Cleavage of ζPKC but Not λ/ιPKC by Caspase-3 during UV-induced Apoptosis*

The stimulation of caspases is a critical event in apoptotic cell death. Several kinases critically involved in cell proliferation pathways have been shown to be cleaved by caspase-mediated mechanisms. Thus, the degradation of δ protein kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments corresponding to their catalytic domains, consistent with the observations that both enzymes are important for apoptosis. In contrast, other kinases reported to have anti-apoptotic properties, such as Raf-1 and Akt, are inactivated by proteolytic degradation by the caspase system. Since the atypical PKCs have been shown to play critical roles in cell survival, in the study reported here we have addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although ζPKC and λ/ιPKC are both inhibited in UV-treated cells, only ζPKC but not λ/ιPKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is enzymatically inactive. The sequence where caspase-3 cleaves ζPKC was mapped, and a mutant resistant to degradation was shown to protect cells from apoptosis more efficiently than the wild-type enzyme.

The stimulation of caspases is a critical event in apoptotic cell death. Several kinases critically involved in cell proliferation pathways have been shown to be cleaved by caspase-mediated mechanisms. Thus, the degradation of ␦ protein kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments corresponding to their catalytic domains, consistent with the observations that both enzymes are important for apoptosis. In contrast, other kinases reported to have anti-apoptotic properties, such as Raf-1 and Akt, are inactivated by proteolytic degradation by the caspase system. Since the atypical PKCs have been shown to play critical roles in cell survival, in the study reported here we have addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although PKC and /PKC are both inhibited in UV-treated cells, only PKC but not /PKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is enzymatically inactive. The sequence where caspase-3 cleaves PKC was mapped, and a mutant resistant to degradation was shown to protect cells from apoptosis more efficiently than the wild-type enzyme.
The two members of the atypical protein kinase C (PKC) 1 subfamily of isozymes (aPKCs), namely PKC and /PKC, have recently been shown to be involved in a number of important cellular functions including cell proliferation and survival (1)(2)(3)(4)(5). The mechanisms whereby the aPKCs control these functions most probably involve their ability to regulate ERK-AP1 (6 -11) and NF-B (10,(12)(13)(14)(15)(16)(17)(18) signaling pathways, both important intermediaries in the cascades controlling cell growth and apoptosis (2, 19 -26). Contrary to the other PKCs, the atypicals are insensitive to diacylglycerol and 12-O-tetradecanoylphorbol-13-acetate but have been proposed to be regulated by ceramide (18,27,28), phosphatidylinositol 3-kinase (10, 29 -34), and Ras (8,35,36). In addition, the aPKCs selectively bind to, and are regulated, by two novel proteins (LIP and Par-4) which seem to be critical components of pathways controlling cell growth and survival. Thus, LIP (for /PKC-interacting protein) potently induces NF-B in a /PKC-dependent manner (14), whereas Par-4 is a potent inhibitor of the atypical PKC enzymatic activity and, interestingly, was initially identified by differential screening in cells that were undergoing growth arrest and apoptosis (3,37). Consistently, the ectopic expression of Par-4 in NIH-3T3 cells induces apoptosis in a manner that is dependent on its ability to block the atypical PKCs (2,3). Recent studies demonstrate that the expression of Par-4 sensitizes prostate cancer and melanoma cells to apoptotic stimuli (38), as well as that it may be a mediator of neuronal apoptosis (39). Overexpression of the atypical PKCs inhibits UV-and drug-induced apoptosis in NIH-3T3 (2, 3) and human leukemia cells (5), respectively. Taken together, these results indicate that the aPKCs are potent anti-apoptotic kinases that can be inactivated by pro-apoptotic regulators such as Par-4.
The activation of the caspase system is a critical event in apoptosis (40 -42), and recent studies demonstrate that a number of signal transduction kinases are subjected to a caspasemediated breakdown (43). Early work reported that the degradation of ␦PKC by caspase-3 generates an active fragment corresponding to its catalytic domain (44), suggesting that ␦PKC activity may be important during apoptotic cell death (45). Consistently, inhibition of ␦PKC impairs UV-induced apoptosis in keratinocytes (46). MEKK-1 is also cleaved during apoptosis by anoikis (47), Fas ligation (48), and genotoxic stress (49). In all three cases, the caspase-mediated degradation of MEKK-1 produced a catalytically active fragment, in keeping with the pro-apoptotic properties of MEKK-1 (47)(48)(49). PKN is another example of a kinase degraded by the caspase system producing a catalytically active fragment (50). However, other kinases, such as Raf-1 and Akt are inactivated by proteolytic degradation by the caspase system during apoptosis (43). This is particularly relevant because both Raf-1 and Akt are antiapoptotic kinases (50 -56). Therefore, a model is emerging whereby the activation of caspases in apoptotic cells leads to the cleavage of different kinases with opposite outcomes, depending on whether the given kinase plays a pro-or an antiapoptotic role. Because the aPKCs control pro-survival signals, in the study reported here we have addressed the potential degradation of the atypical PKCs by the caspase system in HeLa cells exposed to UV irradiation.
