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Signaling and Regulation

The Localization of Protein Kinase C in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, Michigan

Requests for reprints: Chaya Brodie, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, MI 48202. Phone: 313-916-8619; Fax: 313-916-9855. E-mail: chaya{at}brodienet.com

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Protein kinase C (PKC) regulates cell apoptosis and survival in diverse cellular systems. PKC translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKC constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKC affects its apoptotic and survival functions. PKC-Cyto, PKC-Mito, and PKC-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKC-ER. PKC-Cyto and PKC-Mito underwent cleavage, whereas no cleavage was observed in the PKC-Nuc and PKC-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKC-Cyto and PKC-Mito. In contrast to the apoptotic effects of the PKC-Cyto, PKC-Mito, and PKC-Nuc, the PKC-ER protected the cells from tumor necrosis factor–related apoptosis-inducing ligand–induced and etoposide-induced apoptosis. Moreover, overexpression of a PKC kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKC and increased tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. The localization of PKC differentially affected the activation of downstream signaling pathways. PKC-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKC-Nuc increased c-Jun NH₂-terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKC-Cyto, whereas c-Jun NH₂-terminal kinase activation mediated the apoptotic effect of PKC-Nuc. Our results indicate that the subcellular localization of PKC plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways. (Mol Cancer Res 2007;5(6):627–39)

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
The ubiquitously expressed isoform protein kinase C (PKC) has been shown to regulate cell apoptosis and survival in various cells depending on the specific cellular system and apoptotic stimuli (1-3). Most studies report a proapoptotic function of PKC in response to various stimuli, such as H₂O₂ (4), ceramide (5), tumor necrosis factor- (6), and the DNA-damaging agents UV radiation (7), cisplatin (8), and etoposide (9). Different apoptotic stimuli induce caspase-dependent cleavage of PKC, which result in the generation of a constitutively active catalytic fragment (10, 11). The cleavage of PKC has been implicated in its apoptotic function (12, 13), and the expression of the catalytic fragment has been shown to induce cell apoptosis in different cellular systems (14, 15). The apoptotic function of PKC has been associated with the activation of multiple signaling proteins, such as c-Jun NH₂-terminal kinase (JNK; ref. 16), p38 (17), AKT (18), p73 (19), DNA-PK (20), scramblase 3 (21), and lamin B (22).

In addition to its apoptotic functions, PKC has been also reported to exert antiapoptotic effects. Thus, PKC protects macrophages from apoptosis induced by nitric oxide (23) and exerts antiapoptotic effects on glioma cells treated with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; ref. 24) or infected with a virulent strain of Sindbis virus (25). Similarly, PKC promotes survival and chemotherapeutic drug resistance of non–small cell lung cancer cells (26).

One of the factors that may contribute to the diverse effects of PKC on cell apoptosis is its different subcellular localization. Indeed, various apoptotic stimuli induce translocation of PKC to distinct subcellular sites. Thus, PKC translocates to the Golgi in response to ceramide and IFN- (5, 27) to the mitochondria in response to oxidative stress, UV radiation, and phorbol 12-myristate 13-acetate (28, 29) and to the nucleus in response to etoposide, -irradiation, and cytosine arabinoside (9, 30). Translocation of PKC to the Golgi, mitochondria, and nucleus in response to these apoptotic stimuli has been associated with proapoptotic effects of this isoform (5, 9, 28). In addition, PKC has been shown to translocate to the endoplasmic reticulum (ER) in cells infected with Sindbis virus and in glioma cells treated with TRAIL, where PKC exerts antiapoptotic effects (24, 25). Although these studies suggest different functions of PKC in the various subcellular sites, the role of the translocation of PKC in its proapoptotic and antiapoptotic effects and in the activation of downstream signaling pathways is largely not defined.

In this study, we explored the role of the subcellular localization of PKC in its proapoptotic and antiapoptotic functions. Specifically, we asked whether the localization of PKC in the ER provides antiapoptotic signals as opposed to proapoptotic signals in the mitochondria and nucleus and how the localization of PKC in the distinct subcellular sites affects the activation of downstream signaling pathways. For these experiments, we targeted PKC to the cytoplasm, mitochondria, nucleus, and ER using the pShooter vectors, which have been widely used to examine the role of the subcellular localization of various proteins in their specific cellular functions (31-33). We found that overexpression of a constitutively active form of PKC (PKC-CA) in the cytosol, mitochondria, or nucleus resulted in cell apoptosis. In contrast, expression of PKC-CA in the ER had no apoptotic effect but it rather protected glioma and HeLa cells against TRAIL- and etoposide-induced apoptosis. In addition, we found that the subcellular localization of PKC had an important role in the induction of distinct apoptosis-related signaling pathways.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Targeted Localization of PKCCA in Distinct Subcellular Sites of the A172 Glioma Cells
PKC has been associated with the regulation of both cell apoptosis and survival (3, 6, 8, 9, 24, 30). One of the factors that may contribute to the diversity of PKC effects is its ability to localize to different subcellular sites. To examine the role of PKC localization in its proapoptotic and antiapoptotic functions, we used a PKC constitutively active mutant that was targeted to the cytosol (PKC-Cyto), mitochondria (PKC-Mito), nucleus (PKC-Nuc), and ER (PKC-ER) using the pShooter vectors (31-33). A172 cells were transiently transfected with the different pShooter vectors, and the localization of PKC was examined using anti-Myc antibody (Fig. 1A ). In parallel, A172 cells were transfected with the corresponding pShooter vectors encoding green fluorescent protein (GFP), which have been used as positive controls (Fig. 1B). Figure 1 shows immunostaining of A172 cells transfected with the different pShooter vectors using anti-Myc antibody and A172-expressing GFP pShooter vectors. The expression of PKC was selectively localized to the cytoplasm, nucleus, mitochondria, and ER in the PKC-Cyto, PKC-Nuc, PKC-Mito, and PKC-ER, respectively (Fig. 1A). The localization of PKC in the different subcellular sites was similar to that of the corresponding GFP pShooter vectors (Fig. 1B).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1. Subcellular localization of the PKC pShooter vectors. A172 cells were transfected with control vector, PKC-Cyto, PKC-Mito, PKC-Nuc, and PKC-ER. A. Cells were fixed with 4% paraformaldehyde and then incubated with a rabbit anti-Myc antibody for 1 h followed by incubation with an anti-rabbit Alexa Fluor 488 antibody. Cells were visualized by confocal microscopy. In parallel, cells were transfected with the appropriate pShooter vectors encoding GFP, which served as positive controls. B. The localization of the GFP was visualized using confocal microscopy. C. Colocalization of PKC-ER, PKC-Nuc, and PKC-Mito with the specific organelle markers, anti-calnexin, 7-AAD, and MitoTracker Orange, was determined in cells overexpressing the respective pShooter vectors. The localization of PKC is shown in green (left), the ER. Mitochondria and nucleus staining are shown in red (middle) and the merge image (right) is yellow for the overlapping red and green signals. The results are from one representative experiment out of three similar experiments.

