BRIEF DEFINITIVE REPORT:
Proteolytic Activation of Protein Kinase C delta  by an ICE/CED 3-like Protease Induces Characteristics of Apoptosis

By Tariq Ghayur,* Margaret Hugunin,* Robert V. Talanian,* Sheldon Ratnofsky,* Christopher Quinlan,* Yutaka Emoto,Dagger Pramod Pandey,Dagger Rakesh Datta,Dagger Yinyin Huang,Dagger Surender Kharbanda,Dagger Hamish Allen,* Robert Kamen,* Winnie Wong,* and Donald KufeDagger

From * BASF Bioresearch Corporation, Worcester, Massachusetts 01605; and Dagger  Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Summary
Materials and Methods
Results and Discussion
Footnotes
References


Summary

Recent studies have shown that protein kinase C (PKC) delta  is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKCdelta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKCdelta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKCdelta or a kinase inactive PKCdelta fragment had no detectable effect. The findings suggest that proteolytic activation of PKCdelta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.


The protein kinase C (PKC) family consists of multiple subspecies that possess a conserved catalytic domain. The classic or group A isoforms (alpha , beta , and gamma ) require Ca2+ for activity and contain cysteine-rich motifs that confer phospholipid-dependent binding of diacylglycerol (1). The group A PKCs are cleaved at the third variable region (V3) by the neutral proteases, calpains I and II, to catalytically active fragments (2). Recent studies have demonstrated that the Ca2+-independent delta  isoform, and not the group A PKCs, is selectively cleaved at V3 to a catalytically active fragment in cells induced to undergo apoptosis (3, 4). Inhibition of apoptosis by overexpression of Bcl-2 or Bcl-xL is associated with a block of PKCdelta cleavage (3, 4). The finding that PKCdelta is cleaved at a site (DMQD/N) adjacent to aspartic acid has supported the potential involvement of aspartate-specific cysteine proteases which are known to be activated during apoptosis.

The nematode Ced-3 cysteine protease is related to the mammalian interleukin-1beta converting enzyme (ICE) (5, 6). The demonstration that overexpression of Ced-3 or ICE induces apoptosis has provided support for involvement of these cysteine proteases in cell death pathways (7). ICE/ Ced-3 family members include Nedd2/Ich-1, CPP32/ YAMA/apopain, Tx/Ich-2/ICErelII, ICErelIII, Mch2, Mch3/ ICE-LAP3/CMH-1 (reviewed in reference 8), ICE-LAP6 (9), FLICE/Mch5 (10, 11), and Mch4 (11). ICE cleaves the precursor of IL-1beta to the active cytokine (6, 12, 13). Other known substrates of the ICE/Ced-3 family include: (a) poly (ADP-ribose) polymerase (PARP) which is cleaved by CPP32, Mch3 and Ced-3, but not ICE (14); and (b) DNA-dependent protein kinase (DNA-PK), the U1 small nuclear ribonucleoprotein and D4-GDP dissociation inhibitor for the Rho family GTPases (D4-GDI), which are cleaved by CPP32 (17, 18). However, the functional role of these cleavage products in the induction of apoptosis is unclear.

The present results demonstrate that PKCdelta is cleaved by CPP32 and not certain other ICE/Ced-3 family members. We also demonstrate that overexpression of the PKCdelta catalytic fragment is involved in the induction of phenotypic changes that are characteristic of apoptosis.