Cell Culture, Transfections, and Reagents-HeLa cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 g/ml penicillin G, and 100 g/ml streptomycin (FLOW). Subconfluent cells were transfected by the calcium phosphate method (CLONTECH, Inc.). Some experiments were performed in the presence of the ICE inhibitors YVAD-CHO, DEVD-CHO, or z-VAD-fmk (Bachem, United Kingdom). For UV treatment, culture medium was removed, dishes were washed once with phosphate-buffered saline, UVC irradiated (180 J/m 2 ), and fresh medium was added to the cells. The monoclonal anti-/PKC was from Transduction Laboratories. Anti-Myc epitope (9E10) antibody was from UBI.
aPKC Activity-HeLa cells incubated at different times after UV light exposure were extracted with lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM EGTA, and protease inhibitors) and immunoprecipitated with a monoclonal anti-/PKC. Immunoprecipitates were washed seven times with lysis buffer containing 0.5 M NaCl. For in vitro kinase assay, immunocomplexes were incubated with 1 g of myelin basic protein and 5-10 Ci (100 M) of [␥-32 P]ATP in kinase buffer (35 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.5 mM EGTA, 0.1 mM CaCl 2 , 1 mM phenyl phosphate) for 30 min at 30°C in a final volume of 20 l. Reactions were stopped by the addition of concentrated sample buffer. Samples were boiled for 3 min and separated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by exposure and quantitation in an InstantImager (Packard). The activity of transfected Myc-PKC was determined in immunoprecipitates with an anti-Myc antibody (9E10, UBI) as described above.
In Vitro Protease Assays-Wild-type PKC and the different mutants were translated in vitro using a coupled transcription and translation system with T7 polymerase (Promega, Madison, WI). Apoptotic cells extract was prepared as follows. HeLa cells were UV-irradiated for 10 h, washed once in ice-cold phosphate-buffered saline, and resuspended at 2 ϫ 10 8 /ml in lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM dithiothreitol, 0.1% CHAPS, 10% sucrose, and 1 mM phenylmethylsulfonyl fluoride). The lysate was then subjected to four freeze-thaw cycles prior to centrifugation at 10,000 ϫ g for 10 min. Recombinant caspase-3 was from Pharmingen. Cell extracts, in the absence or presence of caspase inhibitors (10 nM; YVAD-CHO or DEVD-CHO), or purified caspase-3 were incubated for 1 h at 37°C with 5 l of 35 S-labeled in vitro translated wild-type or mutant PKC. The reaction was stopped by addition of Laemmli sample buffer and subjected to SDS-PAGE prior to drying and exposure in an InstantImager (Packard).
Apoptosis Assays-␤-Galactosidase co-transfection assays for determination of cell death were performed as described (2,3). TUNEL analysis was performed using the "in situ cell death detection kit" (Boehringer Mannheim).

RESULTS
We initially determined the effect of UV irradiation on the activity and potential cleavage of /PKC and PKC during apoptosis. HeLa cells were UV-irradiated for different times, after which endogenous /PKC was immunoprecipitated and its activity was determined. Parallel extracts were analyzed by immunoblot. Results of Fig. 1A demonstrate that short exposures (5-20 min) to UV irradiation provokes a measurable activation of /PKC activity. However, longer treatments leads to a robust reduction of /PKC activity to below basal levels, that is detectable at 2 h, and maximal at 4 -20 h. Immunoblot analysis reveals that the protein levels of /PKC remain unchanged at all the time points measured. Identical results were obtained when this experiment was performed in HeLa cells that have been transfected with a Myc-tagged /PKC construct and in which the activity and levels of the enzyme were determined with an anti-Myc antibody (not shown). Because there are not reliable antibodies selective for the PKC isoform, we next transfected HeLa cells with a Myc-tagged version of PKC after which cells were UV-irradiated as above. Results of Fig.  1B show that, in contrast to /PKC, short-term irradiation does not affect PKC activity. However, longer exposures to UV light produces a dramatic reduction on PKC activity that precedes its proteolytic degradation that produces a fragment of approximately 50 kDa. Because this PKC construct was Myc-tagged at the COOH terminus, this fragment must include the catalytic domain. Therefore, the anti-Myc immunoprecipitates contained not only the full-length protein but also the fragments generated by UV irradiation (not shown). The fact that the enzymatic activity is reduced in UV-irradiated cells indicates that the PKC 50-kDa fragment generated by UV irradiation is catalytically inactive. TUNEL analysis of parallel cultures indicated that apoptosis becomes detectable at 10 h. At this time point, 12 Ϯ 2% of the cells showed signs of DNA fragmentation. At 20 h, the number of apoptotic cells increased to 30 Ϯ 10%, whereas at 40 h, virtually all cells were apoptotic.