Overexpression of PKC-CA in the Cytoplasm, Mitochondria, and Nucleus, but not in the ER, Induces Cell Apoptosis
We next examined the effect of the organelle-targeted PKC on cell apoptosis. For these experiments, we used both the A172 and the HeLa cells. Following transfection with the various PKC pShooter vectors, the expression of PKC was determined using anti-Myc antibody (Fig. 2A ) and cell apoptosis was determined using propidium iodide staining and fluorescence-activated cell sorting (FACS) analysis and by anti–histone ELISA. As shown in Fig. 2B, transfection of A172 cells with the PKC-Cyto, PKC-Mito, and PKC-Nuc induced a large degree of cell apoptosis in the cells. The most significant effect was induced by the PKC-Nuc and PKC-Mito, whereas a smaller effect was observed with the PKC-Cyto. The apoptotic effect was first observed after 24 h of the transfection, and by 48 h thereafter, the majority of the cells were apoptotic (data not shown). In contrast, cell apoptosis was not induced by the PKC-ER by 24 h (Fig. 2B) or 48 h after transfection (data not shown). Overexpression of the A172 cells with the PKC-ER also did not induce major morphologic changes in the cells, whereas cells overexpressing the PKC-Cyto, PKC-Mito, and PKC-Nuc exhibited apoptotic morphology (Fig. 2C). Similar results were obtained using anti–histone ELISA (Fig. 2D).

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2. Effect of targeting PKC to distinct subcellular sites on cell apoptosis. A172 (A-D) and HeLa (E) cells were transfected with control vector (CV), PKC-Cyto, PKC-Mito, PKC-Nuc, and PKC-ER. A. Following 24 h, the A172 cells were analyzed for the expression of PKC using anti-Myc antibody. CF, catalytic fragment. The A172 (B) and HeLa (E) cells were analyzed for cell apoptosis using propidium iodide staining and FACS analysis. C. The morphology of transfected A172 cells was monitored using a phase-contrast microscope. D. Cell apoptosis was also measured using anti–histone ELISA. B and D. Columns, mean of four separate experiments; bars, SE. A, C, and E. Results are from one representative out of four independent experiments. *, P < 0.001.

Targeting PKC to Distinct Subcellular Sites Results in Differential Cleavage of PKC and Activation of Caspase-3
The induction of cell apoptosis by PKC has been associated with cleavage of this isoform and with the generation of a constitutively active catalytic fragment (4, 9, 13, 30); however, the role of PKC localization in its cleavage is not well characterized. We therefore examined the cleavage of PKC in cells overexpressing the different PKC pShooter vectors. The expression of the ectopic PKC was detected using Western blot analysis and an anti-PKC antibody that preferentially recognizes the rat PKC and not the human kinase. As presented in Fig. 3A , overexpression of the PKC-CA in the cytoplasm and in the mitochondria resulted in cleavage of PKC and in the accumulation of a 40-kDa catalytic fragment, whereas no significant cleavage was observed in PKC-Nuc and PKC-ER. Similar results were obtained using an anti-Myc antibody that recognizes the COOH-terminal Myc tag (Fig. 2A).

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3. Targeting of PKC to distinct subcellular sites results in differential cleavage of PKC and activation of caspase-3. A172 cells were transfected with the different PKC pShooter vectors. A. At 24 h after transfection, cells were assayed for the cleavage of PKC using Western blot analysis and an anti-PKC antibody that recognizes the catalytic domain of the ectopic PKC. B. Caspase-3 activity was measured using the fluorimetric caspase-3 substrate as described in Materials and Methods. C and D. The role of caspase-3 in the apoptosis of the A172 cells induced by the PKC-Cyto, PKC-Mito, and PKC-Nuc was evaluated in cells treated with the caspase-3 inhibitor (DEVD-FMK, 10 µmol/L) following cell transfection. PKC cleavage was determined using Western blot analysis and anti-PKC that recognized the catalytic domain (C), and cell apoptosis was determined using propidium iodide staining and FACS analysis (D). E. Cytochrome c release from the mitochondria was determined in cytosolic fractions. F. Caspase-9 activity was measured using the fluorimetric substrate Ac-LEHD-AFC. The results represent the mean of three independent experiments (B, D, and F) or are from one representative out of four independent experiments (A, C, and E). *, P < 0.001; **, P < 0.05.

