Proteolytic Cleavage and Activation of Protein Kinase C μ by Caspase-3 in the Apoptotic Response of Cells to 1-β-d-Arabinofuranosylcytosine and Other Genotoxic Agents

Protein kinase C (PKC) μ is a novel member of the PKC family that differs from the other isozymes in structural and biochemical properties. The precise function of PKCμ is not known. The present studies demonstrate that PKCμ is cleaved during apoptosis induced by 1-β-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. PKCμ cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-xL protein or the baculovirus p35 protein. Our results demonstrate that PKCμ is cleaved by caspase-3 at the CQND378S site. Cleavage of PKCμ is associated with release of the catalytic domain and activation of its kinase function. We also show that, unlike the cleaved fragments of PKCδ and θ, overexpression of the PKCμ catalytic domain is not lethal. Cells stably expressing the catalytic fragment of PKCμ, however, are more sensitive to apoptosis induced by genotoxic stress. In addition, expression of the caspase-resistant PKCμ mutant partially inhibits DNA damage-induced apoptosis. These findings demonstrate that PKCμ is cleaved by caspase-3 and that expression of the catalytic domain sensitizes cells to the cytotoxic effects of ara-C and other anticancer agents.

Protein kinase C (PKC) 1 is a family of phospholipid-dependent serine/threonine kinases that play a major role in regulating a wide variety of physiological processes (1). Based on their structure and cofactor regulation, the PKC isozymes have been divided into the conventional (cPKC; ␣, ␤, ␥), novel (nPKC; ␦, ⑀, , ), and atypical (aPKC; , /) subclasses (1). In contrast to other PKC isozymes, PKC lacks a region homologous to the typical pseudosubstrate domain and contains a pleckstrin homology (PH) domain and thus represents a distinct PKC subclass (2,3). The recently identified PKC exhibits a high degree of homology to PKC and has been designated to this subclass (4).
Treatment of human tumor cell lines with genotoxic agents is associated with induction of apoptosis (5,6). Efforts to define the role of PKC in apoptosis in part have been complicated by the expression of multiple isoforms in different cell types and their involvement in both pro-and antiapoptotic signaling cascades. Studies have demonstrated that PKC and selectively interact with Par-4 and abrogate its anti-apoptotic effects (7). PKC␣ has been shown to phosphorylate Bcl-2 and suppress apoptosis in Pre-B REH cells (8). Other studies have shown that PKC␦ and are proteolytically cleaved and activated by caspase-3 during apoptosis induced by diverse anticancer agents (9 -11). Caspase-3-mediated cleavage of PKC␦ and occurs in the third variable region, which separates the regulatory and catalytic domains. Cleavage of PKC␦ and isoforms by caspase-3 results in release and activation of the catalytic domain (10,11). The findings that proteolytic cleavage of PKC␦ and is inhibited by overexpression of the anti-apoptotic Bcl-x L protein or of the baculovirus p35 protein have supported their involvement in apoptosis (9,11). Other studies have demonstrated that overexpression of the kinase-active PKC␦ and catalytic domains, but not full length or kinase-inactive fragments, results in induction of certain features characteristic of apoptosis (10,11). Conversely, PKC, which plays a critical role in cell survival, is cleaved and inactivated by caspase-3 during UV-induced apoptosis (12).
In contrast to other PKC isoforms, PKC and its mouse homologue PKD has unique enzymatic features and a distinct substrate specificity (13)(14)(15). These findings have suggested that PKC is involved in novel signaling pathways. PKC is located in the Golgi bodies and is involved in basal transport processes (16). Recent studies have demonstrated that the 14-3-3 protein interacts with PKC and negatively regulates its activity (17). Other studies have shown that overexpression of PKC reduces sensitivity to tumor necrosis factor (TNF)-induced but not ceramide-induced apoptosis (18). However, the precise role of PKC in intracellular signaling cascades during apoptosis remains unclear.
