COMMUNICATION:
Caspase-3-mediated Cleavage of Protein Kinase C theta  in Induction of Apoptosis*

(Received for publication, May 14, 1997)

Rakesh Datta , Hiromi Kojima , Kiyotsugu Yoshida and Donald Kufe

From the Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

The 11 known isoforms of the protein kinase C (PKC)1 family have been divided into the classical (cPKC; alpha , beta , gamma ), novel (nPKC; delta , epsilon , eta , theta , µ), and atypical (aPKC; zeta , lambda ) groups (1). The Ca2+-dependent cPKCs contain the conserved regulatory regions, C1 and C2, while the Ca2+-independent nPKC and aPKC isoforms lack the C2 domain. Cleavage of cPKCs in the third variable (V3) region by calpains I and II deletes the C1 and C2 regulatory regions and results in catalytically active fragments (2). Other studies have shown that the nPKCdelta isoform is activated by the Caspase-3 cysteine protease in cells induced to undergo apoptosis (3-5). Caspase-3-mediated cleavage of PKCdelta at a DMQD/N site in the V3 region deletes the C1 regulatory domain (3-5). Overexpression of the anti-apoptotic Bcl-2 and Bcl-xL proteins blocks PKCdelta cleavage (3, 4). These findings have suggested that PKCdelta is functionally involved in the induction of apoptosis.

The PKCtheta isoform is structurally related to PKCdelta (6-8), although the V3 domain of PKCtheta has no significant homology with that in PKCdelta or the other PKC isoforms. Few insights are available regarding the functional roles of PKCtheta . Whereas PKCdelta transcripts are found ubiquitously, PKCtheta is predominantly expressed in hematopoietic cells and skeletal muscle (6, 8). Studies in T cells have demonstrated that PKCtheta is involved in antigen-specific activation (9). PKCtheta 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 PKCtheta from the cytosolic to membrane fraction after phorbol ester stimulation (12). Unlike the cPKCs and PKCdelta , there are no reports of proteolytic cleavage of the PKCtheta isoform.

The present studies demonstrate that PKCtheta is cleaved to an activated form in cells induced to undergo apoptosis. The results indicate that PKCtheta is cleaved by the Caspase-3 protease. We also show that overexpression of the PKCtheta catalytic fragment induces characteristics of apoptosis.


MATERIALS AND METHODS

Cell culture

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 units/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/liter L-glutamine. U-937 cells overexpressing bcl-xL, CrmA, and p35 were prepared as described (13-15). Cells were treated with 10 µmol/liter 1-beta -D-arabinofuranosylcytosine (ara-C; Sigma), 3 µg/ml etoposide (Bristol-Myers Squibb Co., Princeton, NJ), and 100 µmol/liter cisplatinum (Sigma).

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-PKCtheta 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).

Analysis of DNA Fragmentation

Cells (5 × 106) were harvested, washed, and incubated in 50 µl 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 µl of 10 mM EDTA (pH 8.0) containing 2% (w/v) low-melting-point agarose 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.

In Vitro Translation and Protease Cleavage Assays

The full-length (FL) PKCtheta cDNA (provided by J. Anthony Ware, Beth Israel Hospital, Boston) was cloned into BamHI sites of a modified pSVbeta plasmid (CLONTECH). A PKCtheta (D351A and D354A) mutant was generated in two steps by overlapping primer extension. PARP cDNA was generated by polymerase chain reaction cloning (5). [35S]methionine-labeled proteins (PKCtheta FL, PKCtheta FL(D-A), PARP) were synthesized by coupled transcription and translation reactions (Promega, Madison, WI). Labeled proteins were incubated with 5 µg/ml Escherichia coli-derived Caspase-3beta , 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 µg 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 PKCtheta proteins were prepared by coupled transcription and translation. A vector expressing a PKCtheta fragment (CF; amino acids 355-706) was generated by polymerase chain reaction cloning from the full-length PKCtheta cDNA. A mutant PKCtheta CF 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