To examine the role of caspase-3 activity in the cleavage of PKC in the different subcellular sites and in the apoptosis induced by the PKC-Cyto and the PKC-Mito, we used the caspase-3 inhibitor DEVD-FMK (10 µmol/L). Treatment of the cells with DEVD-FMK following the transfection of the cells with the different pShooter vectors decreased the cleavage of the PKC-Cyto and the PKC-Mito (Fig. 3C). In addition, treatment of the cells with the caspase-3 inhibitor markedly reduced the apoptosis induced by the PKC-Cyto and PKC-Mito and only moderately decreased the apoptotic effect of PKC-Nuc (Fig. 3D).

PKC has been reported to activate the mitochondrial pathway, which eventually leads to the activation of caspase-3 (14, 29). To examine the role of PKC localization in the activation of the mitochondrial pathway, we examined its effect on cytochrome c release and on caspase-9 activation. As presented in Fig. 3E, PKC-Mito significantly increased the release of cytochrome c from the mitochondria to the cytosol, whereas a smaller effect was observed with the PKC-Cyto and no significant effects were observed with the PKC-Nuc and PKC-ER. Similarly, the PKC-Mito significantly increased the activation of caspase-9, whereas smaller increase was obtained in the PKC-Cyto–overexpressing cells (Fig. 3F).

Targeting of PKC to the ER Protects Glioma and HeLa Cells against Cell Apoptosis
Recent studies in our laboratory showed that PKC translocated to the ER in response to TRAIL treatment (24) and Sindbis virus neurovirulent infection (25) and that in both cases PKC acted as a survival kinase. As we have found that PKC-ER does not induce apoptosis in either the A172 or HeLa cells, we considered the possibility that expression of PKC in the ER might actually protect glioma cells against cell apoptosis. We first examined whether the PKC-ER protected the cells against TRAIL-induced apoptosis. A172 cells were transfected with control vector, PKC-Nuc, and PKC-ER, and after 24 h, the cells were treated with TRAIL (100 ng/mL) for an additional 5 h. As presented in Fig. 4A , TRAIL induced 45% cell apoptosis in the control vector A172 cells and increased the apoptotic effect of the PKC-Nuc (data not shown). In contrast, cells overexpressing the PKC-ER exhibited lower levels of cell apoptosis and only 20% of the cells underwent cell apoptosis (Fig. 4A). Similar results were obtained with HeLa cells; in these cells, overexpression of PKC-ER reduced cell apoptosis by 68% compared with control vector cells (Fig. 4B). TRAIL did not induce the cleavage of PKC-ER in either A172 or HeLa cells (data not shown).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4. Localization of PKC in the ER exerts protective effects against cell apoptosis. A172 (A, C, and D) and HeLa (B and C) cells were transfected with control vector and PKC-ER. Following 24 h, A172 cells were treated with TRAIL (100 ng/mL) for 5 h (A) and HeLa cells were treated for 24 h (B). C. In addition, both A172 and HeLa cells were treated with etoposide (50 µg/mL) for 24 h. Cell apoptosis was determined using propidium iodide staining and FACS analysis. D. A172-expressing control vector and PKC-ER were treated with thapsigargin (2 µg/mL) or tunicamycin (4 µmol/L) for 36 h, and cell death was determined using propidium iodide staining and FACS analysis. A172 cells were transfected with control vector and PKC kinase-dead mutants that were targeted to the ER (PKCKD-ER) or the nucleus (PKCKD-Nuc). The expression of the PKCKD-ER and PKCKD-Nuc was determined by Western blot analysis (E) and their subcellular localization was determined by staining with anti-Myc antibody and confocal microscopy (F). G. Following 24 h, cells were treated with TRAIL for 5 h and cell apoptosis was determined using propidium iodide staining and FACS analysis. The results represent the mean of three independent assays (A, B, C, D, and G) or are from one representative experiment out of three similar experiments (E and F). *, P < 0.001; **, P < 0.05.

To further study the role of PKC in the ER, we used a PKC kinase-dead mutant targeted to the ER to interfere with endogenous PKC that localizes in this subcellular site. For these experiments, we used the apoptotic stimulus TRAIL, which induces translocation of PKC to the ER (24). As a control in this experiment, we used the PKCKD-Nuc. Figure 4E and F shows the overexpression of the PKCKD-ER and PKCKD-Nuc using Western blot analysis (Fig. 4E) and their localization in the ER and nucleus, respectively, using anti-Myc antibody staining and confocal microscopy (Fig. 4F). Figure 4G shows that the PKCKD-ER slightly increased the apoptosis of the A172 cells but significantly enhanced the apoptotic effect of TRAIL. In contrast, overexpression of a PKCKD mutant that was targeted to the nucleus did not alter the apoptotic response of the cells to TRAIL. These results suggest that the localization of PKC in the ER exerts an antiapoptotic effect against TRAIL-induced apoptosis and that inhibition of the endogenous PKC in the ER increases the apoptotic effect of TRAIL.

PKC-Cyto and PKC-Nuc Induce the Activation of Different Signaling Pathways
The proapoptotic and antiapoptotic effects of PKC are mediated by activating various downstream signaling pathways (3). To identify signaling pathways that are induced by the compartmentalized PKC, we examined the expression and phosphorylation of various apoptosis-related proteins in cells overexpressing the various PKC pShooter vectors.

