Cell apoptosis was determined using anti-histone ELISA (A and B) or PI staining and FACS analysis (C). The optical densities of etoposide-treated cells (A) or of etoposide-treated CV cells (B) were designated 100% (total apoptosis), and all other values are presented as percent of this total. (A and B) The results represent the means ± SE of triplicate measurements in each of three experiments. (C) Distributions are from a representative experiment. *, P < 0.001, compared to control cells.

Since the in vitro inhibitory effect of rottlerin on PKC{delta} activity has recently been subject to controversy (12, 22, 30), we further explored the role of PKC{delta} in the apoptotic effect of etoposide by employing cells overexpressing a PKC{delta} dominant-negative mutant (K376R) (36). Cells stably expressing the PKC{delta} DN mutant were treated with ZnCl2 for 24 h (to induce overexpression through its MTH promoter), followed by a 48-h treatment with etoposide (50 µM). Overexpression of PKC{delta} DN did not affect the basal level of C6 cell apoptosis; however, it reduced the apoptosis induced by etoposide by 40% (Fig. 2B).

The role of PKC{delta} in the apoptotic effect of etoposide was also demonstrated using cells stably overexpressing PKC{delta}. The expression of PKC{delta} in these cells was seven- to eightfold higher than control vector cells (35). These cells exhibited an increased rate of apoptosis in response to etoposide (45%) compared to cells expressing the control vector (20%; Fig. 2C).

Etoposide induces nuclear translocation of PKC{delta}. Activation of PKC by different stimuli results in distinct patterns of translocation, which are cell and stimulus dependent. To examine the effect of etoposide on the translocation of PKC{delta}, we treated C6 cells with etoposide for various periods of time and followed the expression of PKC{delta} using immunofluorescence and confocal microscopy. In control cells, PKC{delta} was found largely in the cytosol, but with some expression in the nucleus (Fig. 3). Treatment with etoposide induced further translocation of PKC{delta} to the nucleus. Translocation was observed already after 3 h, and the expression of PKC{delta} in the nucleus was observed up to 24 h following treatment. We confirmed that etoposide did not induce translocation of PKC{delta} to the Golgi, endoplasmic reticulum, or the mitochondria, as determined by costaining with these organelle-specific markers (data not shown).



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  		FIG. 3. Translocation of PKC{delta} in C6 cells treated with etoposide. C6 cells were treated with etoposide (50 µM) for 0, 3, 6, and 24 h, and the translocation of PKC{delta} was assessed using immunofluorescence staining. Following fixation with 4% PFA, cells were incubated with a rabbit anti-PKC{delta} antibody for 1 h and with an anti-rabbit antibody conjugated to fluorescein isothiocyanate. Cells were visualized by confocal microscopy. The results are from one representative experiment out of five similar experiments.

No translocation of PKC{alpha}, -ß, -{zeta}, -µ, and -{iota} was observed in etoposide-treated cells, whereas some translocation of PKC{varepsilon} to the perinuclear membrane was observed (data not shown).

Cleavage of PKC{delta} by etoposide. PKC{delta} undergoes cleavage in response to various apoptotic stimuli (21, 49). To examine the effect of etoposide on the cleavage of PKC{delta} in our system, we treated C6 cells with etoposide (50 µM) for various periods of time and analyzed cell lysates by using Western blotting. The level of PKC{delta} decreased following 24 h of etoposide treatment, and a 40-kDa cleavage product of PKC{delta} appeared and started to accumulate (Fig. 4A). No significant cleavage of PKC{alpha}, -ß, -{varepsilon}, -{zeta}, and -µ was observed in response to etoposide, and no cleavage products were detected (data not shown).



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  		FIG. 4. Nuclear cleavage and translocation of PKC{delta} in etoposide-treated cells. C6 cells were treated with etoposide (50 µM) for 24 h, and the cleavage of PKC{delta} was determined by Western blotting using an anti-PKC{delta} antibody (rabbit; Santa Cruz). (A) A 40-kDa cleaved form is marked by an arrow. For the detection of PKC{delta} in the nucleus, extracts from control and etoposide-treated C6 cells (24 h) were fractionated and the nuclear and cytoplasmic fractions (50 µg/ml) were examined for the presence of PKC{delta} and for its cleaved form by using Western blot analysis. (B) The extracts were also examined for the expression of the nuclear marker lamin B. (C) The nuclear translocation of PKC{delta} in C6 cells treated with etoposide for 6 h was monitored by immunofluorescence using anti-PKC{delta} antibody directed against the catalytic domain (fluorescein isothiocyanate) or against the regulatory domain (Alexa FluorTM 546 goat anti-mouse IgG). The results shown represent one of three separate experiments, which yielded similar results.