The present studies demonstrate that PKC is cleaved during apoptosis induced by 1-␤-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents. The results demonstrate that PKC is cleaved by caspase-3 at the CQND 378 S site between regulatory and catalytic domains. Cleavage of PKC results in activation of its kinase function. We also show that overexpression of the cleaved catalytic domain sensitizes cells to the cytotoxic effects of genotoxic agents.

MATERIALS AND METHODS
Cell Culture and Transfection-Human U-937 myeloid leukemia cells (American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. Human osteosarcoma cell lines SAOS2 and U2OS were * This investigation was supported by USPHS Grants GM58200 (to R. D.) and CA29431 (to D. K.) awarded by the DHHS, National Institutes of Health. 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. § These authors contributed equally. ʈ To whom correspondence should be addressed. Tel.: 617-632-2939; Fax: 617-632-2933; E-mail: rakesh_datta@dfci.harvard.edu 1 The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; PH, pleckstrin homology; ara-C, 1-␤-D-arabinofuranosylcytosine; PKD, protein kinase D; TNF, tumor necrosis factor; CF, catalytic fragment; PBS, phosphatebuffered saline; IR, ionizing radiation; GFP, green fluorescence protein.
Immunoblot Analysis-Cell lysates were prepared as described (21). Proteins were subjected to electrophoresis in 10% SDS-polyacrylamide gels and then transferred to nitrocellulose paper. The residual binding sites were blocked by incubating the filters with 5% dry milk in PBST (phosphate-buffered saline (PBS)/0.05% Tween 20). The filters were incubated with anti-PKC polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-caspase-3 (21). After washing twice with PBST, the blots were incubated with anti-rabbit IgG peroxidase conjugate (Amersham Pharmacia Biotech). The antigen-antibody complexes were visualized using chemiluminescence (ECL detection system; Amersham Pharmacia Biotech).
Apoptosis Assays-Analysis of DNA fragmentation was performed as described (21). Briefly, cells (5 ϫ 10 6 ) were harvested, washed, and incubated in 50 l of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K (Sigma) for 6 h at 50°C. The samples were incubated with 50 l of 10 mM EDTA (pH 8.0) containing 2% (w/v) agarose at low melting point and 40% sucrose for 10 min at 70°C. The DNA was separated in 2% agarose gels. After treatment with RNase, the gels were visualized by UV illumination. HeLa cells were suspended at a density of 1 ϫ 10 7 cells per ml and transfected by electroporation (0.22 V, 960 F). Analysis of DNA content was performed by staining ethanol-fixed cells with propidium iodide and monitoring by FACScan (Becton-Dickinson). The number of cells with sub-G 1 DNA content were determined with a MODFIT LT program (Verity Software house, Topsham, ME).
In Vitro Translation and Protease Cleavage Assays-The C-terminal PKC fragment (PKC (349 -912)) generated by polymerase chain reaction from the full length PKC cDNA was cloned into the pcDNA3 vector. PKC (D348A), PKC (D378A), and PKC (D391A) were generated in two steps by overlapping primer extension. [ 35 S]Methioninelabeled PKC wild type, mutants, and PKC (349 -912) were synthesized by coupled transcription and translation reactions (Promega, Madison, WI). Labeled proteins were incubated with 5 g/ml Escherichia coli-derived caspase-3 in 50 mM HEPES (pH 7.5), 10% glycerol, 2.5 mM dithiothreitol, and 0.25 mM EDTA at room temperature for 30 min (22). Cleavage reactions were also performed in the presence of 5 g of cytoplasmic extract from untreated or ara-C-treated cells. The reaction products were analyzed using electrophoresis in 10 or 12% SDS-polyacrylamide gels and then by using autoradiography.
