Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis.

Protein kinase C theta (PKCtheta) is a member of the novel or nPKC family. A functional role for PKCtheta is unknown. The present studies demonstrate that PKCtheta is cleaved in the third variable region (V3) in apoptosis induced by diverse agents. PKCtheta cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-xL or the baculovirus p35 protein. PKCtheta is cleaved by Caspase-3 and by apoptotic cell lysates at a DEVD354/K site. We also show that overexpression of the cleaved kinase-active PKCtheta fragment, but not full-length PKCtheta or a kinase-inactive fragment, results in induction of sub-G1 phase DNA, nuclear fragmentation, and lethality. These findings indicate that proteolytic cleavage of PKCtheta by Caspase-3 induces events characteristic of apoptosis.


Protein kinase C (PKC) is a member of the novel or nPKC family. A functional role for PKC is unknown. The present studies demonstrate that PKC is cleaved in the third variable region (V3) in apoptosis induced by diverse agents. PKC cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x L or the baculovirus p35 protein. PKC is cleaved by Caspase-3 and by apo-
ptotic cell lysates at a DEVD 354 /K site. We also show that overexpression of the cleaved kinase-active PKC fragment, but not full-length PKC or a kinase-inactive fragment, results in induction of sub-G 1 phase DNA, nuclear fragmentation, and lethality. These findings indicate that proteolytic cleavage of PKC by Caspase-3 induces events characteristic of apoptosis.
The PKC isoform is structurally related to PKC␦ (6 -8), although the V3 domain of PKC has no significant homology with that in PKC␦ or the other PKC isoforms. Few insights are available regarding the functional roles of PKC. Whereas PKC␦ transcripts are found ubiquitously, PKC is predominantly expressed in hematopoietic cells and skeletal muscle (6,8). Studies in T cells have demonstrated that PKC is involved in antigen-specific activation (9). PKC interacts with 14-3-3 proteins (10) and is involved in AP-1-mediated transcription (11). Other work has shown that the human immunodeficiency virus Nef protein inhibits translocation of PKC from the cytosolic to membrane fraction after phorbol ester stimulation (12). Unlike the cPKCs and PKC␦, there are no reports of proteolytic cleavage of the PKC isoform.
The present studies demonstrate that PKC is cleaved to an activated form in cells induced to undergo apoptosis. The results indicate that PKC is cleaved by the Caspase-3 protease. We also show that overexpression of the PKC catalytic fragment induces characteristics of apoptosis.
Immunoblot Analysis-Cytoplasmic extracts were prepared and fractionated through Q-Sepharose columns as described (3,4). 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/0.05% Tween 20). The filters were incubated with anti-PKC polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After washing twice with PBST, the blots were incubated with anti-rabbit IgG peroxidase conjugate (Amersham Corp.). The antigen-antibody complexes were visualized by chemiluminescence (ECL detection system; Amersham).
In Vitro Translation and Protease Cleavage Assays-The full-length (FL) PKC cDNA (provided by J. Anthony Ware, Beth Israel Hospital, Boston) was cloned into BamHI sites of a modified pSV␤ plasmid (CLONTECH). A PKC (D351A and D354A) mutant was generated in two steps by overlapping primer extension. PARP cDNA was generated by polymerase chain reaction cloning (5). [ 35 S]methionine-labeled proteins (PKCFL, PKCFL(D-A), PARP) were synthesized by coupled transcription and translation reactions (Promega, Madison, WI). Labeled proteins were incubated with 5 g/ml Escherichia coli-derived Caspase-3␤, Caspase-1, Caspase-2, Caspase-4, Caspase-6, or Caspase-7 in 50 mM Hepes (pH 7.5), 10% glycerol, 2.5 mM dithiothreitol, and 0.25 mM EDTA at room temperature for 30 min (16). Cleavage reactions were also performed in the presence of 5 g of cytoplasmic extract from untreated or ara-C-treated cells and in the presence of recombinant CrmA or p35 (14,15). The reaction products were analyzed by electrophoresis in 10 or 12% SDS-polyacrylamide gels and then autoradiography.
Analysis of Kinase Activity-Recombinant PKC proteins were prepared by coupled transcription and translation. A vector expressing a PKC fragment (CF; amino acids 355-706) was generated by polymerase chain reaction cloning from the full-length PKC cDNA. A mutant PKCCF with Lys-409 substituted by Arg (K-R) was generated by overlapping primer extension. Protein kinase assays were performed as described (PKC assay kit; Life Technologies, Inc.).
Cell Transfections-PKCFL, PKCCF, or PKCCF(K-R) were cloned into the pEGFP-C1 vector (CLONTECH). HeLa cells were suspended at a density of 1 ϫ 10 7 cells/ml and transfected by electroporation (Gene Pulsar, Bio-Rad; 0.22 V, 960 F). At 40 h post-transfection, cells were sorted by FACScan (Becton Dickinson, Mansfield, MA), and * This work was supported by United States Public Health Service Grants CA29431 and CA66996 awarded by the National Cancer Institute, DHHS. 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. 1 The abbreviations used are: PKC, protein kinase C (cPKC, classical PKC; nPKC, novel PKC; aPKC, atypical PKC); ICE, interleukin-1␤converting enzyme; Ced, Caenorhabditis elegans death; PARP, poly-(ADP-ribose) polymerase; FL, full-length; CF, cleaved fragment; DAPI, 4,6-diamidino-2-phenylindole. viability was checked by trypan blue exclusion. Transfected cells were stained with propidium iodide. FACScan was used to determine sub-G 1 content in cells positive for green fluorescence. Chromatin fragmentation was determined by staining methanol-fixed cells with 0.5 g/ml DAPI (Molecular Probes, Eugene, OR).