Since the nuclear translocation of PKC{delta} preceded its caspase-dependent cleavage, we examined if PKC{delta} cleavage occurred in the nucleus. We first performed cell fractionation and separated the nuclear and cytosolic fractions. We found that in untreated cells, PKC{delta} was mainly expressed in the cytosol with some expression in the nucleus. Following etoposide treatment, PKC{delta} translocated to the nucleus and a cleaved form of PKC{delta} accumulated only in the nuclear fraction (Fig. 4B). The nuclear marker lamin B was also detected only in the nuclear fraction. Using anti-PKC{delta} antibodies directed against the regulatory and catalytic domains, we found that the immunostaining of PKC{delta} using these two antibodies resulted in a similar pattern of nuclear translocation (Fig. 4C). The results of these two experiments suggest that PKC{delta} translocated to the nucleus, where it underwent cleavage. Since some PKC{delta} was present in the nucleus of the untreated cells without being cleaved, the cleavage must depend on the etoposide treatment itself as well as on its location.

Caspase 3 is involved in cleavage of PKC{delta} and apoptosis is induced by etoposide. PKC{delta} can be cleaved by a caspase-dependent process (21, 34). We therefore examined the effects of the cell-permeable caspase 3 inhibitor DEVD.FMK (20 µM) on the cleavage of PKC{delta} in the C6 cells. As seen in Fig. 5A, pretreatment of the cells for 1 h with DEVD.FMK significantly reduced the accumulation of the PKC{delta} cleavage product in response to etoposide. Similar results were obtained with another caspase inhibitor, Z-VAD.FMK (data not shown).



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  		FIG. 5. Role of caspase 3 in cleavage of PKC{delta} and C6 cell apoptosis induced by etoposide. C6 cells were treated with etoposide for 24 h in the absence and presence of DEVD (20 µM) and Z-VAD (25 µM). The cleavage of PKC{delta} was determined using Western blot analysis (A), and cell apoptosis was determined using anti-histone ELISA (B). The results are representative of four similar experiments (A) or represent the means ± SE of three separate experiments (B). The expression of active caspase 3 in the nucleus of etoposide-treated cells (24 h) was determined using immunofluorescence staining (C) and Western blot analysis of cytosolic (C) and nuclear (N) fractions (D). *, P < 0.001

We also examined the ability of the caspase inhibitors to block the apoptosis induced by etoposide in C6 cells. Pretreatment of the cells with either DEVD.FMK or Z-VAD.FMK reduced the apoptotic effect of etoposide by approximately 60% (Fig. 5B), suggesting a role for caspase 3 in the apoptotic response. In contrast, the caspase 1 inhibitor YVAD did not affect etoposide effects in these systems (data not shown).

Since the cleavage of PKC{delta} occurred in the nucleus, we examined whether active caspase 3 was also expressed in the nucleus in etoposide-treated cells. Using immunofluorescence staining, we found that the active form of caspase 3 was expressed in the nucleus of etoposide-treated cells, whereas a very low expression of active caspase 3 was detected in control cells (Fig. 5C). Similar results were obtained using Western blot analysis of nuclear and cytosolic fractions (Fig. 5D).

Inhibition of PKC{delta} blocks cleavage of caspase 3 and PKC{delta}. To examine the importance of PKC{delta} activity in its ability to undergo cleavage, we employed the selective PKC{delta} inhibitor rottlerin. Pretreatment of the cells with rottlerin (5 µM) reduced the cleavage of caspase 3 in response to etoposide (Fig. 6A). Similarly, cells overexpressing PKC{delta} DN exhibited a significantly lower caspase 3 activity than control vector cells (Fig. 6B). Finally, the cleavage of PKC{delta} and the accumulation of the PKC{delta} catalytic domain were reduced in rottlerin-treated cells (Fig. 6C), suggesting that the cleavage of both caspase 3 and PKC{delta} is dependent on an active PKC{delta}.



