Infection of Glioma Cells with Sindbis Virus Induces Selective Activation and Tyrosine Phosphorylation of Protein Kinase C δ

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCδ to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCδ inhibitor rottlerin and a PKCδ kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCδ in the apoptosis induced by SVNI we used a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). PKCδ5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCδ. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCδ5 but did not induce cleavage of PKCδ or PKCδ5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCδ exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCδ on specific tyrosines is required for this function.

Sindbis virus (SV) 1 is a single-stranded positive-strand RNA alphavirus that has been used as a model for studying the molecular mechanisms underlying virus-induced apoptosis (1). Infection of cells with SV results in either persistent or lytic infection depending on the cell type and the viral strain (1,2). SV lytic infection induces apoptosis, which can be inhibited by overexpression of anti-apoptotic proteins such as Bcl-2 (3). Similarly, overexpression of anti-apoptotic genes such as bcl-2, beclin, and crmA inhibits mortality of mice following SV infec-tion (4) and converts lytic to persistent infection (3). Because the neurovirulence of the different viral strains correlates with mortality in mice and with the apoptosis of cultured cells, it has been suggested that the apoptosis plays a central role in the pathogenesis of the virus (5). Little is known about the signal transduction pathways involved in the apoptotic effect of SV. In a recent study, SV has been reported to increase the phosphorylation of p38 and Hsp27 (6). In addition, SV induces ceramide formation (7) and activation of double-strand RNA-dependent protein kinase (8) and nuclear factor B (9).
The protein kinase C (PKC) family of serine-threonine kinases plays important roles in signal transduction and in various cellular functions (10 -12). At least 12 isoforms have been described so far showing diversity in their structures and biological functions (13,14). The PKC isoforms are grouped on the basis of their structures and cofactor requirements into three main subclasses, the classical PKCs (␣, ␤1, ␤2, and ␥), the novel PKCs (␦, ⑀, , and ), and the atypical PKCs (PKC and PKC /). The two other members, PKC and PKC, represent a fourth subclass with unique characteristics (15,16). The regulation of PKC activity involves phosphorylation on serine and threonine residues (17,18). In addition, recent studies suggest that phosphorylation on tyrosine residues can modulate the activity and substrate recognition of PKC (19).
The PKCs have been implicated as important regulators of cell apoptosis (20,21). PKC␣, PKC⑀, and PKC have been associated with inhibition of cell apoptosis (22,23), whereas PKC␦, , and have been described as proapoptotic kinases, and their cleavage by caspase 3 has been shown to be important for their action (24,25). Indeed, apoptotic stimuli such as etoposide and ionizing radiation induce the cleavage of these isoforms and the accumulation of active catalytic fragments (25,26). PKC␦ has been reported to play a role in the apoptosis induced by etoposide (27) and in the apoptosis of keratinocytes and LNCaP cells in response to PMA (28,29). Although most studies suggest that PKC␦ is associated with the induction of apoptosis, an anti-apoptotic effect for PKC␦ has been reported for some systems (30).
In a recent study, we reported that etoposide induced apoptosis of C6 cells via activation of PKC␦ and its tyrosine phosphorylation on specific tyrosine residues (31). In the present study, we report that infection of C6 cells with SVNI, a neurovirulent strain of Sindbis virus, also induced selective activation and tyrosine phosphorylation of PKC␦. Furthermore, we found that this phosphorylation was essential for the ability of PKC␦ to protect C6 cells from SVNI-induced apoptosis. Our results emphasize that the action of PKC depends critically on its biological context.

EXPERIMENTAL PROCEDURES
Materials-Monoclonal anti-PKC␦ antibody was obtained from Transduction Laboratories (Lexington, KY), and polyclonal anti-PKC antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor antibodies and the endoplasmic reticulum (ER) marker DiOC 5 (3) were from Molecular Probes (Eugene, OR). An anti-active caspase 3 antibody was obtained from New England Biolabs (Beverly, MA). Cell fragmentation (anti-histone) ELISA and lactate dehydrogenase kits were from Roche Molecular Biochemicals. Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, sodium vanadate, and anti-actin antibody were obtained from Sigma.