As presented in Fig. 5 , the overexpression of the PKC-Cyto selectively increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein (XIAP), whereas the expression of PKC-Nuc induced the phosphorylation of JNK. In contrast, we did not detect changes in the phosphorylation of signal transducers and activators of transcription 1 or in the expression of Bax and Bcl₂ in cells overexpressing the different PKC pShooter vectors.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   FIGURE 5. Targeting of PKC-CA to distinct subcellular sites exerts differential effects on the expression and phosphorylation of various apoptosis-related proteins. A172 cells were transfected with the control vector and the different PKC pShooters. After 24 h, the expression and phosphorylation of various apoptosis-related proteins were analyzed using Western blot analysis. The results are from one representative experiment out of three similar experiments.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   FIGURE 6. Role of p38 and XIAP in the apoptotic effect of PKC-Cyto. A172 cells were transfected with control vector or PKC-Cyto. A. The phosphorylation of MKK3/MKK6 was examined 24 h later by Western blot analysis. Cells overexpressing PKC-Cyto were treated with the p38 inhibitor SB203580 (10 µmol/L) 3 h after transfection. The phosphorylation of p38 by Western blot analysis (B) and cell apoptosis by propidium iodide staining and FACS analysis (C) were determined after 24 h. A172 cells were transfected with control (scramble) and p38 siRNA duplexes. After 48 h, the expression of p38 in the control and p38 siRNA-transfected cells was examined by Western blot analysis (D) and the cells were then transfected with control vector or PKC-Cyto. E. Following an additional 24 h, the cells were analyzed for cell apoptosis. A172 cells were cotransfected with XIAP and control vector or with XIAP and PKC-Cyto. F. Expression of XIAP in the PKC-Cyto cells examined by Western blot analysis. G. Cell apoptosis was determined following 24 h using propidium iodide staining and FACS analysis. The results represent the mean of three independent experiments (C, E, and G) or are from one representative out of four independent experiments (A, B, D, and F). *, P < 0.05; **, P < 0.001.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   FIGURE 7. Role of JNK in the apoptotic effect of PKC-Nuc. A172 cells were transfected with control vector or PKC-Nuc. A. The phosphorylation of MMK4 and MKK7 was determined 24 h later. Cells were transfected with control vector and PKC-Nuc and, after 3 h of transfection, treated with the JNK inhibitor SP600125 (20 µmol/L). B. The phosphorylation of JNK in the presence of SP600125 was determined after 24 h. C. Cell morphology of the PKC-Nuc–transfected cells in the presence and absence of SP600125 was determined using phase-contrast microscopy. D. Cell apoptosis was determined using propidium iodide staining and FACS analysis. E. The specificity of the JNK and p38 pathways in PKC-Nuc and PKC-Cyto effects, respectively, was determined by examining the effects of SP600125 and SB203580 in the PKC-Nuc–overexpressing and PKC-Cyto–overexpressing cells. The results represent the mean of three independent experiments (D and E) or represent one of four similar experiments (A-C). *, P < 0.001; **, P < 0.05.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
PKC has been reported to play a major role in the regulation of cell apoptosis and survival in response to various stimuli (1-3). Although most studies report a proapoptotic function of PKC, there are other studies that show an antiapoptotic effect of this isoform (24, 25). One of the main factors that affect PKC activity and functions and contributes to the diverse effect of PKC is its subcellular localization (34). Indeed, PKC undergoes translocation to distinct subcellular sites in response to various apoptotic stimuli (5, 9, 27-29); however, the role of the subcellular localization of PKC in its apoptotic functions is just beginning to be understood. In this study, we targeted PKC to the cytoplasm, ER, nucleus, and mitochondria and examined the role of the localization of PKC in its proapoptotic and antiapoptotic effects.

The first objective of this study was to find a cellular system that will allow the selective targeting of PKC to the mitochondria, ER, and nucleus in a way that will resemble as much as possible what happens with the endogenous PKC. The pShooter vectors seemed to be the best choice for targeting PKC because these vectors have been widely and successfully used to study the role of the subcellular localization of different proteins in their cellular functions (31-33). Thus, this system has been used to show the role of the mitochondrial localization of mutant superoxide dismutase 1 in cell death in a model of familial amyotrophic lateral sclerosis (32), to study the role of the intracellular localization of transglutaminase in cell death (31), and to examine the functional coupling of chromogranin with the InsP3R for calcium signaling (33). Importantly, targeting of PKC to the mitochondria using the appropriate pShooter vector induced phospholipid scramblase-dependent cell apoptosis similar to the endogenous PKC (21).

We found that the expression of PKC-Cyto, PKC-Mito, and PKC-Nuc induced cell apoptosis, whereas expression of PKC-ER had no apoptotic effect. The role of the nuclear and mitochondrial localization of PKC in its apoptotic function has been shown thus far indirectly by studies that reported an association between the translocation of PKC to the nucleus and mitochondria and its proapoptotic effects. Indeed, the role of the nuclear localization of PKC in its apoptotic effect has been shown in studies showing that PKC translocated to the nucleus in response to apoptotic stimuli, such as etoposide (9, 30), and that the overexpression of a PKC nuclear localization signal (NLS) mutant abrogates the apoptotic effect of this drug (35). Similarly, PKC has been shown to translocate to the mitochondria in response to UV radiation (14), phorbol 12-myristate 13-acetate (28), and oxidative stress (29) and to induce cell apoptosis in response to these stimuli. Our results support these studies and provide direct evidence that the localization of PKC-CA in the cytosol, mitochondria, and nucleus, but not in the ER, promotes cell apoptosis.