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  		FIG. 6. The PKC{delta} inhibitor rottlerin and PKC{delta} DN decrease the activation of caspase 3 and the cleavage of PKC{delta} in etoposide-treated cells. C6 cells were treated with etoposide (50 µM) in the absence or presence of rottlerin (5 µM) for 24 h. The cleavage of caspase 3 (A) and of PKC{delta} (C) was detected using Western blot analysis. The membranes were probed with antibodies against active caspase 3 (17 kDa) (A) or PKC{delta} (C). The results represent one of three separate experiments which yielded similar results. (B) Cells overexpressing PKC{delta} DN were treated with etoposide for 24 h, and the activity of caspase 3 was measured using the Caspase 3 Cellular Activity Assay Kit as described in Materials and Methods. Caspase 3 activity was calculated and expressed as picomoles/minute/microgram of protein, and the percent of maximal effect was determined. The results represent the means ± SE of four experiments. *, P < 0.001

Both regulatory and catalytic domains of PKC{delta} are required for apoptotic effect induced by etoposide. In a recent study, we demonstrated that the regulatory domain of PKC{delta} mediated its inhibitory effect on C6 cell proliferation and on the expression of the astrocytic marker GS (5). The regulatory domain of PKC{delta} was phosphorylated on tyrosines 155 and 187 in response to PMA and PDGF, respectively (5, 35), and the translocation of the PKC{delta}/{alpha} chimera resembled that of PKC{delta} (40). To examine the relative contributions of the regulatory and catalytic domains of PKC{delta} to its apoptotic effects, we used chimeras between the regulatory and catalytic domains of PKC{alpha} and -{delta} combined at the highly conserved hinge region. The expression and activity of C6 cells overexpressing the different chimeras were already described (5).

Cells were pretreated for 24 h with ZnCl2, followed by etoposide treatment (50 µM) for an additional 48 h. Cells overexpressing PKC{delta}/{delta} exhibited levels of apoptosis similar to those of control vector-transfected cells. Treatment of the control vector cells with etoposide induced about 60% apoptosis, similar to the results obtained with the parental C6 cells, whereas cells overexpressing PKC{delta} exhibited increased levels of apoptosis. Cells overexpressing PKC{alpha}/{alpha} exhibited a rate of cell apoptosis similar to that of the control vector cells. Interestingly, cells overexpressing the chimera containing the regulatory domain of PKC{delta} ({delta}/{alpha}) or the chimera containing the catalytic domain of PKC{delta} ({alpha}/{delta}) exhibited a similar level of apoptosis to that of control vector cells and did not resemble cells overexpressing PKC{delta} (Fig. 7). These results suggest that both domains are required for the apoptotic effect of PKC{delta}.



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  		FIG. 7. Apoptosis of C6 cells overexpressing different PKC chimeras in response to etoposide. C6 cells overexpressing PKC{alpha}, PKC{delta}, and the chimeras PKC{alpha}/{delta} and PKC{delta}/{alpha} were treated with etoposide for 48 h. Cell apoptosis was determined using anti-histone ELISA. The optical densities of etoposide-treated PKC{delta}-overexpressing cells were designated 100% (total apoptosis), and all other values are presented as a percent of this total. The results represent the means ± SE of triplicate measurements in each of three experiments.

Etoposide induces tyrosine phosphorylation of PKC{delta}. PKC{delta} has been shown to undergo tyrosine phosphorylation in response to PMA, growth factors, and apoptotic stimuli, such as H2O2 and {gamma}-irradiation (5, 35, 58). To examine the effect of etoposide on tyrosine phosphorylation of PKC{delta}, we treated C6 cells with etoposide for 15 to 180 min. PKC{delta} was immunoprecipitated, and the membrane was blotted with anti-phosphotyrosine antibody. As illustrated in Fig. 8A, a low basal level of tyrosine phosphorylation was observed in untreated cells. Etoposide induced tyrosine phosphorylation of PKC{delta} in a time-dependent manner. Initial phosphorylation was observed after 45 min (data not shown), maximal phosphorylation was obtained after 60 min and was decreased thereafter (Fig. 8A). Tyrosine phosphorylation of PKC{delta} in response to etoposide was lower than that induced by PMA and exhibited slower kinetics (5, 35). The tyrosine phosphorylation induced by etoposide was specific for PKC{delta}. We did not observe tyrosine phosphorylation of any of the other isoforms (data not shown).



