DAP kinase activity is critical for C2-ceramide-induced apoptosis in PC12 cells

Authors


M. Yamamoto, Pharmaceuticals Discovery Laboratory, Yokohama Research Center, Mitsubishi-Tokyo Pharmaceuticals Inc., 1000 Kamoshida, Aoba-ku, Yokohama 227-8502, Japan. Fax: + 81 45 963 3992, Tel.: + 81 45 963 4340, E-mail: mutu@phy.med.kyoto-u.ac.jp

Abstract

Exposure of PC12 cells to C2-ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-γ-induced apoptosis of HeLa cells, in the C2-ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 µm C2-ceramide, both the total (assayed in the presence of Ca2+/calmodulin) and Ca2+/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 µm C2-ceramide, the Ca2+/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C2-ceramide concentration. C8-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C2-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C2- and C8-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C2-, and C8-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.

Abbreviations
DAP kinase

death-associated protein kinase

MLC kinase

myosin light chain protein kinase

CaM

kinase, Ca2+/calmodulin-dependent protein kinase

ZIP

kinase, zipper-interacting protein kinase

DMEM

Dulbecco's modified Eagle's medium

C2-ceramide

N-Acetylsphingosine

C8-ceramide

N-octanoylsphingosine C2-dihydroceramide, N-acetylsphinganine; EGFP, enhanced green fluorescent protein.

Using a functional gene selection approach based on random inactivation of gene expression by antisense cDNA libraries, death-associated protein kinase (DAP kinase) was cloned as a positive mediator of interferon-γ-induced HeLa cell apoptosis [1–3]. DAP kinase contains a calmodulin-binding region and phosphorylates both itself and other substrates in a Ca2+/calmodulin-dependent fashion [1,4]. ΔCaM-DK, a mutant lacking the calmodulin-binding domain, is constitutively active, while mutant K42A-DK, in which a lysine residue within the kinase domain is replaced by an alanine residue, is inactive. The death-promoting function of DAP kinase requires the kinase activity; therefore overexpression of the ΔCaM-DK mutant leads to cell death without any external stimuli, whereas overexpression of the K42A-DK mutant is not cytotoxic [4]. In addition, DAP kinase contains unique domains and motifs, including eight ankyrin repeats, two potential ATP/GTP binding sites (P-loops), a cytoskeleton binding domain, a death domain, and a serine rich C-terminal region [4,5], which are thought to be involved in various biological processes and to modulate DAP kinase function [4,6,7].

We recently found that DAP kinase mRNA is predominantly expressed in the brain and lung. In the developing brain, a high level of DAP kinase mRNA expression is detected in proliferative and postmitotic regions of the cerebral cortex and hippocampus and in cerebellar Purkinje cells, suggesting that DAP kinase may play a pivotal role in neurogenesis; furthermore, the expression of DAP kinase mRNA is increased prior to cell death induced by transient forebrain ischemia [8]. These findings indicate a possible relationship between DAP kinase and physiological or pathological neuronal cell death. However, it is not known whether DAP kinase activity is critical for neuronal cell death.

Ceramide, generated by sphingomyelin hydrolysis, has been proposed as a pleiotropic lipid second messenger regulating cell cycle arrest, differentiation and apoptosis [9,10]. Several reports have shown that exposure to a cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide), induces apoptotic cell death in many types of cells, including neuronal cells, such as primary cultures of mesencephalic neurons [11], sensory neurons [12], immature cerebellar granule cells [13], and the rat pheochromocytoma cell line, PC12 [14].

In this study, we employed PC12 cells, which are extensively used as a model to study mechanisms regulating neuronal survival and apoptotic cell death [15], to study the role of DAP kinase in ceramide-induced apoptosis. DAP kinase was found to be activated in the early response to ceramide exposure, and the activity depended on the ceramide concentration. The proportion of Ca2+/calmodulin-independent activity was also increased during this process. Furthermore, overexpression of wild-type DAP kinase made cells more sensitive to ceramide. In contrast, overexpression of the K42A-DK mutant or of DD-DK, a construct containing the death domain and the C-terminal tail, which acts as another kind of dominant negative mutant [6], protected cells from ceramide-induced apoptosis. This is the first study to quantitatively measure DAP kinase activity and to show that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis.