PKC undergoes cleavage in response to various apoptotic stimuli, such as etoposide (9, 30), cisplatin (13), UV radiation (14), and TRAIL (24). Although the cleavage of PKC and its catalytic fragment are mainly associated with its proapoptotic effects (9, 13, 14, 30), in some systems PKC cleavage does not play a role in its apoptotic effects (36, 37) or it is associated with PKC protective effects (24). We found that PKC-Cyto and PKC-Mito were cleaved to generate a 40-kDa catalytic fragment. In contrast, the PKC-Nuc and PKC-ER did not undergo significant cleavage, although the PKC-Nuc induced a large degree of cell apoptosis. Although most of the studies implicate the catalytic domain of the nuclear PKC as an important mediator of its apoptotic activity (9, 19, 20, 35), our results show that the apoptotic effect of PKC-Nuc was not dependent on its cleavage and catalytic fragment and that the localization of PKC in the nucleus was sufficient to induce cell apoptosis.

We found that PKC-Cyto and PKC-Mito induced activation of caspase-3, whereas only minor activation of caspase-3 was detected in the PKC-Nuc overexpressors. The cleavage of PKC is mainly mediated by caspase-3 (6, 9, 30), which in turn is phosphorylated and activated by PKC (9, 30, 38). Thus, the large increase in caspase-3 activation induced by PKC-Cyto and PKC-Mito is in accordance with their cleavage. Similarly, inhibition of caspase-3 significantly inhibited the apoptosis induced by PKC-Cyto and PKC-Mito, whereas it only slightly abrogated the apoptosis induced by the PKC-Nuc. These results suggest that PKC-Nuc induced cell apoptosis mostly in a caspase-3–independent manner. One of the reasons for the lack of the activation of caspase-3 and PKC cleavage in the PKC-Nuc–overexpressing cells may be the different subcellular localization of PKC relative to that of caspase-3. In a recent study, we reported that etoposide induced colocalization of caspase-3 and PKC in the nucleus and that caspase-3 induced cleavage of PKC in this site (9). However, the localization of caspase-3 in the nucleus was dependent on the activation of caspase-9 by a tyrosine phosphorylated PKC. Indeed, PKC-Nuc did not induce cytochrome c release from the mitochondria or the activation of caspase-9.

PKC-ER did not induce cell apoptosis in either A172 or HeLa cells but rather protected the cells from different apoptotic stimuli. Interestingly, we recently reported that translocation of PKC to the ER was associated with antiapoptotic effects of this isoform. Thus, PKC translocated to the ER in response to Sindbis virus infection (25) and in response to TRAIL treatment (24), and in both cases, PKC protected the cells from the apoptosis induced by these stimuli. These results are further supported by our current findings that targeting PKC to the ER reduced cell apoptosis induced by TRAIL and etoposide in both A172 and HeLa cells. In addition to showing the protective effect of the overexpressed PKC-ER, we also showed that the localization of the endogenous PKC in the ER has similar protective effect. Thus, expression of a PKCKD mutant that was targeted to the ER increased the apoptosis induced by TRAIL by abrogating the protective effect of the ER-localized endogenous PKC. Collectively, these results indicate that PKC that resides in the ER, either ectopically or endogenously, similarly exerts antiapoptotic effects.

In addition to its protective effects against cell apoptosis, the PKC-ER partially inhibited cell death induced by thapsigargin and tunicamycin, suggesting that localization of PKC in the ER had a protective effect also in the ER stress-induced apoptosis. The role of PKC in the ER and the mechanisms involved in its antiapoptotic effects are currently not understood. Localization in the ER has been described for other PKC isoforms, such as PKC (39) and PKC (40), but the functions of these PKC isoforms in the ER have not been reported. Importantly, there are several apoptosis-related proteins, such as caspase-12, Bcl₂, Bax, Bik, and Bak (41, 42), which reside in the ER and play a role in the regulation of cell apoptosis. One possible PKC substrate in the ER is Bcl₂, which has been shown to undergo phosphorylation by PKC (43) and to regulate the cross-talk between the ER and the mitochondria during cell apoptosis (44). Interestingly, a recent study reported that targeting of Bcl₂ to the ER plays an important role in its protective effect (41). Studies exploring the role of Bcl₂ in the antiapoptotic effects of PKC-ER and the identification of novel PKC substrates/partners in the ER are currently under way.

PKC has been shown to activate a large number of downstream signaling pathways in response to various apoptotic stimuli (3). Thus, the activation of signaling proteins, such as c-Abl (45), JNK (16), p38 (17), p73 (19), and DNA-PK (20), has been associated with the proapoptotic effect of PKC, whereas phosphorylation of AKT (24) and HSP25 (46) is associated with its antiapoptotic effects. We found that the localization of PKC affected its ability to activate specific downstream signaling pathways. Thus, the PKC-Cyto induced phosphorylation of p38 and dephosphorylation of AKT and decreased the expression of XIAP, whereas the PKC-Nuc induced phosphorylation of JNK.

PKC has been recently reported to increase the phosphorylation of p38 in various cell types (17, 47). However, the role of the subcellular localization of PKC in this effect was not reported. We found that the PKC-Cyto induced the phosphorylation of MKK3/MKK6, which lies upstream of p38 (48). Although PKC-Cyto activated the p38 pathway, it played only a partial role in the apoptosis induced by the PKC-Cyto because the p38 inhibitor SB203580 and silencing of p38 only moderately inhibited cell apoptosis, suggesting that additional signaling pathways mediate the apoptotic effect of the PKC-Cyto.