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  		FIG. 8. Etoposide induces tyrosine phosphorylation of PKC{delta} in the regulatory domain. Parental C6 cells or cells stably transfected with PKC{delta} or the chimeras PKC{delta}/{alpha} and PKC{alpha}/{delta} were treated with etoposide (50 µM) for various periods of time. Cells were then harvested, and immunoblotting (IB) and immunoprecipitation (IP) of PKC{delta} were performed using anti-PKC{delta} (A) or anti-PKC{varepsilon} (B) antibodies as described in Materials and Methods. Following SDS-PAGE, membranes were stained with anti-phosphotyrosine antibody (anti-PY) or with anti-PKC{delta} antibodies. The results are from one representative experiment out of four separate experiments.

We next examined whether the tyrosine phosphorylation in response to etoposide occurred in the regulatory or catalytic domains. For these experiments, we used the PKC chimeras {alpha}/{delta} and {delta}/{alpha}. Treatment of the chimeras with etoposide for 60 min resulted in the phosphorylation of PKC{delta}/{delta} and of the chimera containing the regulatory domain of PKC{delta} (PKC{delta}/{alpha}). The chimera containing the catalytic domain of PKC{delta} did not exhibit an increase in tyrosine phosphorylation in response to etoposide (Fig. 8B), suggesting that the tyrosine phosphorylation occurred in the regulatory domain.

Tyrosine phosphorylation of PKC{delta} is essential for its apoptotic effect. In previous studies, we identified five putative tyrosine phosphorylation sites in PKC{delta} and generated a PKC{delta}5 mutant, in which these five tyrosine phosphorylation sites were mutated to phenylalanine (5). The expression and activity of C6 cells overexpressing this mutant were already described (5). Overexpression of the PKC{delta}5 mutant in C6 cells abolished the decrease in the expression of the astrocytic marker GS induced by PMA or PDGF and the decrease in cell proliferation induced by PMA. Expression of the PKC{delta}5 mutant also resulted in a lower level of tyrosine phosphorylation of PKC{delta} in response to these treatments (5). Using cells expressing PKC{delta}5, we found that etoposide did not induce a significant increase in tyrosine phosphorylation of PKC{delta}5 (Fig. 9A). Similarly, treatment of these cells with etoposide resulted in low levels of apoptosis similar to the response observed in the control vector cells when measured after 24 h (Fig. 9B) and in lower levels of apoptosis than control vector cells after 48 h of treatment (Fig. 9C). Thus, the tyrosine phosphorylation of PKC{delta} in the regulatory domain induced by etoposide was essential for the apoptotic effect of PKC{delta} in response to this drug.



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  		FIG. 9. Tyrosine phosphorylation and cell apoptosis in response to etoposide in cells overexpressing PKC{delta} and PKC{delta}5. C6 cells overexpressing control vector (CV), PKC{delta}, or PKC{delta}5 were treated with etoposide (50 µM) for 60 min (A), 24 h (B), or 48 h (C). (A) Cells were then harvested, and immunoprecipitation of PKC{delta} was performed using anti-PKC{varepsilon} antibody as described in Materials and Methods. Following SDS-PAGE, membranes were stained with anti-phosphotyrosine antibody (anti-PY) or with an anti-PKC{delta} antibody (rabbit; Santa Cruz). The results represent one of three separate experiments, which yielded similar results. For the measurement of apoptosis, cells were harvested after 24 h (B) or 48 h (C) of treatment and were analyzed using PI staining and FACS analysis (B) or by anti-histone ELISA (C). The optical densities of etoposide-treated PKC{delta}-overexpressing cells were designated 100% (total apoptosis), and all other values are presented as a percent of this total. The results represent the means ± SE of triplicate measurements in each of three experiments. *, P < 0.001.

Cleavage of caspase 3 and PKC{delta} in cells overexpressing PKC{delta}5 mutant. To further explore the role of tyrosine phosphorylation of PKC{delta} in the apoptosis induced by etoposide, we compared the activation of caspase 3 in cells overexpressing control vector, PKC{delta}, and the PKC{delta}5 mutant. Using a specific antibody recognizing the cleaved product (17 kDa) of caspase 3, we found that etoposide induced caspase 3 cleavage in the control vector cells. Cells overexpressing PKC{delta} showed a larger amount of the cleaved product, whereas cells overexpressing the PKC{delta}5 mutant exhibited very low levels of the 17-kDa fragment (Fig. 10A). Similar results were obtained for caspase 3 activity as measured by a caspase 3 colorimetric assay (Fig. 10B). These results suggest that the activation of caspase 3 required a tyrosine phosphorylated form of PKC{delta}.