RESULTS AND DISCUSSION
Treatment of U-937 cells with ara-C and other DNA-damaging agents results in the induction of apoptosis (17,18). Whereas the V3 region of PKC has a DEVD/K site similar to that cleaved in PARP during apoptosis (19,20), we asked whether PKC is also susceptible to cleavage. PKC was detectable as a 78-kDa band in control cells (Fig. 1A). By contrast, ara-C-induced apoptosis was associated with cleavage of PKC to a 40-kDa fragment (Fig. 1A). Similar results were obtained during apoptosis induced by cisplatinum, etoposide, and 20gray ionizing radiation ( Fig. 1B and data not shown).
To determine whether PKC cleavage is associated with induction of apoptosis, we studied cells that overexpress the anti-apoptotic Bcl-x L protein and exhibit resistance to induction of apoptosis (13). Exposure of control U-937/neo cells to ara-C resulted in cleavage of PKC, while there was no apparent effect of this agent on PKC in the U-937/Bcl-x L transfectant ( Fig. 2A). The cowpox protein CrmA (21) and the baculovirus protein p35 (16) block apoptosis in diverse models by directly inhibiting members of the ICE/Ced-3 family of cysteine proteases. Overexpression of CrmA had no effect on ara-Cinduced PKC cleavage or apoptosis (Fig. 2B). By contrast, ara-C treatment of cells that overexpress p35 resulted in no detectable cleavage of PKC or induction of apoptosis (Fig. 2B). These findings indicated that PKC is cleaved by an ICE/Ced-3-like protease.
The Caspase-3 protease is insensitive to CrmA and inhibited by p35 (14,16,22). Whereas Caspase-3 cleaves PARP at DEVD 216 /G (22, 23), we asked whether Caspase-3 induces cleavage at a DEVD/K site in the V3 region of PKC. Fulllength PKC labeled with [ 35 S]methionine was cleaved by purified Caspase-3 to a 40-kDa fragment (Fig. 3A). By contrast, there was no apparent cleavage of PKC with purified ICE (Fig.  3A). Cleavage of PKC at the DEVD/K site predicts the formation of a catalytic domain of 40 kDa that corresponds physically with the PKC fragment identified in apoptotic U-937 cells. Nonetheless, to confirm the Caspase-3-mediated cleavage site in PKC, we mutated the essential P1 and P4 Asp residues with substitution by Ala (D351A and D354A). The finding that Caspase-3 fails to cleave the PKC(D-A) mutant provided support for involvement of the DEVD 354 /K site (Fig. 3A). Caspase-3, Caspase-7, and Ced-3 cleave PARP at the DEVD 216 /G site (22,23). The finding that PKC is cleaved by Caspase-3, and not Caspase-7, provided support for selectivity of the PKC cleavage site (Fig. 3B). There was also no detectable cleavage of PKC with Caspase-2, Caspase-9, or Caspase-6 ( Fig. 3B). Other studies have demonstrated that Caspase-3 is activated in cells treated with ara-C (14,15). Addition of labeled PKC to lysates from ara-C-treated, but not control, cells was associated with cleavage of PKC (Fig. 3C). Preincubation of the active lysate with CrmA had no effect, while p35 blocked PKC cleavage (Fig. 3C). Taken together, these results indicate that PKC is cleaved by Caspase-3 in cells induced to undergo apoptosis.
To assess whether cleavage of PKC at the DEVD/K site is associated with activation of kinase function, we assayed recombinant PKC proteins for phosphorylation of the pseudosubstrate region of PKC (amino acids 19 -31) with replacement of Ala-25 by Ser. The [A25S]PKC(19 -31) peptide serves as a substrate for PKC (24). The PKC-cleaved fragment (CF; amino acids 355-706) was over 6-fold more active than PKC full-length (FL) and the PKCFL(D-A) mutant (Table I). Moreover, a mutant of the cleaved fragment with Lys-409 in the ATP binding site mutated to Arg (K409R; designated K-R) had little if any activity above that found for control bacterial lysates (Table I). These findings demonstrate that cleavage at the DEVD/K site results in PKC activation.
To determine if PKC contributes to apoptosis, we transfected HeLa cells with PKCFL, PKCCF, or PKCCF(K-R) cloned into vectors expressing the green fluorescence gene. Positive transfectants were selected by flow cytometry, reseeded in medium, and assayed for viability by trypan blue exclusion. Over 90% of the PKCFL transfectants were viable, while only 10 -15% of the PKCCF-transfected cells survived (Fig. 4A). The finding that over 90% of the PKCCF(K-R) transfectants were viable provided further support for the selective effects of PKCCF expression (Fig. 4A). To assess whether transfection of PKCCF induces apoptosis, we monitored the appearance of green fluorescence-positive cells with sub-G 1
Recent studies have demonstrated that the aPKCs (PKC and ) interact with Par-4 and abrogate the ability of Par-4 to induce apoptosis (26). These findings have suggested that the aPKCs exhibit an anti-apoptotic function. By contrast, the present results and previous work on PKC␦ (3,4) support a potential role for at least certain nPKCs in promoting apopto-sis. The absence of detectable cleavage of PKC␣, -␤, -⑀, andfurther supports the selective involvement of PKC and -␦ in apoptosis (3,4). Previous studies have demonstrated that Caspase-3 cleaves PARP (22,23), DNA-PK (27, 28), D4-GDI (29), U1 small nuclear riboprotein (27), and PKC␦ (5). We show that PKC is also cleaved by Caspase-3 and that Bcl-x L functions upstream to this event. The finding that the cleaved fragment of PKC induces characteristics typical of apoptosis further supports a role for PKC in mediating apoptotic events and not simply a bystander effect of Caspase-3 activation.

Activation of PKC in Apoptosis
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