Materials and Methods

In Vitro Cleavage of PKCdelta and PARP. The full-length PKCdelta cDNA was cloned into the SpeI and BamH1 sites of a modified pSVbeta plasmid (Clontech, Palo Alto, CA). PKCdelta (D327A/D330A) was generated in two steps by overlapping primer extension. PARP cDNA was generated by PCR cloning. The proteins were labeled with [35S]methionine by coupled transcription and translation reactions (Promega, Madison, WI). Labeled proteins were incubated with 5 µg/ml Escherichia coli-derived CPP32beta in 50 mM Hepes (pH 7.5), 10% glycerol, 2.5 mM DTT, and 0.25 mM EDTA at room temperature for 30 min. The reaction products were analyzed by electrophoresis in 10-20% SDS-polyacrylamide gels and then autoradiography. For the kinase assays, full-length PKCdelta , PKCdelta (D327A/D330A), PKCdelta catalytic fragment (CF), and PKCdelta CF(K-R) were prepared by coupled transcription and translation. PKCdelta and PKCdelta (D327A/D330A) were incubated with 5 µg/ml CPP32beta at room temperature for 30 min. Protein kinase assays using MBP as a substrate were performed as described (PKC Assay Kit; GIBCO BRL, Gaithersburg, MD).

Analysis of Peptide Proteolysis. Peptides were synthesized and purified to ~95% by standard methods and confirmed by mass spectrometry. Reaction mixtures (810 µl) contained: 100 mM Hepes (pH 7.5), 20% (vol/vol) glycerol, 5 µM dithiothreitol, 0.5 mM EDTA, and 380 ng N-His CPP32 (19). Peptide substrates were added to final concentrations of 10 µM. The reaction mixtures were incubated at 30°C. Aliquots were removed at 10 min intervals for 60 min and added to vials containing 3 M HCl to stop the reactions. The amount of substrate remaining at each time was quantitated by reverse phase HPLC. Data were fit to the equation (St/So) = e-kt, where k is the decay rate constant equal to Vmax/Km. Observed Vmax/Km values were normalized to 1.00 for the PARP peptide.

Cell Transfections. Cells were seeded at a density of 1.7 × 105 in each well of 6-well dishes 24 h before transfection. For each well, 2 µg DNA construct and 0.5 µg pSvbeta plasmid containing beta -gal were coprecipitated with calcium phosphate. Cells were incubated with the coprecipitate for 30 h at 37°C and then analyzed by X-gal staining. Cells (1.7 × 105/well) were also transfected with 2 µg DNA construct for 30 h at 37°C, fixed with 4% paraformaldehyde, postfixed with 5% acetic acid in ethanol and then stained with 5 µg/ml Hoechst dye. For sub-G1 DNA content, cells transfected by lipofectamine were stained with propidium iodide and monitored by FACScan®. Chromatin condensation was assessed by staining with acridine orange and ethidium bromide (20).


Results and Discussion

To determine whether PKCdelta is cleaved by one of the known ICE-like proteases, full-length 78-kD PKCdelta labeled with [35S]methionine was incubated with purified recombinant proteases. Cleavage of PKCdelta to a 40-kD fragment was observed with purified CPP32beta (14) (Fig. 1 A). In contrast, ICE failed to cleave PKCdelta at concentrations up to 600 U/µl (3). The related Ich-1, Ich-2, Mch2, Mch3, and ICErelIII proteases also failed to cleave PKCdelta (data not shown). Because PKCdelta is cleaved at DMQD330N in vivo (3, 4), we asked whether this site is responsible for CPP32mediated cleavage in vitro. CPP32 may prefer peptidic substrates with aspartic acid at the P1 and P4 positions (15). Consequently, we prepared a PKCdelta mutant with substitution of D327A and D330A. Incubation with CPP32 resulted in no detectable CPP32-mediated cleavage of this mutant to the 40-kD catalytic fragment, while there was partial digestion to a species of ~55 kD (not observed with wild-type substrate) (Fig. 1 A). Recombinant CPP32 also cleaved the 116-kD full-length PARP to the predicted 85-kD fragment (14, 15) (Fig. 1 A). Using peptides derived from the cleavage sites of PARP and PKCdelta in proteolytic assays, we found that CPP32 cleaves both substrates and not a peptide spanning the IL-1beta maturation site (Table 1). These findings confirm that PKCdelta , like PARP, is a substrate for CPP32.