In addition to the phosphorylation of the proapoptotic protein p38, the PKC-Cyto induced dephosphorylation of AKT, which regulates cell survival in a variety of cellular systems (49). The survival effects of AKT are exerted by phosphorylating proteins, such as BAD, caspase-9, the forkhead transcription factors (49), or XIAP (50). Indeed, XIAP has been recently identified as a downstream target of AKT and as an important regulator of AKT survival effects. AKT phosphorylates XIAP on Ser⁸⁷ and this phosphorylation stabilizes XIAP and prevents its ubiquitination and degradation (50). Our results indicate that the PKC-Cyto reduced the expression of XIAP and that overexpression of XIAP significantly abrogated the apoptotic effect of PKC-Cyto. Thus, the decrease in XIAP expression contributed to the apoptotic effect of PKC-Cyto in addition to the moderate effect of p38.

In contrast to the effect of the PKC-Cyto on the AKT and p38 signaling pathways, overexpression of PKC-Nuc selectively increased the phosphorylation of JNK, whereas it did not affect the phosphorylation of AKT, p38, and signal transducers and activators of transcription 1 or the expression of Bax, Bcl₂, or XIAP. To further characterize the effect of PKC-Nuc on JNK activation, we examined the phosphorylation of two JNK kinases: MKK4 and MKK7. We found that the PKC-Nuc induced the phosphorylation of MKK7, whereas it did not affect the activation of MKK4. PKC has been previously shown to activate MKK7 (51); however, this is the first report showing the role of nuclear PKC in this effect. The JNK pathway played a major role in the apoptotic effect of PKC-Nuc because inhibition of JNK significantly decreased the apoptotic effect of the PKC-Nuc. JNK has been implicated in the regulation of cell apoptosis in response to various stimuli (51, 52) and has been shown to act downstream of PKC (16, 51, 53, 54). A role for the nuclear PKC in the activation of JNK is further supported by recent studies that showed that the nuclear translocation of PKC by etoposide preceded the activation of JNK in salivary gland acinar cells (30, 53). Thus, JNK may represent another nuclear PKC substrate in addition to the already well-characterized substrates, p73 (19) and DNA-PK (20).

Interestingly, PKC-Mito did not induce any significant changes in the expression or phosphorylation of the different signaling proteins that were examined in this study, whereas it induced the release of cytochrome c from the mitochondria and the activation of caspase-9. Thus, targeting of PKC to the mitochondria seems to activate the mitochondrial pathway probably via phosphorylation of mitochondrial-associated proteins, such as scramblase 3, which has been recently identified as a substrate of PKC in the mitochondria (21).

Various studies have shown the importance of the subcellular localization of signaling proteins in their biological functions. Indeed, the targeting of Bcl₂ to the mitochondria induces cell apoptosis, whereas its targeting to the ER promotes its protective function (55). In addition, the localization of extracellular signal-regulated kinase 2 determines its protective effects against different apoptotic stimuli (56). With regard to PKC, numerous studies showed that the subcellular localization of PKC has major roles in the activity, functions, and diverse effects of the different PKC isoform effects (34, 57, 58). Less is known, however, about the role of the subcellular localization of a specific isoform in its diverse functions in a given cellular system. Our results show that the subcellular localization of PKC has an important role in its proapoptotic and antiapoptotic functions; proapoptotic effects when localized in the cytosol, mitochondria, and nucleus and antiapoptotic effect in the ER. Moreover, the localization of PKC differentially activates different downstream signaling pathways. Thus, the results of this study have important implications for our understanding of the role of PKC localization in its different effects and may provide the basis for the identification of novel PKC substrates/partners in the different subcellular sites and for the development of inhibitors that can selectively block distinct functions of PKC in a specific cellular system.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Materials
An affinity-purified polyclonal anti-PKC antibody (C-17) was purchased from Santa Cruz Biotechnology, and anti-Myc antibody was from Upstate USA, Inc. Anti-calnexin was from Stressgen, and the nuclear marker 7-AAD and the mitochondria marker MitoTracker Orange were from Molecular Probes, Invitrogen. Human TRAIL was from PeproTech, and anti–phosphorylated AKT, AKT, p38, phosphorylated p38, phosphorylated MKK3/MKK6, MKK3, phosphorylated JNK, JNK, phosphorylated MKK7, MKK7, phosphorylated MKK4, MKK4, signal transducers and activators of transcription 1, phosphorylated signal transducers and activators of transcription 1, XIAP, active caspase-3, Bax, and Bcl₂ antibodies were obtained from Cell Signaling Technology. The caspase-3 inhibitor DEVD-FMK, the p38 mitogen-activated protein kinase inhibitor SB202190, and the JNK inhibitors SP600125 and JNK inhibitor III (HIV-TAT_47-57-gaba-c-Jund_33-57) were from Calbiochem. Etoposide, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and sodium vanadate were obtained from Sigma Chemical Co.

Cell Culture
The glioma cell lines A172 and HeLa were obtained from the American Type Culture Collection. Cells were grown on tissue culture dishes in medium consisting of DMEM containing 10% heat-inactivated FCS, 2 mmol/L glutamine, 50 units/mL penicillin, and 0.05 mg/mL streptomycin. Medium was changed every 3 to 4 days and cultures were passaged using 0.25% trypsin.