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  		FIG. 10. Activation of caspase 3 and cleavage of PKC{delta} in cells overexpressing PKC{delta} and PKC{delta}5. C6 cells overexpressing control vector (CV), PKC{delta}, or PKC{delta}5 were treated with etoposide (50 µM) for 24 h, and the activation of caspase 3 (A and B) and cleavage of PKC{delta} (C) were determined. Cells were harvested and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with active caspase 3 antibody (A) or with anti-PKC{varepsilon} that recognizes the {varepsilon} tag which is located in the catalytic domain of the PKC{delta} constructs (C). The results represent one of three separate experiments which yielded similar results. The activity of caspase 3 was measured using the Caspase 3 Cellular Activity Assay Kit as described in Materials and Methods (B). Caspase 3 activity was calculated and expressed as picomoles/minute/microgram of protein, and the percent of maximal effect was determined. The results represent the means ± SE of three experiments.

We then examined the cleavage of PKC{delta} and PKC{delta}5 in response to etoposide. Using the {varepsilon} tag, we were able to detect the cleaved catalytic domain of the exogenous PKC{delta} and PKC{delta}5. Using the anti-PKC{varepsilon} antibody, we found no detectable cleaved product in the etoposide-treated control vector cells. Accumulation of the catalytic fragment was observed in cells overexpressing PKC{delta}, whereas cells overexpressing the PKC{delta}5 mutant displayed no detectable cleavage (Fig. 10C). Since the kinetics of PKC{delta} phosphorylation is faster than the cleavage and is transient, the phosphorylation presumably plays a role upstream from the actual cleavage.

Translocation of PKC{delta} and the PKC{delta} 5 mutant in response to etoposide. One possible explanation for the differential effects of PKC{delta} and the PKC{delta}5 mutant on cell apoptosis is their differential translocation following etoposide treatment. We therefore examined the translocation of PKC{delta} and the PKC{delta}5 mutant in response to etoposide. For these experiments, we used GFP-tagged PKC{delta} wild type or the PKC{delta}5 mutant. Cells were transiently transfected with the specific construct, and the response of the cells to etoposide was monitored after 6 and 24 h.

We found that etoposide induced a similar pattern of translocation for both PKC{delta} and PKC{delta}5 (Fig. 11). Thus, stimulation of the cells with etoposide for 6 h induced translocation of PKC{delta}-GFP to the nucleus in about 90 to 95% of the cells, similar to the results obtained using immunofluorescent staining of the endogenous PKC{delta}. Likewise, PKC{delta}5-GFP translocated to the nucleus in response to etoposide in about 90% of the cells. A similar pattern of nuclear translocation of PKC{delta} and PKC{delta}5 was also obtained after 24 h of etoposide treatment (data not shown).



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  		FIG. 11. Translocation of PKC{delta} and PKC{delta}5 in etoposide-treated C6 cells. Cells were transiently transfected with GFP-PKC{delta} or GFP-PKC{delta}5. After 48 h, cells were treated with etoposide (50 µM) for 6 h and cells were viewed using confocal microscopy. Cells shown are representative of four independent experiments

Role of single tyrosine mutants in apoptotic response of etoposide. To identify the specific tyrosines that are involved in the inhibitory effect of PKC{delta} on cell apoptosis, we examined the role of the single tyrosines 52, 64, 155, 187, and 565. C6 cells were stably transfected with the different PKC{delta} mutants in which each one of these tyrosines was individually mutated to phenylalanine. The expression and activity of C6 cells overexpressing the different PKC mutants were previously described (35).

The apoptotic effect of etoposide was examined in cells overexpressing the different mutants using PI staining and FACS analysis. We found that cells overexpressing PKC{delta}Y52F, PKC{delta}Y155F, and PKC{delta}Y565F exhibited an enhanced apoptotic response to etoposide similar to that of cells overexpressing PKC{delta}. In contrast, treatment with etoposide of cells overexpressing PKC{delta}Y64F or PKC{delta}Y187F resulted in a lower apoptotic response, similar to the response observed with cells overexpressing the PKC{delta}5 mutant (Fig. 12A). Similar results were obtained with anti-histone ELISA (Fig. 12B).