Fig. 1. PKCdelta is proteolytically activated by CPP32 in vitro. (A) PKCdelta (full-length: FL), PKCdelta (D327A/D330A) and PARP were labeled with [35S]methionine and incubated with recombinant CPP32beta . The reaction products were analyzed by SDS-PAGE and autoradiography. The kinase active PKCdelta catalytic fragment (CF) and the kinase inactive PKCdelta CF(K-R) were labeled with [35S]methionine and analyzed under similar conditions. (B) Recombinant PKCdelta and PKCdelta (D327A/D330A) were incubated with CPP32 and then assayed for protein kinase activity using MBP as substrate.
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Table 1. CPP32 Proteolysis of Peptides Spanning the PARP, PKCdelta , and IL-1beta Cleavage Sites


Substrate Sequence Relative Vmax/Km

PARP Ac-WGDEVD216-GVDEVW-NH2 1.00
PKCdelta Ac-GEDMQD330NSGTYW-NH2 0.42
IL-1beta Ac-NEAYVHD116APVRSLY-NH2 0.00

We also asked whether cleavage of PKCdelta by CPP32 is associated with activation of the kinase function. Fulllength PKCdelta exhibited a low level of myelin basic protein (MBP) phosphorylation, while incubation with CPP32 resulted in a greater than sixfold increase in kinase activity (Fig. 1 B). In contrast, CPP32 had no detectable effect on kinase function of the PKCdelta (D327A/D330A) mutant (Fig. 1 B). A recombinant 40-kD CF of PKCdelta (amino acids 331676) exhibited constitutive kinase activity, while a mutant of the fragment with K-378 in the ATP binding site mutated to R (K378R; designated K-R) yielded background levels of MBP phosphorylation found with control bacterial lysates (Figs. 1, A and B). These findings collectively demonstrate that CPP32-mediated cleavage of the DMQD330N site activates PKCdelta .

To study the role of PKCdelta in apoptosis, we used the transient HeLa cell transfection system previously found to demonstrate induction of apoptosis by ICE-like proteases (7). Cotransfection of the kinase inactive PKCdelta CF(K-R) mutant with the beta -galactosidase (beta -gal) marker gene had little effect on HeLa cell morphology (Fig. 2 A). Most of the blue X-gal positive cells remained flat and attached to the dish (Fig. 2 A). Cotransfection of the kinase active PKCdelta CF and beta -gal resulted in condensed, small blue cells (Fig. 2 B), consistent with the induction of apoptosis (7). Similar findings were obtained with NIH3T3 cells (Figs. 2, C and D). Overexpression of PKCdelta in both cell types also resulted in detachment of non-viable cells into the culture medium.


Fig. 2. Transfection of the PKCdelta catalytic fragment (CF) induces morphologic changes characteristic of apoptosis. HeLa (upper panels) and NIH3T3 (lower panels) cells were cotransfected with pSvbeta -beta -gal and vectors expressing: (A and C) kinase inactive PKCdelta CF(K-R) and (B and D) kinase active PKCdelta CF. Transfection was determined by X-gal staining and apoptotic cells were identified by their condensed morphology.
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Hoechst staining of HeLa cells transfected with a vector that expresses full-length PKCdelta had no detectable changes in nuclear morphology (Fig. 3 A), but overexpression of PKCdelta CF resulted in fragmented nuclei (Fig. 3 B). Transfection of kinase inactive PKCdelta CF(K-R) was associated with a normal nuclear morphology (Fig. 3 C). The changes observed with expression of the PKCdelta CF were also compared to those found upon exposure to 1-beta -D-arabinofuranosylcytosine (ara-C), a DNA-damaging agent that induces proteolytic cleavage of PKCdelta and apoptosis (4). Treatment of HeLa cells with ara-C resulted in a similar pattern of nuclear fragmentation (Fig. 3 D).