Plasmid Construction
The constitutive active PKC mutant contains double point mutations of alanine to arginine at positions 144 and 145 in the inhibitory pseudosubstrate sequences within the regulatory domain as described (59). The pShooter vectors pCMV/myc/cyto, pCMV/myc/ER, pCMV/myc/mito, and pCMV/myc/nuc were purchased from Invitrogen. The inserts, rat PKC-CA (constitutively active, DR144/145A) or PKCKD (kinase-dead, K376A; ref. 9), were digested from the PKC-CA-EGFP-N1 and PKCKD plasmids using the XhoI and MluI sites and subcloned into the XhoI/NotI site of the pShooter vectors. The non–organelle-targeted vector was designated PKC-Cyto, the mitochondria-targeted vector with the mitochondria signal was designated PKC-Mito, the ER-targeted vectors that contain the ER retention signal were designated PKC-ER and PKCKD-ER, and the nucleus-targeted vectors that contain the nuclear localization signal were designated PKC-Nuc and PKCKD-Nuc. Confirmation of proper ligation was done by DNA sequencing. The pCDNA3-XIAP-Myc plasmid (Addgene plasmid 11833) was kindly provided by Dr. Guy Salvesen (University of California, San Diego, CA).

Cell Transfection
A172 cells were transfected either with the control vector or with the different PKC pShooter vectors by electroporation using the Nucleofector device, protocol number U29 (Amaxa Biosystems). Transfection efficiency using nucleofection was between 70% and 90%.

The attachment of the cells was not significantly altered by the electroporation, and the attachment of the cells overexpressing the different pShooter vectors was similar to that of the control vector.

HeLa cells were transfected using LipofectAMINE (Invitrogen) according to the manufacturer's instructions.

siRNA Transfection
Control (scrambled sequence) and p38 siRNA duplexes were synthesized and purified by Dharmacon. Transfection of siRNAs was done using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Experiments were done 72 h after transfection.

Preparation of Cell Homogenates and Immunoblot Analysis
Cell pellets (10⁶ cells/mL) were resuspended in 100 µL of lysis buffer [25 mmol/L Tris-HCl (pH 7.4), 50 mmol/L NaCl, 0.5% sodium deoxycholate, 2% NP40, 0.2% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 50 µg/mL aprotinin, 50 µmol/L leupeptin, 0.5 mmol/L Na₃VO₄] on ice for 15 min. Sample buffer (2x) was added and the samples were boiled for 5 min. Lysates (30 µg protein) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS and subsequently probed 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 enhanced chemiluminescence Western blotting detection kit (Amersham).

Immunofluorescence Staining
Transfected cells were 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, incubated with an anti-Myc antibody. Following washes in PBS, cells were incubated with an anti-rabbit Alexa Fluor 488 antibody for additional 60 min and mounted in FluoroGuard antifade reagent. Cells were viewed and photographed using confocal microscopy with x63 magnification at an excitation wavelength of 488 nm. For the visualization of the ER, cells were incubated with mouse anti-KDEL antibody followed by incubation with anti-mouse Alexa Fluor 546 antibody. Visualization of the nucleus and the mitochondria was done using labeling of the cells with the nuclear marker 7-AAD (10 mg/mL) and with the mitochondria marker MitoTracker Orange (50 nmol/L), respectively.

Cells transfected with the GFP pShooter vectors were fixed in 4% paraformaldehyde for 20 min and mounted in FluoroGuard antifade reagent.

Measurements of Cell Apoptosis
Cell apoptosis was measured using propidium iodide staining and analysis by flow cytometry as described previously (9, 24). Briefly, transfected cells (1 x 10⁵/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 PBS, and treated for 15 min with RNase (50 µmol/L) at room temperature. Cells were then stained with propidium iodide (5 µg/mL) and analyzed on a Becton Dickinson cell sorter.

Cell apoptosis was also measured using anti–histone ELISA (Cell Death Detection ELISA kit, Roche Applied Science). For these experiments, 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 a wavelength of 405 nm.

Caspase-3 and Caspase-9 Activity Assays
Cells were lysed with caspase assay lysis buffer containing 0.5% NP40 and 5 mmol/L EDTA in 50 mmol/L Tris (pH 7.5). Following 20-min incubation on ice, the cells were centrifuged at 20,000 x g at 4°C. The supernatants were placed in a 96-well plate containing reaction mixture with DEVD-AMC or Ac-LEHD-AFC, fluorimetric substrates of caspase-3 and caspase-9, respectively. Following 60 min of incubation at 37°C, fluorescence was measured at a wavelength of 380 nm.

Cytochrome c Release
Cytochrome c release from the mitochondria was determined in the cytosolic fraction. Mitochondrial and cytosolic fractions were isolated using the ApoAlert Cell Fractionation kit (Clontech, BD Biosciences) as described previously (24). Briefly, cells were centrifuged at 600 x g for 5 min at 4°C. Cell pellets were resuspended with 0.8 mL of ice-cold fractionation buffer and incubated on ice for 10 min. Cells were then homogenized with an ice-cold Dounce homogenizer and centrifuged at 700 x g for 10 min. The supernatants were then centrifuged at 10,000 x g for 25 min at 4°C and the supernatants (cytosolic fraction) and pellets (mitochondrial fraction) were collected. Cytochrome c was identified in the cytosolic fraction by using a rabbit anti–cytochrome c antibody.

Statistical Analysis
The results are presented as the mean values ± SE. Data were analyzed using ANOVA and a paired Student's t test to determine the level of significance between the different groups.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Grant support: NIH grant RO1CA109196 and William and Karen Davidson Fund, Hermelin Brain Tumor Center.

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

Note: R. Gomel and C. Xiang contributed equally to this work.

Received 8/14/06; revised 3/14/07; accepted 3/22/07.