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  		FIG. 12. Apoptosis and tyrosine phosphorylation in C6 cells overexpressing different PKC{delta} tyrosine mutants. C6 cells overexpressing PKC{delta} or the PKC{delta} mutants were plated in the absence and presence of etoposide (50 µM). (A) Cell apoptosis was measured by PI staining and FACS analysis after 24 h. The results are from one representative experiment out of four separate experiments. (B) Cell apoptosis was also determined after 48 h using anti-histone ELISA. The optical densities of etoposide-treated PKC{delta}-overexpressing cells were designated 100% (total apoptosis), and all other values are presented as percent of this total. The results represent the means ± SE of triplicate measurements in each of five experiments. Tyrosine phosphorylation of PKC{delta}, PKC{delta}5, PKC{delta}Y64F, and PKC{delta}Y187F was determined following 1 h of etoposide treatment. (C) Cells were harvested, and immunoprecipitation of PKC{delta} was performed using anti-PKC{varepsilon} antibody as described in Materials and Methods. Following SDS-PAGE, membranes were stained with anti-phosphotyrosine antibody (anti-PY) or with an anti-PKC{delta} antibody. The results represent one of three separate experiments which yielded similar results.

We also found that etoposide did not induce a significant increase in the tyrosine phosphorylation of PKC{delta}Y64F and PKC{delta}Y187F, similar to the results obtained with PKC{delta}5 (Fig. 12C). Finally, we found no detectable cleaved product of PKC{delta} in the etoposide-treated PKC{delta}Y64F and PKC{delta}Y187F overexpressors (data not shown).

Apoptosis induced by etoposide is not inhibited by Src-kinase inhibitors PP1 and PP2. In a previous study, we showed that Fyn and Lyn can associate with PKC{delta} and that Fyn associates with PKC{delta} via tyrosine 187 (35). To examine the role of Src-related kinases in the apoptotic response of etoposide, we employed the Src-kinase inhibitors PP1 and PP2. Pretreatment of the cells with either PP1 (10 µM) or PP2 (10 µM) did not affect the apoptotic response of etoposide (data not shown), suggesting that Src-related kinases are probably not involved in the tyrosine phosphorylation of PKC{delta} and in the apoptotic response of etoposide.


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DISCUSSION
 
In this study, we explored the importance of PKC{delta} and its tyrosine phosphorylation for the induction of apoptosis in C6 glioma cells upon treatment with etoposide. We found that etoposide induced apoptosis of C6 cells, that this response was partially inhibited by the PKC{delta} inhibitor rottlerin and PKC{delta} DN, and that overexpression of PKC{delta} enhanced the apoptotic effect of etoposide. Etoposide, an inhibitor of topoisomerase II, has been reported to induce apoptosis in a broad range of different cells (29), and PKC{delta} has previously been implicated in the induction of apoptosis (34, 49). Thus, our results demonstrate that C6 glioma cells exhibit a response to etoposide similar to those of other tumor cells.

Etoposide induced translocation of PKC{delta} to the nucleus within 3 h of treatment. Various PKC isoforms can be found in the nucleus and in subnuclear compartments (7), and PKC isoforms can undergo nuclear translocation in response to various stimuli, including phorbol esters (55), growth factors (39, 43), and apoptotic stimuli, such as {gamma}-irradiation (58). In addition, increases in the levels of nuclear PKC isoforms were reported in the context of cell proliferation and differentiation (16, 31), suggesting a role of nuclear PKC in these processes. It is presently unclear how PKC{delta} is transported to the nucleus since PKC isoforms do not contain any known nuclear localization signal, but PKC-binding proteins probably play a role in this process (7). Indeed, in a recent study, it was reported that PKC{delta} associates with c-Abl via its SH3 domain and undergoes tyrosine phosphorylation and nuclear translocation in complex with c-Abl in response to {gamma}-irradiation (58).