Viruses-The variant of Sindbis virus used in this study was described previously (32). Briefly, the neurovirulent strain SVNI was isolated by serial passages of an SV strain in brains of suckling and weanling mice.
Virus Plaque Assay in Vero Cells-Growth of SVNI was quantified on Vero cells by plaque formation as described previously (33). Briefly, a dilution of virus was added to Vero cell monolayers in Petri dishes and incubated at 37°C for 1 h to permit viral absorption. The monolayer was overlaid with 2ϫ minimum Eagle's medium and 2% tragacanth (gum tragacanth grade III; Sigma) containing 2% fetal bovine serum and 2.4% NaHCO 3 . Cultures were incubated for 48 h, and plaques were counted after staining with 0.05% neutral red.
Site-directed Mutagenesis of PKC␦-Site-directed mutagenesis of PKC␦ was performed using the Transformer site-directed mutagenesis kit from CLONTECH (Palo Alto, CA). Conversion of tyrosine residues at sites 52, 64, 155, 187, and 565 into phenylalanine was performed as described previously (34). PKC␦ and the PKC␦ mutants were subcloned into the metallothionein promoter-driven eukaryotic expression vector (⑀MTH).
C6 Glial Cultures and Cell Transfection-C6 cells were grown in medium consisting of Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (50 units/ml), and streptomycin (0.05 mg/ml). The cells were transfected with either the empty vectors or with the PKC␦ and PKC␦ mutant expression vectors using LipofectAMINE PLUS (Invitrogen) as described (34). For the translocation studies, cells were transiently transfected with the PKC␦-GFP fusion protein as described (34). Experiments were carried out routinely on two clones of the transfected cells.
Measurements of Cell Apoptosis-Cell apoptosis was measured by flow cytometry after propidium iodide staining, by Hoechst staining, and by ELISA using anti-histone antibodies. Cells were infected with SVNI for 24 or 36 h. Detached cells and trypsinized adherent cells were The channels corresponding to a sub-G 1 DNA content, reflecting apoptosis, are indicated by the arrows. The results are from one representative experiment of five similar experiments. The morphology of the cells (36 h) was monitored under a phase contrast light microscope (B). The results are representative of four similar experiments. For Hoechst staining cells were stained as described under "Experimental Procedures" and were then visualized under a fluorescent microscope (C). Apoptosis using anti-histone ELISA was performed as described under "Experimental Procedures" (D). The optical density values of SVNI-infected cells (36 h) were designated as 100% (total apoptosis), and the other values were expressed relative to this. The results represent the means Ϯ S.E. of triplicate measurements in each of three experiments. *, p Ͻ 0.001. collected, fixed in 70% ethanol for 1 h on ice, washed with PBS, and treated for 15 min with RNase (200 g/ml) at room temperature. Cells were stained with propidium iodide (5 g/ml) and analyzed on a BD PharMingen cell sorter. For Hoechst staining, cells were fixed with methanol and incubated for 10 min with 1 g/ml Hoechst 33258. Cells were then viewed and counted under UV illumination for the visualization of the Hoechst-stained nuclei.
For anti-histone ELISA, fragmented DNA was extracted from the control and infected cells and was incubated in 96-well plates coated with anti-histone antibodies for 2 h. Plates were incubated with anti-DNA antibodies conjugated to peroxidase for an additional 2 h. Substrate solution was added, and absorbance was measured at 405 nm.
Lysates (30 g of protein) were resolved by SDS-PAGE and were transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in phosphate-buffered saline 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), and the immunoreactive bands were visualized by the ECL Western blotting detection kit (Amersham Biosciences).
Immunoprecipitation-Immunoprecipitation was performed as described previously (34). Briefly, C6 cells were infected with SVNI for different periods of time. The samples were pre-absorbed with 25 l of protein A/G-Sepharose for 10 min, and immunoprecipitation was performed using 4 g/ml of anti-PKC␦ or anti-PKC⑀ antibody for 1 h at 4°C followed by incubation with 30 l of A/G-Sepharose for an additional hour. Following washes, the pellets were resuspended in 25 l of SDS sample buffer and boiled for 5 min. The samples were subjected to Western blotting.