Analysis of Kinase Activity-Full length PKC proteins prepared by coupled transcription and translation were incubated with caspase-3 alone or in the presence of recombinant p35 (20,21). Protein kinase assays were performed as described (PKC assay kit; Life Technologies, Inc., Gaithersburg, MD) using glycogen synthase as a substrate. Proteins prepared from U-937 and U-937/CF cells were subjected to immunoprecipitation with anti-PKC antibody. Immune complex kinase assays were performed by incubating the immunoprecipitates in kinase buffer (50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 4 mM MgCl 2 , 50 M ATP, 1 Ci of [␥-32 P]ATP, and 5 g of glycogen synthase substrate) for 10 min at 30°C. An equal volume of the reaction was spotted on phosphocellulose paper. The filters were then washed twice with 1% phosphoric acid and once with 95% ethanol. The amount of radiolabeled phosphate incorporated into the peptide was quantified by liquid scintillation counting.

RESULTS
Previous studies have demonstrated that ara-C induces apoptosis of human U-937 myeloid leukemia cells (6). To determine whether PKC is cleaved during apoptosis, we treated U-937 cells with ara-C and harvested the cells at various times. Immunoblot analysis of the lysates with an anti-PKC antibody demonstrated time-dependent decreases in the 110-kDa PKC protein and increases in a 60-kDa cleaved fragment (Fig.   1). The kinetics of cleavage of PKC coincided with the activation of caspase-3 and the appearance of internucleosomal DNA fragmentation (Fig. 1). Because ara-C incorporates into DNA and induces DNA strand breaks (23,24), we studied the effects of other classes of DNA-damaging agents on proteolytic cleavage of PKC. Ionizing radiation (IR) induces DNA strand breaks either by direct interaction with DNA or through the formation of reactive oxygen intermediates (25). Cisplatin induces DNA intrastrand cross-links (26), whereas etoposide induces DNA strand breaks as a result of forming a complex with topoisomerase II and the DNA 5Ј terminus (27). Treatment of cells with IR, cisplatin, or etoposide was associated with the cleavage of PKC to a 60-kDa fragment ( Fig. 2A). As shown with ara-C, cleavage of PKC coincided with the induction of DNA fragmentation by these agents (Fig. 2A). Treatment of osteosarcoma cell lines SAOS2 and U2OS with cisplatin also resulted in cleavage of PKC to a 60-kDa fragment (Fig. 2B). These results demonstrate that treatment of different cell types with diverse DNA-damaging agents is associated with the cleavage of PKC during apoptosis.
U-937 cells that overexpress Bcl-x L (U-937/Bcl-x L ) exhibit resistance to induction of apoptosis by blocking release of mitochondrial cytochrome c and activation of caspase-3 (28). Although exposure of U-937/neo cells to ara-C resulted in cleavage of PKC, there was no detectable cleavage in ara-C-treated U-937/Bcl-x L cells (Fig. 3). The cowpox virus protein CrmA and baculovirus protein p35 have been shown to prevent apoptosis by inhibiting caspases. Previous studies have demonstrated that apoptosis induced by genotoxic agents is mediated by a CrmA-insensitive and p35-sensitive mechanism (20, 21). Using the previously characterized U-937 transfectants stably overexpressing CrmA or p35, we found that CrmA expression has FIG. 1. Proteolytic cleavage of PKC by ara-C. U-937 cells were treated with 10 M ara-C for the indicated times. Lysates were subjected to immunoblot analysis using anti-PKC antibody (upper panel) and anti-caspase-3 antibody (middle panel). DNA was analyzed for fragmentation in agarose gels (lower panel). no effect on ara-C-induced proteolysis of PKC (Fig. 3). By contrast, cleavage of PKC was inhibited following ara-C treatment of U-937/p35 cells (Fig. 3). These findings indicated that PKC is cleaved by a p35-sensitive caspase-like protease.