PKCtheta FL, PKCtheta CF, or PKCtheta CF(K-R) were cloned into the pEGFP-C1 vector (CLONTECH). HeLa cells were suspended at a density of 1 × 107 cells/ml and transfected by electroporation (Gene Pulsar, Bio-Rad; 0.22 V, 960 µF). At 40 h post-transfection, cells were sorted by FACScan (Becton Dickinson, Mansfield, MA), and viability was checked by trypan blue exclusion. Transfected cells were stained with propidium iodide. FACScan was used to determine sub-G1 content in cells positive for green fluorescence. Chromatin fragmentation was determined by staining methanol-fixed cells with 0.5 µg/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 PKCtheta has a DEVD/K site similar to that cleaved in PARP during apoptosis (19, 20), we asked whether PKCtheta is also susceptible to cleavage. PKCtheta was detectable as a 78-kDa band in control cells (Fig. 1A). By contrast, ara-C-induced apoptosis was associated with cleavage of PKCtheta to a 40-kDa fragment (Fig. 1A). Similar results were obtained during apoptosis induced by cisplatinum, etoposide, and 20-gray ionizing radiation (Fig. 1B and data not shown).


Fig. 1. Proteolytic cleavage of PKCtheta by DNA damaging agents. U-937 cells were treated with 10 µM ara-C (A) or 3 µg/ml etoposide (ETOPO) or 10 µM cisplatinum (CDDP) (B) for 6 h. Lysates were subjected to immunoblot analysis using anti-PKCtheta antibody (upper panel). DNA fragmentation was assessed by electrophoresis in 2% agarose gels (lower panel). FL, full-length; CF, cleaved fragment.
[View Larger Version of this Image (40K GIF file)]

To determine whether PKCtheta cleavage is associated with induction of apoptosis, we studied cells that overexpress the anti-apoptotic Bcl-xL protein and exhibit resistance to induction of apoptosis (13). Exposure of control U-937/neo cells to ara-C resulted in cleavage of PKCtheta , while there was no apparent effect of this agent on PKCtheta in the U-937/Bcl-xL 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-C-induced PKCtheta cleavage or apoptosis (Fig. 2B). By contrast, ara-C treatment of cells that overexpress p35 resulted in no detectable cleavage of PKCtheta or induction of apoptosis (Fig. 2B). These findings indicated that PKCtheta is cleaved by an ICE/Ced-3-like protease.


Fig. 2. Effects of overexpression of bcl-xL, CrmA, or p35 on ara-C-induced cleavage of PKCtheta . U-937 and U-937/bcl-xL (A) or U-937, U-937/CrmA, and U-937/p35 (B) cells were treated with ara-C for 6 h. Immunoblot analysis of the lysates was performed with anti-PKCtheta antibody (upper panel). DNA was analyzed for fragmentation in agarose gels (lower panel). FL, full-length; CF, cleaved fragment.
[View Larger Version of this Image (47K GIF file)]

The Caspase-3 protease is insensitive to CrmA and inhibited by p35 (14, 16, 22). Whereas Caspase-3 cleaves PARP at DEVD216/G (22, 23), we asked whether Caspase-3 induces cleavage at a DEVD/K site in the V3 region of PKCtheta . Full-length PKCtheta labeled with [35S]methionine was cleaved by purified Caspase-3 to a 40-kDa fragment (Fig. 3A). By contrast, there was no apparent cleavage of PKCtheta with purified ICE (Fig. 3A). Cleavage of PKCtheta at the DEVD/K site predicts the formation of a catalytic domain of 40 kDa that corresponds physically with the PKCtheta fragment identified in apoptotic U-937 cells. Nonetheless, to confirm the Caspase-3-mediated cleavage site in PKCtheta , 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 PKCtheta (D-A) mutant provided support for involvement of the DEVD354/K site (Fig. 3A). Caspase-3, Caspase-7, and Ced-3 cleave PARP at the DEVD216/G site (22, 23). The finding that PKCtheta is cleaved by Caspase-3, and not Caspase-7, provided support for selectivity of the PKCtheta cleavage site (Fig. 3B). There was also no detectable cleavage of PKCtheta 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 PKCtheta to lysates from ara-C-treated, but not control, cells was associated with cleavage of PKCtheta (Fig. 3C). Preincubation of the active lysate with CrmA had no effect, while p35 blocked PKCtheta cleavage (Fig. 3C). Taken together, these results indicate that PKCtheta is cleaved by Caspase-3 in cells induced to undergo apoptosis.