Experimental procedures

Drugs and reagents

N-Acetylsphingosine (C2-ceramide) (Sigma Chemical Co., St Louis, MO), N-octanoylsphingosine (C8-ceramide) (Calbiochem-Novabiochem Corp., San Diego, CA) and N-acetylsphinganine (C2-dihydroceramide) (ICN Pharmaceuticals, Inc., Costa Mesa, CA) were dissolved in ethanol. The final ethanol concentration in the culture medium in all experiments was less than 0.25%. [γ-32P]ATP (6000 Ci·mmol−1) and ATP were purchased from Amersham Pharmacia Biotech (Little Chalfont Buckinghamshire, UK) and AMRESCO Inc. (Solon, OH, USA), respectively.

Cell culture

PC12 cells were maintained at 37 °C on collagen-coated dishes (Iwaki Glass Co., Ltd, Tokyo, Japan) in normal growth medium composed of Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc., Grand Island, NY, USA) supplemented with 5% fetal bovine serum (Life Technologies, Inc.), 5% horse serum (Life Technologies, Inc.), 15 mm Hepes (pH 7.4; Life Technologies, Inc.), and 50 µg·mL−1 of gentamycin (Sigma) in a humidified atmosphere containing 5% CO2.

DNA fragmentation assay

PC12 cells were collected by centrifugation and lysed at 50 °C for 2 h in 200 mm Tris/HCl (pH 8.0), 100 mm EDTA, 1% SDS, and 0.1 mg·mL−1 of proteinase K (Nacalai tesque, Inc., Kyoto, Japan). Potassium acetate (5.0 m) was then added to a final concentration of 1.0 m and the lysate centrifuged at 15 000 g for 15 min at 4 °C. The DNA in the supernatant was extracted with an equal volume of phenol/chloroform, precipitated with ethanol, and suspended in 10 mm Tris/HCl (pH 8.0) and 1 mm EDTA containing 20 µg·mL−1 of RNaseA (Nacalai tesque, Inc.). The DNA (2 µg) was then electrophoresed on a 2% agarose gel, and visualized under UV light after staining with ethidium bromide.

Kinase assay

PC12 cells were collected by centrifugation, washed twice with ice-cold NaCl/Pi, then lysed in lysis buffer [50 mm Tris/HCl (pH 7.5), 70 mm NaCl, 1% Triton X-100, 5 mm EDTA, 5 mm EGTA, 2 mm phenylmethylsulfonyl fluoride (Sigma), 1 µg·mL−1 aprotinin (Roche Diagnostics GmbH, Mannheim, Germany), 1 µg·mL−1 leupeptin (Roche Diagnostics GmbH), 100 nm okadaic acid (Calbiochem-Novabiochem Corp.), 50 mm NaF (Nacalai tesque, Inc.), 10 mm Na3VO4, 30 µm mycalolide B (Wako Pure Chemical Industries, Ltd, Osaka, Japan)][16]. After insoluble material was removed by centrifugation (15 000 g for 5 min at 4 °C), the lysates were immunoprecipitated with monoclonal anti-(DAP kinase) Ig (Sigma) absorbed on protein G–Sepharose 4FF (Amersham Pharmacia Biotech). After three washes with kinase assay buffer [50 mm Tris/HCl (pH 7.5), 8 mm MgCl2, 0.01% BSA, 100 nm okadaic acid, 0.5 mm dithiothreitol], the beads were incubated for 10 min at 30 °C in kinase assay buffer containing 100 µm skeletal muscle myosin light chain kinase (MLC kinase) substrate peptide (AKRPQRATSNVFS) (ICN Pharmaceuticals, Inc.), 200 µm[γ-32P]ATP (1 Ci·mmol−1) and either 0.5 mm CaCl2 and 1 µm bovine calmodulin (Sigma) (total activity) or 1 mm EGTA (Ca2+/calmodulin independent activity) [17]. Incorporation of 32P into MLC kinase substrate peptide was measured essentially as described by DeRemer et al. [18]. After incubation, aliquots of reaction mixture were spotted onto P81 phosphocellulose paper (Whatman Inc., Clifton, NJ, USA), which was then washed three times with 75 mm H3PO4, rinsed with ethanol, dried, and subjected to liquid scintillation counting. Kinase activity was normalized to the amount of DAP kinase in the immunoprecipitants, determined by immunoblotting using monoclonal anti-(DAP kinase) Ig (1 : 250 dilution; Transduction Laboratories, Lexington, KY, USA) as primary antibody and horseradish peroxidase-conjugated goat anti-(mouse IgG) Ig (1 : 1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as secondary antibody with quantification on an Imaging Scanner (ES-8000; Epson, Tokyo, Japan) with nih image 1.61 software.