In the next series of experiments the involvement of the caspase system in the degradation of PKC was determined. Thus, HeLa cells transfected with Myc-tagged PKC were UV- irradiated either in the absence or presence of z-VAD, a broadspecific inhibitor of caspases, and the activity and degradation of PKC was determined. Results of Fig. 2A demonstrate that the presence of z-VAD completely abrogated the UV-induced cleavage of PKC with little or no effect on its inhibition. Of note, the presence of z-VAD did not affect the changes in activity of /PKC produced by UV irradiation (Fig. 2B). Collectively these results indicate that PKC is degraded by a caspase-mediated process during UV irradiation but that its inhibition is caspase-independent.
To further explore the possible breakdown of PKC by caspases, 35 S-labeled in vitro translated PKC or /PKC were incubated in vitro with extracts from cell cultures that were either untreated or exposed to UV light for 10 h. Interestingly, a fragment of identical size to that produced in vivo and most probably corresponding to the catalytic domain of the enzyme was observed when PKC but not /PKC was incubated with extracts from UV-irradiated cells (Fig. 3A). In addition, another fragment of about 20 -30 kDa was also detected which most probably corresponds to the regulatory domain of PKC (Fig. 3A). The cleavage of PKC is prevented by the incubation with z-VAD or DEVD, but not by YVAD (Fig. 3B). Because DEVD is relatively specific for caspase-3 whereas YVAD is for caspase-1, these results strongly suggest that PKC may be a substrate of caspase-3. This is confirmed in the results of Fig.  3C demonstrating that recombinant caspase-3 efficiently cleaves PKC but not /PKC in vitro.
The size of the PKC fragments generated by the caspase action suggests that the cleavage must occur at the hinge region linking the regulatory and the catalytic domains. In addition, because /PKC is resistant to caspase-mediated breakdown, the caspase cleavage site(s) should be present in a region of PKC with no sequence homology with /PKC. The hinge domain in the atypical PKCs is, together with the V1 region, where the major differences exist between PKC and /PKC (57). Interestingly, the alignment of the hinge regions of PKC and /PKC reveals the existence of three potential caspase sites in PKC that are absent in /PKC. These sites are underlined in Fig. 4 and named as 1, 2, and 3. In order to determine which of these potential sites are important for PKC breakdown, different PKC mutants that abrogate the caspase consensus sequence were constructed, translated in vitro, and incubated with extracts from UV-irradiated cells. The individual disruption of sites 1, 2, and 3, produced no effect on the degradation of PKC (Fig. 5). However, although the double 1/2 mutant was also cleaved, the 2/3 and 1/2/3 mutants were completely resistant to degradation (Fig. 5). This indicates that sites 2 and 3 are the targets of the caspase-mediated breakdown of PKC. In order to explore the role of sites 2 and 3 in vivo, Myc-tagged PKC 2/3 mutant (myc-PKC 2/3 ) was transfected into HeLa cells, after which they were UV-irradiated for different times and the activity and levels of this PKC mutant were determined. Results of Fig. 6 demonstrate that although myc-PKC 2/3 is completely resistant to degradation, its enzymatic activity is dramatically inhibited by UV irradiation. Collectively these findings indicate that UV irradiation inhibits both /PKC and PKC activities. This is consistent with previous data from this laboratory and may be due to the interaction of these PKCs with the selective protein inhibitor Par-4 (2, 3). A previously unreported observation is the degradation of PKC mediated by caspase-3. This generates a fragment that is identical to the catalytic domain of PKC, that should be permanently active because it lacks the regulatory region which keeps the enzyme blocked through the binding of the catalytic domain to the pseudosubstrate sequence (57). However, the data presented in this study (Fig. 1B), suggest that the catalytic fragment generated by UV irradiation is inactive, since at 20 h its enzymatic activity still is inhibited despite the fact that all the PKC has been broken-down to its catalytic fragment. To more firmly demonstrate this possibility, HeLa cells were transfected with a GST-tagged version of the catalytic domain of PKC (GST-PKC CAT ) encompassing residues from site 3 to the end of the protein. Afterward, cells transfected with the catalytic PKC were UV irradiated for different times, and the levels and activity of the GST-PKC CAT construct were determined. Results of Fig. 7 demonstrate that UV irradiation produces a robust inhibition of the catalytic construct, indicating that some signal is generated by this stress treatment that not only inhibits the full-length protein, as previously reported (2) and shown in Fig. 1B, but also the catalytic fragment that is generated during the UV-irradiation process.