In some systems, the apoptotic effect of PKC{delta} has been associated with a cleavage of the catalytic domain from the regulatory domain (34, 49). Cleavage of the catalytic domain of PKC{delta} by caspases has been reported for cells treated with ionizing radiation, tumor necrosis factor alpha, and etoposide (34, 38, 49). We found that etoposide induced cleavage of PKC{delta} in C6 cells and that this cleavage occurred in the nucleus. The absolute extent of cleavage was somewhat lower than that reported for parotid cells (49), presumably reflecting differences between these cell types, including a lower sensitivity of the C6 cells to etoposide. Both the cleavage of PKC{delta} by etoposide and its apoptotic effect were partially inhibited by the caspase inhibitor DEVD.FMK, suggesting a role for caspase 3 in these effects, as was reported for other systems (21, 49). Similarly, we found that the inhibition of PKC{delta} blocked the cleavage of caspase 3 and the cleavage of PKC{delta} was induced by etoposide. These results suggest the presence of a positive loop between PKC{delta} and caspase 3, which is dependent on PKC{delta} activity. Consistent with these results, recent studies demonstrated that caspases, including caspase 3, translocate and act in the nucleus following apoptotic stimuli (15). The partial inhibition of the caspase inhibitors on the apoptotic effect of etoposide was reported for other systems as well (44), and it is probably due to the presence of additional, non-caspase-dependent mechanisms involved in the effects of etoposide (50).

Both the regulatory and catalytic domains of PKC{delta} were important for the apoptotic effects of PKC{delta} in response to etoposide. These results are different from those of our previous studies, in which the regulatory domain of PKC{delta} was responsible for the effects of this isoform on C6 cell proliferation and on GS expression (5, 35). In various studies, it was suggested that, once cleaved, the catalytic domain of PKC{delta} mediates the apoptotic effect of this isoform since overexpression of the catalytic fragment alone was able to induce apoptosis (21). Taken together, our results suggest that the regulatory domain contains signals that are important for the cleavage and activation of the catalytic domain.

Etoposide induced tyrosine phosphorylation of PKC{delta} in the regulatory domain. Tyrosine phosphorylation of PKC{delta} has been reported in the response to PMA and PDGF in C6 cells (5), in EGF-stimulated keratinocytes (13), in response to activation of the IgE receptor in RBL-2H3 cells (25, 54), and in response to apoptotic stimuli, such as {gamma}-irradiation (58) and H2O2 (32). Phosphorylation of tyrosine residues occurs in either the regulatory or catalytic domains of PKC{delta}. Thus, PDGF and PMA induced tyrosine phosphorylation on tyrosines 187 and 155, respectively (35). Similarly, activation of the IgE receptor induced tyrosine phosphorylation on tyrosine 52 (54). In contrast, stimulation of the cells with H2O2 and {gamma}-irradiation induced phosphorylation on tyrosines in the catalytic domain and in the hinge region (32, 33).

The tyrosine phosphorylation of PKC{delta} by etoposide appeared to be essential for the apoptosis induced by PKC{delta}, since the increase in cell apoptosis obtained in cells overexpressing PKC{delta} was absent in cells overexpressing the PKC{delta}5 mutant. These results are similar to our recent findings, which showed a role for tyrosine phosphorylation of PKC{delta} in the inhibitory effect of this isoform on the expression of the astrocytic marker GS (5) and on C6 cell proliferation (35). Thus, the PKC{delta}5 mutant also appears to act in an opposite way to PKC{delta} in its effect on cell apoptosis.

We found that caspase 3 was cleaved in response to etoposide and that this cleavage was enhanced in cells overexpressing PKC{delta} but inhibited in cells overexpressing the PKC{delta}5 mutant. The mechanisms involved in the enhanced cleavage of caspase 3 in response to overexpression of PKC{delta} are not known. PKC{delta} could act directly on caspase 3 to induce its phosphorylation or act indirectly via the phosphorylation of upstream caspases, such as caspase 8 or caspase 9 or other unknown upstream proteins. Indeed, a study by Martins et al. (41) reported that etoposide induced phosphorylation of caspases in HL-60 cells. Similarly, caspase 9, which is upstream of caspase 3, has been shown to be phosphorylated and directly regulated by phosphorylation (8, 20). Whatever the mechanism is for the effects of PKC{delta}, our data suggest that tyrosine phosphorylation of PKC{delta} is essential for the ability of this isoform to cause activation of caspase 3. In accordance with our data and with other studies suggesting that the cleavage of PKC{delta} is mediated by caspase 3 (21, 49), we found that the cleavage of the PKC{delta}5 mutant in response to etoposide was also inhibited. The difference in the ability of the PKC{delta}5 mutant to markedly inhibit etoposide-induced caspase-dependent cleavage and caspase 3 activity compared to its partial inhibition of etoposide-induced cell apoptosis suggests that tyrosine phosphorylation of PKC{delta} mediates the caspase-dependent component of the apoptosis induced by etoposide, whereas it is not involved in the caspase-independent pathways.