DNA transfection

PC12 cells were plated at a density of 2.6 × 104 cells per cm2 in normal growth medium in collagen-coated culture dishes and incubated at 37 °C for 24 h. The cells were cotransfected with 0.04 µg·cm−2 of pQBI25 containing enhanced green fluorescent protein (EGFP) cDNA (Quantum Biotechnologies Inc., Montreal, Canada) and with 0.36 µg·cm−2 of mock expression plasmid (pcDNA3; Invitrogen Corp., Carlsbad, CA, USA) or the DAP kinase expression plasmids, pDK-wt, pDK-ΔCaM, pDK-K42A, or pDK-DD, carrying, respectively, cDNA coding for wild-type DAP kinase, the ΔCaM-DAP kinase␣mutant, the K42A-DAP kinase mutant, or the DD-DAP kinase mutant (aspartic acid 1299 to arginine 1430), all with an hemagglutinin (HA)-tag, in pcDNA3 [5,6] using 2 µL·cm−2 of SuperFect reagent (QIAGEN Inc., Valencia, CA, USA) according to the␣manufacturer's instructions.

Immunoblotting

Forty-eight hours after transfection, the PC12 cells were washed with ice-cold NaCl/Pi, harvested, and lysed with a buffer consisting of 50 mm Tris/HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 2 mm phenylmethanesulfonyl fluoride, 1 µg·mL−1 aprotinin, 1 µg·mL−1 leupeptin, and 30 µm mycalolide B. After the insoluble material was removed by centrifugation (15 000 g for 5 min at 4 °C), the protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (2 µg) were then separated by electrophoresis on 10% or 15% SDS/polyacrylamide gels, and electrophoretically transferred onto poly(vinylidene difluoride) (PVDF) membranes (Millipore Corp., Bedford, MA, USA). To detect the exogenous DAP kinase or actin used as the internal control, the membranes were blocked by incubation for 30 min at room temperature with 2% dried skimmed milk in NaCl/Pi containing 0.1% Tween-20, (NaCl/Pi/Tween) then incubated for 1.5 h at room temperature with rat monoclonal anti-HA Ig (1 : 5000 dilution; Roche Diagnostics GmbH) or polyclonal anti-actin Ig (1 : 5000 dilution; Sigma Chemical Co.). After washing with NaCl/Pi/Tween, the blots were incubated with horseradish peroxidase-conjugated goat anti-(rat IgG) Ig (1 : 1000 dilution; Santa Cruz Biotechnology, Inc.) or horseradish peroxidase-conjugated goat anti-(rabbit IgG) Ig (1 : 5000 dilution; Santa Cruz Biotechnology, Inc.) and the reactive bands visualized using an enhanced chemiluminescence system (ECL plus; Amersham Pharmacia Biotech.) as indicated in the manufacture's protocol.