Fig. 3. Expression of PKCdelta CF results in nuclear fragmentation. HeLa cells were transfected with vectors that express: (A) full-length PKCdelta ; (B) kinase active PKCdelta CF; and (C) kinase inactive PKCdelta CF(K-R). (D) Cells were exposed to 2 µM ara-C. The cells were fixed with paraformaldehyde and then stained with Hoechst dye. Cotransfection of PKCdelta FL, PKCdelta CF and PKCdelta CF(K-R) with pSvbeta -beta -gal demonstrated transfection efficiencies of 50, 42, and 43%, respectively. Percentage of apoptotic cells for the PKCdelta FL, PKCdelta CF, and PKCdelta CF(K-R) transfected populations was 2, 26, and 4%, respectively. Bar, 15 µM.
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To confirm that the nuclear changes induced by PKCdelta CF are associated with induction of apoptosis, we assessed the effects of transfection on the appearance of HeLa cells with sub-G1 DNA content. Transfection of the empty vector, full-length PKCdelta or PKCdelta CF(K-R) resulted in 10-15% of cells with sub-G1 DNA (Fig. 4 A and data not shown). By contrast, transfection of PKCdelta CF was associated with 30- 35% of cells with sub-G1 DNA (Fig. 4 A). Cells were also stained with acridine orange and ethidium bromide to assess chromatin condensation (20). Transfection of PKCdelta CF, but not PKCdelta CF(K-R), resulted in the appearance of bright yellow-green nuclear staining of condensed chromatin (Fig. 4 B).


Fig. 4. Overexpression of PKCdelta CF induces sub-G1 DNA and chromatin condensation. (A) HeLa cells were transfected with PKCdelta FL, PKCdelta CF, or PKCdelta CF(K-R). Cells were assessed for DNA content by flow cytometry at 48 h after transfection. The small triangle denotes G0/G1 DNA. (B) HeLa cells transfected with PKCdelta CF(K-R) (left) and PKCdelta CF (right) were assessed for chromatin condensation after staining with acridine orange and ethidium bromide.
[View Larger Version of this Image (40K GIF file)]

To quantify the effects of PKCdelta CF expression on cell viability, we cotransfected PKCdelta CF or PKCdelta CF(K-R) and the green fluorescence gene (Clontech) into HeLa cells. Positive transfectants were selected by flow cytometry, reseeded in culture medium and assayed at 24 h for viability by trypan blue exclusion. Less than 5% of the PKCdelta CF transfectants were viable, while over 90% of the kinase inactive PKCdelta CF(K-R) transfectants were viable and attached to the dish. Viability of 90-95% was observed after transfection of the null vector and sorting. We conclude that the kinase active catalytic domain of PKCdelta induces characteristics typical of cells undergoing apoptosis: (a) size reduction and round morphology; (b) nuclear fragmentation; (c) chromatin condensation; (d) sub-G1 DNA content; and (e) detachment and loss of viability.

Multiple events that lead to destruction of nuclear and cytoplasmic integrity are probably required for apoptosis. Activation of ICE-family proteases may be a central trigger, resulting in the cleavage of substrates such as PARP (21), lamin B1 (22, 23), topoisomerase 1 (23), D4-GDI (18), DNA-PK, and the U1 small nuclear ribonucleoprotein (17). PKCdelta , but not PKCalpha , beta , epsilon , or zeta , is also cleaved at the onset of apoptosis (3, 4). Little is known about the physiological function of PKCdelta (24, 25). We demonstrate that PKCdelta is cleaved by CPP32 and not other ICE/Ced-3 family members in vitro. The results also demonstrate that expression of the PKCdelta catalytic fragment induces morphologic changes characteristic of apoptosis. We propose that the proteolytic cleavage of PKCdelta is a key mediator of nuclear fragmentation and cell death, and not a bystander effect of protease activation. Moreover, the finding that proteolytic activation of PKCdelta is blocked by Bcl-2 and Bcl-xL suggests that these anti-apoptotic proteins act upstream to this event (3). Elucidation of the substrates phosphorylated as a consequence of PKCdelta cleavage should provide insights into the pathways activated by the catalytic fragment.


Footnotes

Address correspondence to Donald W. Kufe, Division of Cancer Pharmacology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115.

Received for publication 13 August 1996

   This investigation was supported by Public Health Service grants CA66996, CA55241, and CA29431 awarded by the National Cancer Institute, DHHS.

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Copyright © 1996 by The Rockefeller University Press.