The effects of tyrosine phosphorylation on the activity of PKC{delta} or on its function are complex and dependent on the specific system and stimulus. Tyrosine phosphorylation of PKC{delta} in various systems has been reported to reduce or increase PKC{delta} activity and may do so in a substrate-specific manner (13, 25). Thus, the phosphorylation of PKC{delta} in response to etoposide may act by changing the affinity of PKC{delta} towards specific caspases or upstream proteins.

One of the factors that could provide the basis for the different effects of PKC{delta} and PKC{delta}5 is a distinct pattern of translocation. Translocation of PKC to specific cellular compartments could lead to different effects due to the phosphorylation of different substrates and to the association of PKC{delta} with specific proteins present in these locations. One determinant of the localization of PKC following its activation is association with receptors for activated C kinases (RACKs) (42). It is presently not clear to what extent tyrosine kinases can act as RACKs and affect the translocation of PKC isoforms. In a recent study, we found that mutations in tyrosine residues did not alter the translocation of PKC{delta} in response to PMA or PDGF (35). In contrast, Ron et al. suggested that Fyn might act as a RACK of PKC{theta} (51). We found that etoposide induced nuclear translocation of both PKC{delta} and PKC{delta}5. Thus, the nuclear translocation of PKC{delta} does not depend on its phosphorylation, and the differential effects of PKC{delta} and PKC{delta}5 on cell apoptosis in response to etoposide do not appear to reflect their different translocation following activation.

PKC{delta} has been reported to associate with different tyrosine kinases. Thus, p60Src (4, 53, 54, 59), Lyn (54), Fyn (35), and c-Abl (58) can associate with PKC{delta} in either a phosphorylation-dependent or -independent manner. In addition, various reports indicate that PKC{delta} can undergo tyrosine phosphorylation on specific tyrosine residues by Src, Fyn, and c-Abl. The ability of PKC{delta} to be tyrosine phosphorylated on more than one tyrosine suggests that PKC{delta} can associate with different tyrosine kinases. In a recent study, we found that PKC{delta} underwent tyrosine phosphorylation on tyrosines 155 and 187 which was associated with the inhibitory effects of PKC{delta} on cell proliferation and the expression of GS, respectively (35). Our present results indicate that phosphorylation of PKC{delta} on tyrosines 64 and 187 is essential for its apoptotic effect. Our results suggest that Src-related kinases which are involved in the tyrosine phosphorylation of PKC{delta} in response to PDGF are not involved in the apoptosis induced by etoposide. The tyrosine kinase which phosphorylates PKC{delta} in etoposide-treated cells remains to be identified.

In summary, we demonstrated that the apoptosis induced by etoposide involves activation of an as-yet-unidentified tyrosine kinase(s) which phosphorylates PKC{delta} in the regulatory domain. PKC{delta} is then translocated to the nucleus where it directly or indirectly activates caspase 3. Caspase 3 in turn cleaves PKC{delta} and releases the {delta} catalytic domain, which can phosphorylate known apoptosis-related proteins, such as lamin B (9) or DNA-PK (3). It is presently not clear whether the effect of the tyrosine-phosphorylated PKC{delta} on the apoptosis in C6 cells is mediated via altered affinity of PKC{delta} towards downstream PKC substrates or via activation of a tyrosine kinase(s) that is associated with PKC{delta}. However, the results of this study further extend our previous findings that tyrosine phosphorylation plays an important role in the regulation of PKC{delta} activity and in determining the nature of its effects.


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ACKNOWLEDGMENTS
 
This work was supported by the James S. McDonnell Foundation 21st Century Scientist Award/Brain Cancer Research (C.B.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Phone: 972-3-5318266. Fax: 972-3-5350234. E-mail: chaya{at}mail.biu.ac.il. Back


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Molecular and Cellular Biology, January 2002, p. 182-195, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.182-195.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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