Results

Apoptosis induced by ceramide

Recent reports indicate that exposure to high concentrations (10–50 µm) of exogenous C2-ceramide results in apoptosis of neuronal cells and that the apoptotic effect depends on the concentration of C2-ceramide and the cell plating density [11–14]. We therefore initially determined the optimal experimental conditions leading to apoptosis in PC12 cells. PC12 cells were plated at a density of 5.0 × 104 cells per cm2 in normal growth medium, then, after 2 days, the culture medium was changed to DMEM containing 1% horse serum, 50 µg·mL−1 gentamycin (serum reduced medium), and 0, 3, 10, 20, 30, or 50 µm C2-ceramide. After incubation at 37 °C for 12 h, DNA was extracted and chromatin fragmentation examined by electrophoresis. No DNA ladder was seen in PC12 cells treated with 0, 3, 10, or 20 µm C2-ceramide, but a typical DNA ladder was detected in the presence of 30 or 50 µm C2-ceramide (Fig. 1A), showing that concentrations of C2-ceramide greater than 30 µm induced apoptosis in PC12 cells under these conditions. Similar results were obtained using C8-ceramide, an active ceramide analog with a longer 8-carbon fatty acid chain (Fig. 1B). In contrast, an inactive ceramide analog, C2-dihydroceramide, which lacks the C4–5 trans double bond in the sphingolipid backbone that is required for the biological effects of ceramide [19], failed to induce apoptosis (Fig. 1C). Typical apoptotic molphological changes, such as shrinkage, rounding up, and loss of adherence, and staining of the nuclei by propidium iodide, were seen in PC12 cells exposed to 30 µm C2- (Fig. 1D) and C8-ceramide (data not shown), but not in those exposed to 30 µm C2-dihydroceramide (Fig. 1E).

Figure 1.

C2-ceramide and C8-ceramide induce apoptosis in PC12 cells. PC12 cells were cultured in serum-reduced medium containing 0 µm (lane 1), 3 µm (lane 2), 10 µm (lane 3), 20 µm (lane 4), 30 µm (lane 5), or 50 µm (lane 6) C2-ceramide (A), C8-ceramide (B), or C2-dihydroceramide (C). After 12 h, 2 µg of DNA was prepared and examined for the presence of a DNA ladder as described in Experimental procedures. The size markers are indicated on the left. Morphological appearance of PC12 cells exposed to 30 µm C2-ceramide (D) or 30 µm C2-dihydroceramide (E). PC12 cells were stained with 3 µg·mL−1 propidium iodide (PI) (Dojindo, Kumamoto, Japan) and observed under a phasecontrast (BF) or a fluorescence microscope (PI) (Carl Zeiss, Esslingen, Germany).

Ceramide-induced apoptosis is accompanied by DAP kinase activation

DAP kinase, a Ca2+/calmodulin-regulated serine/threonine kinase, is a positive mediator of apoptosis [1–3]. As Western blotting showed that DAP kinase was endogenously expressed in PC12 cells (Fig. 2, lane 1), we examined whether the ceramide-induced apoptosis in PC12 cells was accompanied by DAP kinase activation.

Figure 2.

Immunoblot analysis of DAP kinase in immunoprecipitates. Monoclonal anti-(DAP kinase) Ig (Sigma) was used to immunoprecipitate endogenous DAP kinase from extracts of PC12 cells for 10 min in the absence of ceramide (lane 1) or in the presence of 30 µm C2-ceramide (lane 2), 30 µm C8-ceramide (lane 3), or 30 µm C2-dihydroceramide (lane 4). The immunoprecipitates were analyzed by immunoblotting using another monoclonal anti-(DAP kinase) Ig (Transduction Laboratories).

The physiological substrate of DAP kinase has not been identified. In a previous report [4], purified myosin light chain was used as a substrate, as the catalytic domain of DAP kinase is highly homologous to that of MLC kinase. Zipper-interacting protein kinase (ZIP kinase), with a sequence 81% identical to that of the kinase domain of DAP kinase [20–22], also phosphorylates the regulatory light chain of myosin II the phosphorylation sites being threonine 18 and serine 19 [22]. In this study, the synthetic peptide, skeletal myosin light chain kinase substrate peptide, which contains the regulatory light chain of myosin II site phosphorylated by ZIP kinase, was used as substrate to measure DAP kinase activity.