Immunofluorescence Staining-Cells were infected with SVNI for 1-6 h. Cells were then washed with PBS and fixed in 4% paraformaldehyde for 15 min. Subsequently, cells were washed in PBS and, after blocking with staining buffer (2% bovine serum albumin and 0.1% Triton X-100 in PBS) for 30 min at room temperature, were incubated with the specific anti-PKC antibodies followed by incubation with an anti-rabbit Alexa Fluor 488 or with anti-mouse Alexa Fluor 546 antibodies for an additional hour. Cells were mounted using FluoroGuard antifade reagent and were viewed and photographed using confocal microscopy with ϫ63 magnification at excitation wavelengths of 543 and 488 nm. For ER staining, fixed and permeabilized cells were incubated for 30 min with the ER marker, DiOC 5 (3).
Statistical Analysis-The results are presented as the mean values Ϯ S.E. Data were analyzed using analysis of variance and a Student's t test. Sindbis Virus Induces a Selective Translocation of PKC␦-PKC has been implicated as an important regulator of cell apoptosis (20,21). To study the possible involvement of PKC in the apoptosis induced by SVNI, we first examined the effect of SVNI on the translocation of the different PKC isoforms expressed in the C6 cells. For these experiments we infected C6 cells with SVNI for 1, 3, and 6 h and followed the translocation of the different PKC isoforms using immunofluorescence and confocal microscopy. SVNI induced translocation of PKC␦, as is evident both in its depletion from the nucleus and in its accumulation in the perinuclear region ( Fig. 2A). Translocation was already observed after 1 h, reached maximal levels after 3 h, and returned to control levels after 6 h. PKC␣, -␤, -, -, anddid not show detectable translocation after infection of the cells with SVNI (data not shown).

Sindbis Virus Induces Apoptosis of C6 Cells-SVNI
To further characterize the translocation of PKC␦ in response to SVNI infection we visualized the ER using the ER marker DiOC 5 (3). As shown in Fig. 2B, the ER marker DiOC 5 (3) accumulated in the perinuclear region in a similar pattern to that of PKC␦ in SVNI-treated cells. Merged images showed FIG. 2. SVNI induces translocation of PKC␦ to the ER. C6 cells were infected with SVNI (m.o.i. 5) for 0, 1, 3, and 6 h, and the translocation of PKC␦ was examined using immunofluorescent staining (A). Fixed cells were incubated with a rabbit anti-PKC␦ antibody for 1 h and with an anti-rabbit Alexa Fluor 488 antibody. Cells were visualized by confocal microscopy. For colocalization studies, cell infected with SVNI for 3 h were first stained with anti-PKC␦ antibody as described above followed by incubation with DiOC 5 (3) for 30 min (B). The localization of PKC␦ is shown in red (left), the ER localization is shown in green (middle), and the merge image (right) is yellow for the overlapping red and green signals. The results are from one representative experiment of five similar experiments. Cells were transiently transfected with GFP-PKC␦ and were either infected with SVNI for 3 h or treated with 100 nM PMA for 30 min (C). Cells were then fixed and viewed using confocal microscopy. Images shown are representative of three independent experiments (C).
clearly colocalization of the fluorescence of the DiOC 5 (3) and of the Alexa Fluor 546 used to visualize anti-PKC␦, suggesting that SVNI induced translocation of PKC␦ to the ER.
The pattern of translocation of PKC␦ obtained using GFPtagged PKC␦ was similar to that determined by immunofluorescence. Thus, SVNI infection of cells transiently transfected with GFP-PKC␦ induced translocation of PKC␦ to the perinuclear region (Fig. 2C). It should be noted that this pattern of translocation of PKC␦ contrasts with a different pattern of translocation upon treatment of the cells with PMA. As has been described elsewhere (39), PMA induced translocation of PKC␦ to the plasma and nuclear membranes (Fig. 2C).