	References

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 

1. Gutcher I, Webb PR, Anderson NG. The isoform-specific regulation of apoptosis by protein kinase C. Cell Mol Life Sci 2003;60:1061–70.[Medline]
2. Jackson DN, Foster DA. The enigmatic protein kinase C: complex roles in cell proliferation and survival. FASEB J 2004;18:627–36.[Abstract/Free Full Text]
3. Brodie C, Blumberg PM. Regulation of cell apoptosis by protein kinase c. Apoptosis 2003;8:19–27.[CrossRef][Medline]
4. Kaul S, Anantharam V, Yang Y, Choi CJ, Kanthasamy A, Kanthasamy AC. Tyrosine phosphorylation regulates the proteolytic activation of protein kinase C in dopaminergic neuronal cells. J Biol Chem 2005;280:28721–30.[Abstract/Free Full Text]
5. Kajimoto T, Shirai Y, Sakai N, et al. Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase C in the Golgi complex. J Biol Chem 2004;279:12668–76.[Abstract/Free Full Text]
6. Emoto Y, Manome Y, Meinhardt G, et al. Proteolytic activation of protein kinase C by an ICE-like protease in apoptotic cells. EMBO J 1995;14:6148–56.[Medline]
7. Chen N, Ma W, Huang C, Dong Z. Translocation of protein kinase C and protein kinase C to the membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis. J Biol Chem 1999;274:15389–94.[Abstract/Free Full Text]
8. Basu A, Woolard MD, Johnson CL. Involvement of protein kinase C- in DNA damage-induced apoptosis. Cell Death Differ 2001;8:899–908.[CrossRef][Medline]
9. Blass M, Kronfeld I, Kazimirsky G, Blumberg PM, Brodie C. Tyrosine phosphorylation of protein kinase C is essential for its apoptotic effect in response to etoposide. Mol Cell Biol 2000;22:182–95.
10. Ghayur T, Hugunin M, Talanian RV, et al. Proteolytic activation of protein kinase C by an ICE/CED 3-like protease induces characteristics of apoptosis. J Exp Med 1996;184:2399–404.[Abstract/Free Full Text]
11. Datta R, Banach D, Kojima H, et al. Activation of the CPP32 protease in apoptosis induced by 1-ß-D-arabinofuranosylcytosine and other DNA-damaging agents. Blood 1996;88:1936–43.[Abstract/Free Full Text]
12. D'Costa AM, Denning MF. A caspase-resistant mutant of PKC- protects keratinocytes from UV-induced apoptosis. Cell Death Differ 2005;12:224–32.[CrossRef][Medline]
13. Persaud SD, Hoang V, Huang J, Basu A. Involvement of proteolytic activation of PKC in cisplatin-induced apoptosis in human small cell lung cancer H69 cells. Int J Oncol 2005;27:149–54.[Medline]
14. Denning MF, Wang Y, Tibudan S, Alkan S, Nickoloff BJ, Qin JZ. Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C. Cell Death Differ 2002;9:40–52.[CrossRef][Medline]
15. Sitailo LA, Tibudan SS, Denning MF. Bax activation and induction of apoptosis in human keratinocytes by the protein kinase C catalytic domain. J Invest Dermatol 2004;123:434–43.[CrossRef][Medline]
16. Panaretakis T, Laane E, Pokrovskaja K, et al. Doxorubicin requires the sequential activation of caspase-2, protein kinase C, and c-Jun NH₂-terminal kinase to induce apoptosis. Mol Biol Cell 2005;16:3821–31.[Abstract/Free Full Text]
17. Tanaka Y, Gavrielides MV, Mitsuuchi Y, Fujii T, Kazanietz MG. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway. J Biol Chem 2003;278:33753–62.[Abstract/Free Full Text]
18. Li L, Sampat K, Hu N, Zakari J, Yuspa SH. Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes. J Biol Chem 2006;281:3237–43.[Abstract/Free Full Text]
19. Ren J, Datta R, Shioya H, et al. p73ß is regulated by protein kinase C catalytic fragment generated in the apoptotic response to DNA damage. J Biol Chem 2002;277:33758–63375.[Abstract/Free Full Text]
20. Bharti A, Kraeft SK, Gounder M, et al. Inactivation of DNA-dependent protein kinase by protein kinase C: implications for apoptosis. Mol Cell Biol 1998;18:6719–28.[Abstract/Free Full Text]
21. Liu J, Chen J, Dai Q, Lee RM. Phospholipid scramblase 3 is the mitochondrial target of protein kinase C-induced apoptosis. Cancer Res 2003;63:1153–6.[Abstract/Free Full Text]
22. Cross T, Griffiths G, Deacon E, et al. PKC- is an apoptotic lamin kinase. Oncogene 2002;19:2331–7.
23. Jun CD, Oh CD, Kwak HJ, et al. Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways. J Immunol 1999;162:3395–401.[Abstract/Free Full Text]
24. Okhrimenko H, Lu W, Xiang C, et al. Roles of tyrosine phosphorylation and cleavage of protein kinase C in its protective effect against tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis. J Biol Chem 2005;280:23643–52.[Abstract/Free Full Text]
25. Zrachia A, Dobroslav M, Blass M, et al. Infection of glioma cells with Sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase C. Implications for Sindbis virus-induced apoptosis. J Biol Chem 2002;277:23693–701.[Abstract/Free Full Text]
26. Clark AS, West KA, Blumberg PM, Dennis PA. Altered protein kinase C (PKC) isoforms in non-small cell lung cancer cells: PKC promotes cellular survival and chemotherapeutic resistance. Cancer Res 2003;63:780–6.[Abstract/Free Full Text]