Fig. 3. PKCtheta is proteolytically cleaved by Caspase-3 in vitro. A, [35S]methionine-labeled PKCtheta FL, PKCtheta FL(D-A) and PARP were incubated with recombinant Caspase-3beta or Caspase-1. B, [35S]methionine-labeled PKCtheta FL was incubated with Caspase-3, Caspase-2, Caspase-4, Caspase-6, or Caspase-7. PARP labeled with [35S]methionine was incubated with Caspase-7 as a control. C, [35S]methionine-labeled PKCtheta FL or PKCtheta FL(D-A) was incubated with cytoplasmic extracts from untreated or ara-C-treated U-937 cells in the presence or absence of recombinant CrmA or p35. Arrowheads denote PARP cleavage fragments.
[View Larger Version of this Image (33K GIF file)]

To assess whether cleavage of PKCtheta at the DEVD/K site is associated with activation of kinase function, we assayed recombinant PKCtheta 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 PKCtheta (24). The PKCtheta -cleaved fragment (CF; amino acids 355-706) was over 6-fold more active than PKCtheta full-length (FL) and the PKCtheta FL(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 PKCtheta activation.

Table I. Kinase activity of recombinant PKCtheta proteins

FL, full length. CF, cleaved fragment (amino acids 355-706).

PKCtheta Activitya

FL 1.6  ± 0.28
FL(D-A) 1.2  ± 0.51
CF 10.3  ± 2.1
CF(K-R) 1.0  ± 0.25

a Results (pmol/min) are expressed as mean ± SE of three separate determinations.

To determine if PKCtheta contributes to apoptosis, we transfected HeLa cells with PKCtheta FL, PKCtheta CF, or PKCtheta CF(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 PKCtheta FL transfectants were viable, while only 10-15% of the PKCtheta CF-transfected cells survived (Fig. 4A). The finding that over 90% of the PKCtheta CF(K-R) transfectants were viable provided further support for the selective effects of PKCtheta CF expression (Fig. 4A). To assess whether transfection of PKCtheta CF induces apoptosis, we monitored the appearance of green fluorescence-positive cells with sub-G1 DNA content. Transfection of PKCtheta FL or PKCtheta CF(K-R) was associated with 7-10% of cells with sub-G1 DNA (Fig. 4B). Significantly, transfection of kinase-active PKCtheta CF resulted in 48% of cells with sub-G1 DNA (Fig. 4B). Cells were also stained with DAPI to assess nuclear morphology (25). Transfection of PKCtheta CF, but not PKCtheta FL or PKCtheta CF(K-R), was associated with nuclear fragmentation (Fig. 4C).


Fig. 4. Overexpression of PKCtheta CF induces sub-G1 DNA and morphological changes characteristic of apoptosis. HeLa cells were transfected with vectors expressing the green fluorescence gene and PKCtheta FL, PKCtheta CF or PKCtheta CF(K-R). A, positive cells selected by flow cytometry were assayed for trypan blue exclusion at 40 h after transfection. B, total cell population was assayed for DNA content by flow cytometry at 40 h after transfection. C, transfectants were stained with DAPI and assessed for nuclear morphology.
[View Larger Version of this Image (44K GIF file)]

Recent studies have demonstrated that the aPKCs (PKC zeta  and lambda ) 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 PKCdelta (3, 4) support a potential role for at least certain nPKCs in promoting apoptosis. The absence of detectable cleavage of PKCalpha , -beta , -epsilon , and -zeta further supports the selective involvement of PKCtheta and -delta 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 PKCdelta (5). We show that PKCtheta is also cleaved by Caspase-3 and that Bcl-xL functions upstream to this event. The finding that the cleaved fragment of PKCtheta induces characteristics typical of apoptosis further supports a role for PKCtheta in mediating apoptotic events and not simply a bystander effect of Caspase-3 activation.


FOOTNOTES

*   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. The 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-1beta -converting enzyme; Ced, Caenorhabditis elegans death; PARP, poly(ADP-ribose) polymerase; FL, full-length; CF, cleaved fragment; DAPI, 4,6-diamidino-2-phenylindole.

ACKNOWLEDGEMENTS

We are grateful to Robert Talanian (BASF Bioresearch Corp., Worcester, MA) for providing recombinant proteases and protease inhibitors. We thank Tariq Ghayur (BASF Bioresearch Corp.) for helpful discussions.


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