DAP kinase activity was measured 0, 3, 10, 30, and 90 min after changing to serum-reduced medium containing 30 µm C2-ceramide, C8-ceramide, or C2-dihydroceramide in the presence (total activity) or in the absence (Ca2+/calmodulin independent activity) of Ca2+ and calmodulin [17]. When the cells were exposed to C2- and C8-ceramide, the Ca2+/calmodulin-independent DAP kinase activity was increased 3.6- and 2.9-fold, respectively, at 3 min, showed a maximal increase of 12.2- and 11.6-fold at 10 min, then reduced to 5.6- and 6.4-fold at 30 min; a 3.4- and 3.6-fold increase was still apparent after 90 min (Fig. 3A). As shown in Fig. 3B, in the presence of C2- and C8-ceramide, the total DAP kinase activities also increased 2.1- and 1.8-fold at 3 min, showed a maximal increase of 5.0- and 4.8-fold at␣10 min, then gradually decreased to 1.7- and 1.8-fold at 90 min The Ca2+/calmodulin independent activity/total activity ratio is shown in Fig. 3C. This ratio, which was 0.22 in the absence of ceramide (0 min), increased to 0.60 and 0.64 after 30 min exposure to C2- and C8-ceramide, respectively, then declined gradually. In contrast, 30 µm C2-dihydroceramide caused no increase in either total or Ca2+/calmodulin-independent DAP kinase activity, and no change in the ratio (Fig. 3A–C).

Figure 3.

Time course of ceramide-induced DAP kinase activation. PC12 cells were exposed to 30 µm C2-ceramide, C8-ceramide, or C2-dihydroceramide for 0, 3, 10, 30, or 90 min, then the cells were solubilized in lysis buffer, and DAP kinase immunoprecipitated with monoclonal anti-(DAP kinase) Ig. The kinase reaction was performed in the absence (Ca2+/calmodulin-independent activity, A) or presence (total activity, B) of Ca2+ and calmodulin. The relative DAP kinase activity was determined by comparison with the value measured in the absence of ceramide (0 min). The data are presented as the mean ± SD of four measurements. (C) Shows the ratio obtained by dividing the actual value for Ca2+/calmodulin-independent activity by that for the total activity.

We next determined whether the increase in DAP kinase activity was dependent on the ceramide concentration by exposing PC12 cells to 0, 10, 20, 30, or 50 µm C2-ceramide, C8-ceramide, or C2-dihydroceramide for 10 min. As shown in Fig. 4A, Ca2+/calmodulin-independent DAP kinase activity was slightly stimulated by 1.4- and 1.3-fold by exposure to 10 µm C2- and C8-ceramide, and greater activity was seen with increasing concentrations of these reagents, the increase at 50 µm C2- and C8-ceramide being 12.2- and 11.6-fold, respectively. Total DAP kinase␣activity␣also␣increased with increasing concentration of ceramide, the increase at 50 µm C2- and C8-ceramide being 6.5- and 6.1-fold, respectively (Fig. 4B). The Ca2+/calmodulin-independent activity/total activity ratio also increased with ceramide concentration, reaching a plateau of 0.54 and 0.55 at 30 µm C2- and C8-ceramide, respectively (Fig. 4C). In contrast, C2-dihydroceramide was ineffective in activating DAP kinase and in changing the Ca2+/calmodulin-independent activity/total activity ratio at any concentration tested (Fig. 4A–C).

Figure 4.

Concentration dependency of DAP kinase activation by ceramide. PC12 cells were exposed for 10 min to 0, 10, 20, 30, or 50 µm C2-ceramide, C8-ceramide, or C2-dihydroceramide, then solubilized in lysis buffer, and DAP kinase immunoprecipitated with monoclonal anti-(DAP kinase) Ig. The kinase reaction was performed in the absence (Ca2+/calmodulin-independent activity, A) or presence (total activity, B) of Ca2+ and calmodulin. The relative activity of DAP kinase was determined by comparison with that in the absence of ceramide (0 µm). The data are the␣mean ± SD of four measurements. (C)␣Shows the ratio obtained by dividing the actual value for Ca2+/calmodulin-independent activity by that for the total activity.