PKC␦ Protects C6 Cells from SVNI-induced Apoptosis-Because SVNI induced a selective translocation of PKC␦, we examined the role of this PKC isoform in the apoptotic effect of SVNI. As an initial approach, we used the PKC␦ selective inhibitor rottlerin. Treatment of C6 cells with rottlerin (5 M) or with the vehicle control Me 2 SO (data not shown) did not affect the basal level of cell apoptosis. However, rottlerin increased the apoptotic effect of SVNI by ϳ30 -40%, suggesting that PKC␦ may exert a protective effect on SVNI-infected C6 cells (Fig. 3A). Because the specificity of rottlerin for PKC␦ has been questioned (36 -38), we explored further the role of PKC␦ in the apoptotic effect of SVNI by using a PKC␦ kinase-dead mutant (PKC␦ KD; K376R). Cells stably expressing the PKC␦ KD mutant were infected with SVNI for 24 h. Infection of the PKC␦ KD-expressing cells induced increased cell apoptosis compared with CV cells as observed by cell morphology (Fig.  3B) and by anti-histone ELISA (Fig. 3C).
The role of PKC␦ in the apoptotic effect of SVNI was examined further using cells stably overexpressing PKC␦. The expression of PKC␦ in these cells was about 6-fold higher than the level of PKC␦ in the control vector cells (39). As determined by PI staining, cells overexpressing PKC␦ exhibited a lower rate of apoptosis in response to SVNI (16.3 Ϯ 1.9% apoptotic cells) as compared with control vector cells (34.8 Ϯ 4.5) (Fig. 3D). We conclude that PKC␦ protected C6 cells from apoptosis induced by SVNI, and, conversely, that abrogation of PKC␦ functions either with rottlerin or through expression of the PKC␦ KD enhanced apoptosis induced by SVNI. Infection of C6 Cells with SVNI Induces Tyrosine Phosphorylation of PKC␦-PKC␦ undergoes tyrosine phosphorylation in response to various apoptotic stimuli (31,35,40). To examine whether infection with SVNI induced tyrosine phosphorylation of PKC␦, we infected C6 cells with SVNI for 1-4 h. PKC␦ was immunoprecipitated, and the membrane was blotted with antiphosphotyrosine antibody. As shown in Fig. 4A, SVNI induced tyrosine phosphorylation of PKC␦ in a time-dependent manner, and maximal phosphorylation was obtained after 2 h. We did not detect tyrosine phosphorylation of PKC⑀ (Fig. 4B) or of the other PKC isoforms (data not shown).
Tyrosine Phosphorylation of PKC␦ Is Necessary for Its Protective Effect on SVNI-induced Apoptosis-To explore the importance of the tyrosine phosphorylation of PKC␦ in the apoptosis induced by SVNI, we employed a PKC␦ mutant in which five putative tyrosine phosphorylation sites, 52, 64, 155, and 187 in the regulatory domain and tyrosine 565 in the catalytic domain, were mutated to phenylalanine (34). The expression of this mutant in C6 cells and its effects on cell proliferation, glutamine synthetase expression, and cell apoptosis in response to etoposide have been described (31,34,39). We found that SVNI did not induce tyrosine phosphorylation of the PKC␦5 mutant (Fig. 5A). In contrast to the results observed in response to etoposide, PKC␦5-overexpressing cells exhibited a significantly increased cell apoptosis, compared with control vector cells and with cells overexpressing PKC␦, both as determined by PI staining (Fig. 5B) and by the morphological appearance of the cells (Fig. 5C).
Replication of SVNI in Cells Overexpressing PKC␦ and PKC␦5-To determine whether the differential effect of SVNI on the apoptosis of cells overexpressing PKC␦ and PKC␦5 is because of changes in virus replication, we measured viral replication by plaque assay. Cells were infected with SVNI (m.o.i. 5) for 9, 24, and 48 h. Using plaque assay, we found that the replication of SVNI was similar in control cells and in cells overexpressing PKC␦ and PKC␦5 (Table I).