DNA damage-induced apoptosis is associated with the activation of caspase-3 (20,21). Other studies have shown that caspase-3 is insensitive to CrmA but is inhibited by p35 (21,22,29). To determine whether PKC is cleaved by caspase-3, PKCFL labeled with [ 35 S]methionine was incubated with purified recombinant caspase-3. PKC was cleaved to 60-and 50-kDa fragments by caspase-3 (Fig. 4A). In addition, incuba-tion of PKC with cytosol from ara-C-treated apoptotic U-937 cells resulted in the appearance of similarly cleaved PKC fragments, whereas lysates from untreated cells had little, if any, effect (Fig. 4A). The finding that the apparent molecular masses of the two cleaved fragments (60 and 50 kDa) were together approximately equal to the full length PKC suggested that one site in PKC is sensitive to protease cleavage. Caspase-3 prefers a DXXD-like substrate with an Asp residue at both the P1 and P4 positions (30). PKC has two DXXD sites between the cysteine-rich and PH domains, either of which can yield cleaved fragments of approximately the same size as those observed on immunoblots ( Fig. 4B; schematic of PKC structure). To define the caspase-3-mediated cleavage site in PKC, we constructed a C-terminal (349 -912) PKC fragment (Fig. 4B). Incubation of in vitro translated C-terminal PKC fragment (m349 -912) with purified recombinant caspase-3 resulted in cleavage to a 60-kDa fragment (Fig. 4C). Similar findings were obtained with lysates from ara-C-treated cells but not after incubation of the C-terminal PKC fragment with control lysates (Fig. 4C). Immunoblot analysis of caspase-3-cleaved 349 -912 with an antibody reactive at the C terminus (amino acids 893-912) demonstrated detection of the 60-kDa cleaved fragment (data not shown). These findings indicated that the PKC cleavage site is located at the N terminus of 349 -912. Consequently, to identify the cleavage site in PKC, we generated three PKC mutants with substitution of Asp residues in DDND 348 S, CQND 378 S, and DHED 391 S by Ala (D348A, D378A, and D391A). Incubation with caspase-3 resulted in cleavage of wild type, D348A and D391A mutants to the predicted size fragment (Fig. 4D). By contrast, there was no detectable caspase-3-mediated cleavage of the D378A mutant. Taken together, these findings demonstrate that PKC is cleaved by caspase-3 at the CQND 378 S site (Fig. 4D).
Cleavage of PKC at the CQND 378 S Site Is Associated with Separation of the Regulatory and Kinase Domains-To determine whether cleavage of PKC is associated with activation of the kinase function, we incubated in vitro translated PKCFL with recombinant caspase-3 and assessed activity by phosphorylation of glycogen synthase peptide. The results demonstrate that cleavage of PKCFL with caspase-3 is associated with increases in the PKC kinase function (Fig. 5A). By contrast, preincubation of recombinant caspase-3 with p35, which inhibits PKC cleavage, blocked the increase in kinase activity (Fig.  5A). To define the functional significance of PKC cleavage, we transfected HeLa cells with PKCCF cloned into a vector expressing the green fluorescence protein (GFP). GFP-positive transfectants were selected by flow cytometry and assayed for sub-G 1 DNA content. There were no apparent effects of PKCCF expression on growth or apoptosis (data not shown). U-937 cells were also transfected with the PKCCF cDNA inserted in the pEF-Neo expression plasmid. Cells transfected with pEF-PKCCF overexpressed PKCCF protein as compared with cells transfected with vector alone (Fig. 5B). Anti-PKC immunoprecipitates from U-937/neo and U-937/CF cells were assayed for phosphorylation of the glycogen synthase peptide. The results demonstrate over a 2-fold increase in PKC activity in U-937/CF as compared with U-937/neo cells (Fig. 5C). Taken together, these findings support activation of PKC activity by caspase-3-mediated cleavage of PKCFL to PKCCF.