Approximately equal amounts of DAP kinase were immunoprecipitated irrespective of the test exposure time or the concentration of C2-ceramide, C8-ceramide, or C2-dihydroceramide. Figure 2 shows the results of a typical immunoblotting experiment after 10 min exposure to 30 µm C2-ceramide, C8-ceramide, or C2-dihydroceramide.

Protection from ceramide-induced apoptosis by DAP kinase inhibition

To determine whether DAP kinase activity was critical for C2- and C8-ceramide-induced apoptosis, we investigated the viability of PC12 cells overexpressing the wild-type DAP kinase, the ΔCaM-DAP kinase mutant (a constitutively active kinase mutant), the K42A-DAP kinase mutant (a catalytically inactive mutant displaying dominant negative features), or a construct encompassing the death domain and the serine-rich C-terminal tail (another kind of dominant negative mutant) (Fig. 5A). PC12 cells were cotransfected with pcDNA3, pDK-wt, pDK-ΔCaM, pDK-K42A, or pDK-DD, and pQBI25 containing EGFP cDNA as a marker to visualize the transfected cells. The transfection efficiency was determined by counting the number of EGFP-positive cells at 8 h after DNA transfection and was verified to be about 20%, similar in all experiments. After 48 h, ectopic expression of the wild-type and mutant DAP kinase proteins was confirmed by immunoblotting using monoclonal anti-HA Ig (Fig. 5B). As expected, the specific band of DAP kinase with an apparent molecular mass of ≈ 165 KDa was seen in PC12-wtDK and PC12-K42A-DK cells, a slightly lower molecular mass band (160 KDa) was detected in PC12-ΔCaM-DK cells, and a band at ≈ 16 KDa was seen in PC12-DD-DK cells. The band intensity in PC12-ΔCaM-DK cells was weaker than that in the PC12-wtDK and PC12-K42A-DK cells, suggesting that overexpression of the ΔCaM-DAP kinase mutant resulted in some growth disadvantage for PC12 cells. No band was seen in PC12 cells transfected with pcDNA3 vector alone.

Figure 5.

Expression of wild-type and mutated DAP kinase proteins in transiently transfected PC12 cells. (A) Schematic presentation of the human DAP kinase mutant proteins used in these experiments. The ΔCaM-DK mutant lacks 47 amino acid residues (proline 277 to leucine 323) containing the calmodulin-binding domain. In the K42A-DK mutant, a lysine residue at position 42 in the kinase domain (asterisk) is replaced by an alanine. The DD-DK construct contains the death domain and the serine-rich tail (aspartic acid 1299 to arginine 1430). All expressed proteins have an HA-tag sequence (cross) at the N-terminal. (B) PC12 cells were transfected with pcDNA3 (control), pDK-wt (wt-DK), pDK-ΔCaM (ΔCaM-DK), pDK-K42A (K42A-DK), or pDK-DD (DD-DK) and proteins were prepared as described in Experimental procedures. Approximately 2 µg of each preparation was separated on a 10% SDS-polyacrylamide gel for control, wt-DK, ΔCaM-DK and K42A-DK, and on a 15% SDS/polyacrylamide gel for DD-DK and actin. After electrophoretic transfer onto poly(vinylidene difluoride) membrane, the blot was reacted with anti-HA Ig to detect ectopic expression of DAP kinase and the mutants (upper panel) and with anti-actin Ig (lower panel) to quantitate the loaded protein amounts. In the left panel, the arrow on the right indicates the wt-DK and K42A-DK bands at ≈ 165 KDa and the arrowhead indicates the ΔCaM-DK band with a slightly lower molecular mass (160 KDa). In the right panel, the arrow indicates the DD-DK band at ≈ 16 KDa.