Cleavage of Caspase-3, PKC␦, and PKC␦5 in Response to SVNI Infection-The apoptosis induced by SVNI is mediated by caspases (41). In a recent study we demonstrated that tyrosine-phosphorylated PKC␦ regulated the cleavage of caspase 3, which in turn cleaved PKC␦ to release the active catalytic domain (31). To further explore the role of tyrosine phospho-rylation of PKC␦ in the apoptosis induced by infection of SVNI, we compared the cleavage of caspase 3 in cells overexpressing control vector, PKC␦, and the PKC␦5 mutant. Using a specific antibody that recognizes the cleaved product (17 kDa) of caspase 3, we detected a cleaved fragment of caspase 3 in SVNI-infected control vector cells. Cells overexpressing PKC␦ exhibited very low levels of cleaved caspase 3, whereas large amounts of the cleaved product were obtained in cells overexpressing PKC␦5 (Fig. 6A).
To examine whether SVNI infection induced cleavage of PKC␦ or PKC␦5, we infected C6 cells overexpressing PKC␦ and PKC␦5 with SVNI for 12 and 24 h and analyzed cell lysates by Western blotting. Using the ⑀ tag, which could detect the cleaved catalytic domain of the exogenous PKC␦ and PKC␦5, we found no detectable cleaved product in either of the infected cells (Fig. 6B), suggesting that although SVNI infection induced activation of caspase 3, caspase 3 did not cleave PKC␦ or PKC␦5.
Role of Specific Tyrosine Mutants in the Apoptosis Induced by SVNI-To examine the role of the specific tyrosine residues in the effect of PKC␦ on cell apoptosis induced by SVNI, we used C6 cells stably transfected with different PKC␦ mutants in which each one of the tyrosines (52, 64, 155, 187, 565) was mutated individually to phenylalanine. The expression of these mutants in C6 cells was described previously (39).
We found that cells overexpressing PKC␦Y52F, PKC␦Y64F, and PKC␦Y155F exhibited an enhanced apoptotic response to SVNI albeit to a lesser extent than cells overexpressing PKC␦5. In contrast, infection with SVNI of cells overexpressing PKC␦Y187F or PKC␦Y565F resulted in a lower apoptotic response, similar to the response observed in cells overexpressing PKC␦ (Fig. 7A).
We also found that infection with SVNI induced a smaller increase in the tyrosine phosphorylation of PKC␦Y52F, PKC␦Y64F, and PKC␦Y155F as compared with PKC␦ (Fig. 7B). The lowest degree of the tyrosine phosphorylation was obtained in cells overexpressing the PKC␦Y155F mutant, as compared with the other single tyrosine mutants. Correspondingly, this mutant exhibited the highest degree of cell apoptosis in response to SVNI (Fig. 7B).

DISCUSSION
In this study we explored the role of PKC in the apoptosis induced by Sindbis virus infection. We found that infection of the cells with SVNI induced a selective translocation and tyrosine phosphorylation of PKC␦ and that the phosphorylated PKC␦ exerted a protective effect against SVNI-induced apoptosis.
SVNI, a neurovirulent strain of Sindbis virus, has been reported to induce apoptosis in a variety of cell types (1,2). The mechanisms involved in the apoptotic effect of SVNI have been described mainly with regard to the roles of apoptotic-related proteins such as beclin (4), Bcl2, Bax (42,43), and caspases (41). Thus, SVNI induces high expression of Bax (42), and its apoptotic effects are blocked by high expression of Bcl2 (3). In addition, various studies indicate that the effects of SVNI are mediated by caspase activation, because CrmA (41), caspase 3, and caspase 8 inhibitors (41,44) blocked the apoptotic effect of the virus. In contrast, only a few reports exist with regard to the signal transduction pathways activated by SV infection. SV was reported to induce activation of sphingomyelinase and ceramide release in N18 cells (7) and to activate p38 and the small heat shock protein HSP27 in Vero cells (6). Similarly, SV infection was reported to activate the double-strand RNA-dependent protein kinase (9) and nuclear factor B (8).
We found that SVNI induced a selective translocation of PKC␦ to the ER within 3 h of infection, whereas it did not affect the translocation of the other PKC isoforms. PKC␦ has been reported to undergo differential translocation to distinct cellular compartments depending on the specific stimulus and on the specific cell type. Thus, in C6 cells PKC␦ translocates to the plasma and nuclear membranes in response to PMA, in contrast to its translocation to the ER in response to SVNI. In keratinocytes, PKC␦ undergoes translocation to the mitochondria in response to PMA (29) and in HeLa cells PKC␦ translocates to the Golgi in response to ceramide (45). Various PKC isoforms are also found in the nucleus and in subnuclear compartments (46). It is currently not clear what is the function of PKC␦ in the ER and which proteins can be phosphorylated by PKC␦ in the ER membrane.