The results of Table I demonstrate that the cleavage of PKC by caspase-3 is a critical event for apoptosis. To address this, we used a ␤-galactosidase cell viability assay. HeLa cells were transfected with either a control expression vector or plasmids for wild-type or caspase-resistant PKC, after which the cultures were UV-irradiated and the number of ␤-galactosidasepositive (blue) cells were scored 36 h thereafter. Interestingly, the expression of caspase-resistant PKC protected HeLa cells from UV-induced apoptosis more efficiently than the wild-type enzyme (Table I). These results suggest that the caspase-mediated cleavage of PKC significantly contributes to apoptotic cell death.

DISCUSSION
Caspase activation is a critical and ultimate step in apoptotic cell death. A number of substrates have been reported to be the target of these proteolytic enzymes (40 -42). Among those, considerable attention has recently been focused on the caspasemediated degradation of enzymes involved in cell growth and survival. Thus, the caspase-mediated cleavage seems to trigger the activation of pro-apoptotic kinases, such as MEKK-1 or PKN (46 -49), and the inactivation of anti-apoptotic or proliferative kinases, such as Raf-1 or Akt (43). Regarding PKC, the ␦ isoform has been shown to be cleaved by caspases, which results in the generation of a catalytically active fragment (44,45). This suggests that ␦PKC activation by proteolytic degra- dation may be considered a critical step in apoptosis. Actually, expression of the catalytic fragment of ␦PKC is sufficient to induce programmed cell death (44,45).
In this study we demonstrate a differential regulation of PKC and /PKC during UV-induced apoptosis. Thus, /PKC is stimulated at early times after UV-irradiation whereas PKC is not (Fig. 1). Recent studies from this laboratory show that /PKC can be selectively activated by the novel regulator LIP (14). It is possible that an increased interaction with this protein after UV irradiation may account for the selective activation of /PKC by this stress stimulus. 2 Perhaps more interesting from the point of view of the role of the atypical PKCs in apoptosis, is our observation that both aPKCs are inhibited in UV-irradiated cells, but that only PKC is proteolytically cleaved. This cleavage seems to be produced by the direct action of caspase-3 on two sites in the hinge region of PKC. However, although the degradation of PKC generates a fragment that under normal conditions is more active than the full-length protein, in UV-irradiated cells this fragment is completely inactive. This is in marked contrast to what has been reported for ␦PKC and is in keeping with the notion that PKC is a pro-survival enzyme that needs to be blocked for apoptosis to proceed (1-3, 44, 45). The observation that a caspase-resistant mutant of PKC protects cells from apoptosis more efficiently than the wild-type enzyme, indicates that the proteolytic degradation of PKC is an additional and critical way to modulate UV-induced apoptosis in HeLa cells contributing to the inevitability of the apoptotic cell death. We have previously shown that Par-4 induction during UV irradiation is a mechanism for the inactivation of the atypical PKCs (2, 3). However, Par-4 cannot interact with the catalytic domain of those kinases (3); therefore, the inhibition of the enzymatic activity of the catalytic fragment in the UV-irradiated cells, detected in this study, indicates that mechanisms in addition to the binding of Par-4 to the regulatory domain must operate for the blockage of PKC during apoptosis. These mechanisms will probably target the catalytic domain of the kinase, but its precise nature remains to be determined. However, we are tempted to speculate that the activation of phosphatases may promote the de-phosphorylation of critical residues in the activation loop of PKC that are required for its basal activity (58,59). These residues have been reported to be phosphorylated by PDK-1, a phosphatidylinositol 3-kinase-dependent enzyme (30,31). Although there is no published evidence that phosphatidylinositol 3-kinase is inactivated in UV-irradiated HeLa cells, it is blocked in other forms of apoptosis, such as in anoikis (56), in ceramide-treated cells (60), or following genotoxic stress (61). Therefore, it is possible that the inactivation of phosphatidylinositol 3-kinase by stress leads to the de-phosphorylation of residues important in the activation loop of PKC which, together with the binding of Par-4, may be the critical events on its inhibition.

TABLE I
Enhanced protection from UV-induced apoptosis by the caspase-resistant PKC mutant HeLa cells were transfected with pCMV-␤gal (2.5 g) and either 2 or 10 g of either plasmid control pCDNA3 (control) or expression vectors for wild-type (PKC) or caspase-resistant (PKC 2/3 ) PKC. Twenty-four h post-transfection cells were induced to undergo apoptosis by UV irradiation and 36 h later they were fixed and stained with 5-bromo-4-chloro-3-indoyl ␤-D-galactoside. Results (number of blue cells per 35-mm dish) are the mean Ϯ S.D. of three independent experiments with incubations in duplicate. Statistical differences from control untreated cell cultures.