Ectopic expression of DAP kinase in PC12 cells (PC12-wtDK) did not cause cell death in the absence of stimuli. This is in agreement with the results of previous studies using COS cells [1,4], murine Lewis (3LL) and CMT64 lung carcinoma cells [23], but conflicts with results in HeLa cells and SV80 human fibroblasts [1,4]. The transfected PC12 cells were cultured for 48 h, then exposed for 24 h to 30 µm C2-ceramide, C8-ceramide or C2-dihydroceramide. To determine cell viability, we counted the number of morphologically intact EGFP-positive cells under a fluorescent microscope (Fig. 6). The viability of PC12-wtDK cells was markedly decreased by exposure to 30 µm C2- and C8-ceramide, but not by exposure to 30 µm C2-dihydroceramide. Furthermore, PC12-wtDK cells were more sensitive to C2- and C8-ceramide than PC12 cells transfected with pcDNA3 mock vector. For PC12-ΔCaM-DK cells, viable cell numbers were much lower even in the absence of ceramide or in the presence of 30 µm C2-dihydroceramide. In contrast, PC12-K42A-DK and PC12-DD-DK cells were significantly resistant to 30 µm C2- and C8-ceramide-induced apoptosis. These findings clearly demonstrate that DAP kinase activity is critical for C2- and C8-ceramide-induced apoptosis in PC12 cells.

Figure 6.

Viability of PC12 cells expressing wild-type and DAP kinase mutants after ceramide exposure. Resistance to ceramide-induced apoptosis was quantified by counting the number of morphologically intact EGFP-positive cells under a fluorescence microscope. PC12 cells were transfected with expression plasmids. The transfection efficiency, about 20%, is confirmed to be similar in all experiments by counting the number of EGFP-positive cells at 8 h after DNA transfection. Then, after 48 h, cells were incubated for 24 h in the absence of ceramide (white column) or in the presence of 30 µm C2-ceramide (black column), C8-ceramide (grey column), or C2-dihydroceramide (striped column) and the number of the surviving EGFP-positive cells were counted. The percentage of cell viability is caluculated by dividing the number of EGFP-positive cells in each experimental conditions by the number of EGFP-positive cells transfected with pcDNA3 alone in the absence of ceramide. The data are the mean ± SD of seven randomly selected fields in three independent experiments.

Discussion

DAP kinase is a Ca2+/calmodulin-regulated serine/threonine kinase that participates in apoptosis induced by a variety of signals [1,6]. We recently showed that the expression of DAP kinase mRNA in brain is increased prior to cell death induced by transient forebrain ischemia [8]. These findings indicate a possible relationship between DAP kinase and neuronal cell death, including apoptosis and necrosis. However, it is not known whether the kinase activity is involved in apoptosis. In the present study, we employed PC12 cells, extensively used as a model to study mechanisms regulating apoptosis [15], to investigate the role of DAP kinase activity in ceramide-induced apoptosis.

The results of the DNA fragmentation assay and morphological observation of cells using propidium iodide showed that, in agreement with a previous report [14], C2-, and C8-ceramide both induced apoptosis in PC12 cells. Although DAP kinase was expressed in PC12 cells, both the total and Ca2+/calmodulin-independent DAP kinase activities were very low under normal growth conditions. However, in the presence of C2- or C8-ceramide, the total and Ca2+/calmodulin-independent activities were increased in a concentration-dependent manner by a maximum of 13-fold and fivefold, respectively, after 10 min of exposure, then gradually decreased, although still showing a twofold to fourfold increase after 90 min of exposure. In addition, the Ca2+/calmodulin-independent activity/total activity ratio also increased from 0.2 to 0.6 following exposure to C2- or C8-ceramide, these increases being maintained at 0.4–0.5 even after 90 min. Similar results have been obtained with Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV), one of the member of the CaM kinase family [24]. It is thought that CaM kinase IV requires phosphorylation by a CaM kinase kinase, which is Ca2+/calmodulin-responsive, for full activation. A previous study using Jurkat T lymphocytes showed that both the total and Ca2+/calmodulin-independent CaM kinase IV activities were increased by treatment with anti-CD3 Ig, which triggers an intracellular Ca2+ increase, and that the Ca2+/calmodulin-independent activity/total activity ratio was increased. These results suggest that CaM kinase IV is activated through a CaM kinase kinase cascade triggered by an increase in intracellular Ca2+ levels, and as CaM kinase IV activated by CaM kinase kinase has significant Ca2+/calmodulin-independent activity, it has the potential for prolonged activation. It is tempting to speculate that ceramide-activated DAP kinase, which shows significant Ca2+/calmodulin-independent activity, has the potential for prolonged activation and subsequently results in apoptosis in PC12 cells. This idea was supported by the results of transfection studies showing that the K42A-DAP kinase mutant failed to induce apoptosis in the presence of ceramide, that the ΔCaM-DAP kinase mutant induced apoptosis even in the absence of ceramide, and that PC12 cells overexpressing DAP kinase showed higher sensitivity to ceramide than untransfected PC12 cells. Thus, the intrinsic kinase activity of DAP kinase is critical for apoptosis.