SVNI induced tyrosine phosphorylation of PKC␦. This phosphorylation appeared to be essential for the protective effect of PKC␦ against SVNI-induced apoptosis, because cell apoptosis was decreased in cells overexpressing PKC␦ and was increased significantly in cells overexpressing the PKC␦5 mutant. Indeed, the PKC␦5 mutant acts in an opposite way to PKC␦ in its effect on cell apoptosis in response to SVNI. Likewise, PKC␦ and PKC␦5 induced opposite effects on the expression of the astrocytic marker GS (34), on cell proliferation (39), and on cell apoptosis in response to etoposide (31). In contrast to the effect on apoptosis, production of progeny virus was similar in cells expressing PKC␦, PKC␦5, and CV, suggesting that the differential apoptotic response observed in cells overexpressing PKC␦ and PKC␦5 cannot be attributed to differences in virus replication.
Of the five sites of phosphorylation mutated in PKC␦5, we found that phosphorylation on tyrosines 52, 64, and 155 in the regulatory domain was essential for the protective effect of PKC␦. Phosphorylation of PKC␦ on tyrosine residues occurs in response to a large number of stimuli including PMA (34), platelet-derived growth factor (39,47), epidermal growth factor (48), activation of the IgE receptor (19,49), and apoptotic stimuli such as ␥-irradiation (40), H 2 O 2 (35), and etoposide (31). Tyrosine phosphorylation of PKC␦ occurs on different tyrosine residues. Thus, platelet-derived growth factor and PMA induced tyrosine phosphorylation on tyrosines 187 and 155, respectively (39), and activation of the IgE receptor induced phosphorylation on tyrosine 52 (19). In addition, apoptotic stimuli such as etoposide (31) and H 2 O 2 (35) induce tyrosine phosphorylation in either the regulatory or the catalytic domain, respectively, of PKC␦. It is not known currently why phosphorylation in the regulatory domain of PKC␦ is essential for its protective effect against apoptosis induced by SVNI. Changes in the catalytic activity and cellular localization via binding to specific scaffold proteins could provide the basis for this effect.
A striking finding was that PKC␦ played opposite roles in apoptosis induced by SVNI and by etoposide, although tyrosine phosphorylation of PKC␦ was essential for both effects. The opposite effects of PKC␦ on cell apoptosis in response to SVNI and etoposide and the differential role of the tyrosine phosphorylation in PKC␦ effects may be attributed to a number of factors. First, PKC␦ underwent differential translocation by etoposide and SVNI. Thus, distinct translocation of PKC␦ to the nucleus by etoposide (31) or to the ER by SVNI could lead to different effects because of the phosphorylation of different substrates and to the association of PKC␦ with specific proteins present in these locations. Second, etoposide and SVNI induced phosphorylation of different tyrosine residues on PKC␦. The effects of tyrosine phosphorylation on the activity of PKC␦ or on its function are dependent on the specific system and stimulus; however, it is believed currently that tyrosine phosphorylation can alter the affinity of PKC␦ toward its different substrates (19). Thus, the phosphorylation of PKC␦ on specific residues might confer differential affinity of PKC␦ for distinct apoptoticrelated proteins. Consequently, the activation of different down-stream pathways including caspases or other apoptosisrelated proteins by these two stimuli could provide the basis for the divergent effects of PKC␦ on cell apoptosis.
Apoptosis by SV infection has been reported to involve caspase activation (41,44). Indeed, we found that SVNI induced activation of caspase 3. This activation was increased further in cells overexpressing PKC␦5 and was decreased in cells overexpressing PKC␦. These results are in contrast to our recent studies, which demonstrated that etoposide induced an increased activation of caspase 3 in cells overexpressing PKC␦ and a decreased response in cells overexpressing PKC␦5 (31). One explanation for these differences could be a differential activation of upstream caspases by PKC␦ and PKC␦5 in response to etoposide and SVNI, which will eventually converge in the activation of caspase 3.