Because exposure of cells to genotoxic agents induces cleavage of PKC, we asked if PKCCF affects the sensitivity of cells to DNA damage-induced apoptosis. The U-937/neo and U-937/CF cells were treated with 100 nM ara-C and assayed for DNA fragmentation. In U-937/neo cells, internucleosomal DNA cleavage was observed at 24 h after ara-C treatment (Fig.  6A). However, exposure of U-937/CF cells to ara-C resulted in induction of DNA cleavage, which was detectable as early as 6 h (Fig. 6A). To assess whether the expression of PKCCF sensitizes cells to other genotoxic agents, we treated the transfectants with 20 ng/ml etoposide or 10 M cisplatin and assayed for DNA fragmentation. Although there was no apparent effect of etoposide or cisplatin on U-937/neo cells at 14 h, these agents induced characteristic DNA ladders in U-937/CF cells (Fig.  6B). Apoptosis was also monitored by analyzing cells for sub-G 1 DNA content. Treatment of U-937/CF cells with ara-C, cisplatin, or etoposide was associated with increases in the percentage of cells with sub-G 1 DNA as compared with that found following transfection of empty vector (Table I). Similar results were obtained in another cell population, designated U-937/ CF -1, which expresses a lower level of PKCCF and kinase activity (Table I and data not shown). To define further the role of PKC in apoptosis, we transfected HeLa cells with wild type PKC or the caspase-3-resistant PKCD 378 A mutant and vector expressing GFP. After 24 h, the transfected cells were exposed to cisplatin and incubated for additional 14 h. GFP-positive cells were then analyzed for sub-G 1 DNA content. Compared with cells transfected with wild type PKC, overexpression of PKCD 378 A partially inhibited cisplatin-induced apoptosis (Fig. 6C). Taken together, these results indicate that the cleavage of PKC contributes to DNA damage-induced apoptosis and that PKCCF expression sensitizes cells to the apoptotic effects of diverse genotoxic drugs. DISCUSSION PKC isoforms function in signal transduction pathways that regulate cell growth, differentiation, and apoptosis (1,31). The classic, novel, and atypical PKCs all possess a highly conserved catalytic domain (1). The catalytic domains of PKC and its mouse homologue PKD, however, exhibit little similarity to the other PKC family members (2,3). PKC and PKD also exhibit a distinct substrate specificity (13)(14)(15). Nonetheless, PKC contains a tandem-repeat of cysteine-rich, zinc-finger-like motifs that bind phorbol esters (2). In addition, PKC, like members of the PKC family, is activated by phorbol esters and phospholipids (14,32). The activity of most PKC family members is controlled by a pseudosubstrate region in the regulatory domain that functions as an inhibitor of the active site in the catalytic domain (1). PKC/PKD, however, lacks the typical pseudosubstrate region, and in contrast to the other PKC isoforms, contains a PH domain (2,3). Diverse signaling and cytoskeletal proteins contain PH domains that regulate their subcellular localization and activation (33). Studies of PKC have shown that mutants with deletions or amino acid substitutions in the PH domain exhibit increased basal kinase activity (34). These findings have indicated that the PKC PH domain functions as a negative regulator of the catalytic domain. The present studies demonstrate that PKC is cleaved between the cysteine-rich and PH domains during induction of apoptosis. The results also demonstrate that the C-terminal cleavage product, which contains the catalytic domain, exhibits an increased basal kinase activity. Together, these results suggest that cleavage in this region relieves the inhibitory effects of PH domain on the catalytic function.