Interestingly, and in contrast to the results obtained with etoposide (31), SVNI infection did not induce caspase-dependent cleavage of PKC␦ or PKC␦5. In various systems, the apoptotic effect of PKC␦ has been associated with a cleavage of the catalytic domain from the regulatory domain (27). Cleavage of the catalytic domain of PKC␦ by caspases has been reported in cells treated with ionizing radiation, tumor necrosis factor-␣,  and etoposide (25,27,50), whereas no cleavage of PKC␦ was observed in LNCaP cells undergoing apoptosis in response to PMA (28). Indeed, our recent studies (31) and others (27) have suggested the presence of a positive loop between PKC␦ and caspase 3 in etoposide-treated cells, which was not observed in SVNI-treated cells. Because caspase 3 has been reported recently to undergo nuclear translocation in response to apoptotic stimuli (51), it is possible that the lack of PKC␦5 cleavage by caspase 3 is because of a different cellular localization of the two proteins.
One of the interesting issues remaining to be investigated is the identity of the tyrosine kinases and phosphatases that phosphorylate and dephosphorylate PKC␦ in response to SVNI. PKC␦ has been reported to associate and undergo tyrosine phosphorylation by different tyrosine kinases such as Src, Lyn (19,49), Fyn (39,47), and c-Abl (40). We found differential phosphorylation of tyrosines 52, 64, and 155 in response to SVNI and differential sensitivity of these tyrosine mutants to SVNI-induced apoptosis. The ability of PKC␦ to be phosphorylated on multiple tyrosine residues suggests that PKC␦ can associate with different tyrosine kinases via distinct tyrosine residues, which can then lead to diverse cellular outcomes.
Moreover, it appears that phosphorylation at one tyrosine residue may influence phosphorylation at the others. The identity of the tyrosine kinases involved in the phosphorylation of PKC␦ in SVNI-treated cells is currently being studied. Our results so far suggest that Fyn, which is involved in the tyrosine phosphorylation of PKC␦ in response to platelet-derived growth factor, is probably not involved in the apoptosis induced by either etoposide (31) or SVNI. 2 The mechanisms by which SVNI induces translocation and tyrosine phosphorylation of PKC␦ are not yet understood, but several options can be considered. The translocation and phosphorylation of PKC␦ induced by SVNI were observed following 1-3 h of infection, suggesting that early events such as binding of the virus to its cellular receptor or its entrance via the plasma membrane activated this signaling pathway. Indeed, infection with SVNI induces a rapid activation of ceramide (7) and p38 (6). Moreover, it was reported recently that the entrance of the virus to the cells is sufficient for the induction of apoptosis by the virus (52).
Apoptosis induced by viral infection is a defense mechanism of the host cells, because it limits virus production and prevents the infection of neighboring cells (53). Most viruses inhibit or delay early apoptosis by inducing the expression of endogenous anti-apoptotic cellular proteins or by using their own antiapoptotic genes (53). Thus, the tyrosine phosphorylation of PKC␦ by SVNI may represent a mechanism by which the virus inhibits cell apoptosis, whereas activation of tyrosine phosphatases that dephosphorylate PKC␦ may reflect a cell response that aims at inducing cell apoptosis.
In summary, we demonstrated that the apoptosis induced by SVNI infection involves tyrosine phosphorylation and translocation of PKC␦ to the ER. The tyrosine-phosphorylated PKC␦ protected the cells from the apoptosis induced by SVNI, 2 Unpublished data. whereas the dephosphorylated PKC␦ rendered the cells more sensitive to the apoptosis induced by SVNI. The effect of the tyrosine phosphorylation of PKC␦ on the apoptosis in C6 cells in response to SVNI may be mediated by altering the affinity of PKC␦ toward downstream apoptosis-related substrates or by altering the activity of a tyrosine kinase that is associated with PKC␦. Thus, the differential phosphorylation of specific tyrosine residues and the distinct localization of PKC␦ may provide the basis for the anti-and pro-apoptotic functions of this kinase.