Previous work has demonstrated that the PKC␦ and PKC, but not the classic or atypical, isoforms are cleaved in apoptotic cells (9,11). The Ca 2ϩ -dependent classic PKCs contain the conserved regulatory regions C1 and C2, whereas the Ca 2ϩindependent novel PKCs, including PKC␦ and PKC, lack the C2 domain. Cleavage of the classic PKCs in the third variable (V3) region by calpains I and II deletes the C1 and C2 regulatory regions and results in catalytically active fragments (35). By analogy, cleavage of PKC␦ in the V3 region by the caspase-3 cysteine protease deletes the C1 regulatory region and releases an active catalytic fragment (10). Similar findings have been reported for caspase-3-mediated cleavage of the PKC V3 region (11). The present results demonstrate that PKC is cleaved in cells induced to undergo apoptosis by ara-C and other genotoxic agents. The finding that expression of the baculovirus p35 protein blocks cleavage of PKC supported involvement of a cysteine protease. In addition, the demonstration that CrmA expression had no effect on PKC cleavage indicated lack of involvement of a caspase-1-like protease. In this context, previous work has demonstrated that DNA damageinduced apoptosis is mediated by the CrmA-insensitive, p35sensitive pathway (20,21). We also found that overexpression of the anti-apoptotic Bcl-x L protein blocks PKC cleavage.
Bcl-x L inhibits cytochrome c release from mitochondria in response to genotoxic stress (28). Cytochrome c activates caspase-9 by an Apaf-1-dependent mechanism and thereby activation of caspase-3 (36). Taken together, our findings in cells treated with genotoxic agents suggested that PKC, like PKC␦ and PKC, is cleaved by a caspase-3-dependent mechanism.
Caspases have an absolute requirement for an Asp residue at the P1 position in their substrates. Moreover, caspase-3 prefers an Asp residue at both the P1 and P4 positions and cleaves most known substrates at DXXD motifs (30). Previous studies have shown that PKC␦ and PKC are cleaved by caspase-3 at DMQD 330 N and DEVD 354 K, respectively (10,11). Based on these findings, we predicted that PKC would be cleaved by caspase-3 at one or both of the two consensus DXXD sites (DDND 348 S and DHED 391 S). However, mutation of Asp residues at the P1 positions and incubation of mutant proteins with caspase-3 revealed that PKC is not cleaved at these two consensus DXXD motifs. Subsequent site-directed mutagenesis studies showed that caspase-3 cleaves PKC at an unconventional CQND 378 S site. In concert with these results, recent studies have demonstrated caspase-3-mediated cleavage of other proteins, such as DNA topoisomerase I, amyloid-␤-precursor protein, and p21-activated protein kinase, also occurs at unconventional sites (37)(38)(39). The present findings thus demonstrate that, despite the presence of two DXXD motifs, PKC is cleaved by caspase-3 at the CQND 378 S site.
The available evidence indicates that PKC is involved in diverse cellular events. In B cells, PKC activity is up-regulated after cross-linking of CD19 with the B cell receptor complex (40). PKC associates with the Syk tyrosine kinase, phospholipase C␥ (40), type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase (15). Other studies have demonstrated that PKC is negatively regulated by the 14-3-3 signaling protein (17). The findings that mouse PKC localizes to Golgi and functions downstream of the ␤␥ subunits of heterotrimeric G proteins have also suggested that PKC is involved in protein secretion (16,41). The present results demonstrate that PKC is cleaved in the apoptotic response to genotoxic stress. The functional significance of PKC cleavage is supported by the demonstration that cells expressing the PKC catalytic fragment are more sensitive to the apoptotic effects of genotoxic agents. Studies in cells ex-  pressing full-length PKC have demonstrated that TNF-induced apoptosis is inhibited by enhanced expression of NF-Bdependent protective genes, including the inhibitor of apoptosis protein 2 (18). These findings in cells expressing full length PKC or the catalytic fragment suggest that caspase-3-mediated cleavage of PKC reverses a protective function and confers sensitivity to an apoptotic response. In studies of PKC␦ and PKC, cleavage by caspase-3 results in the release of catalytic fragments that contribute to induction of apoptosis (10,11). By contrast, although cleavage of PKC is not sufficient to induce apoptosis, our findings indicate that expression of PKCCF sensitizes cells to DNA damage-induced apoptosis. Thus, the findings with PKC␦, PKC, and PKC collectively support a pro-apoptotic response involving caspase-3-mediated cleavage and expression of catalytic domains that are active in the absence of lipid second messengers.