The mechanism of DAP kinase activation has not been clear. DAP kinase undergoes phosphorylation and the intrinsic activity is stimulated by Ca2+/calmodulin [1,4]. However, in accordance with a previous observation in C6-ceramide-treated U937 cells [25], permeant-ceramide-treated Jurkat T cells [26] and foreskin fibroblasts [27], we could not detect any C2- or C8-ceramide-induced increase in intracellular Ca2+ level using calcium fluorometry and a fura-2 fluorescent probe (data not shown). This is also consistent with the result that ceramide-treated PC12 cells had significant Ca2+/calmodulin-independent DAP kinase activity. Thus, one possible mechanism of DAP kinase activation is a change in phosphorylation state (i.e. phosphorylation and dephosphorylation). In the ceramide-induced apoptosis pathway, other molecules that are Ca2+/calmodulin-nonresponsive and activate DAP kinase in a ceramide-dependent fashion may exist. Further experiments will be required to elucidate the DAP kinase activation mechanism.

In addition, the apoptotic function of DAP kinase is thought to be modulated through other functional domains. DAP kinase contains eight ankyrin repeats, two potential ATP/GTP binding sites (P-loops), a cytoskeleton-binding domain, a death domain, and a serine-rich C-terminus tail [4,5]. A previous study showed that deletion of the death domain abrogates the apoptotic function and that overexpression of the death domain protects HEK293 and HeLa cells from TNF-α-, Fas-, and FADD/MORT1-induced apoptosis [6]. In the present study, ectopic expression of the death domain or the K42A-DAP kinase mutant in PC12 cells suppressed ceramide-induced apoptosis; however, the protection effect of the death domain was weaker than that of the K42A-DAP kinase mutant. This result is in agreement with previous observations using HEK293 and HeLa cells [6].

Treatment with ceramide results in activation of the CD95 (APO-1/Fas) signaling cascade [28], the stress-activated protein kinase (SAPK/JNK) signaling cascade [29], and the p53 signaling cascade [30,31]. However, the mechanism involved in C2-ceramide-induced apoptosis is controversial. One possible explanation is as follows. As C2-ceramide is cell-permeable, it probably perturbs the membrane structure and increases membrane permeability [32–34]. Changes in membrane permeability, particularly in mitochondria, are involved in the triggering of cell death. In mitochondria, C2-ceramide triggers the formation of reactive oxygen species, the disruption of electron transport and energy metabolism, and the release of caspase-activating proteins, cytochrome c, and apoptosis-inducing factor, and then activates the caspase family of proteases [35]. Recent studies have demonstrated that C2-ceramide-induced apoptosis is required for caspase activation [36–38]. It has also emerged that C2-ceramide-induced apoptosis in PC12 cells is prevented not only by a caspase inhibitor, but also by neurotrophic factors, such as nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). These protective effects exerted by NGF and bFGF are independent of either the extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3 kinase (PI3 kinase) cascade, both of which are known to be cell survival-promoting signal pathways [36]. Thus, it is feasible that an as yet undescribed apoptotic pathway, which may include DAP kinase, is antagonized in neurotrophic factor-dependent rescue from C2-ceramide-induced apoptosis.

Mitocondrial permeability transitions and the subsequent activation of caspases also take place under ischemic conditions [39,40]. We have previously demonstrated that the expression of DAP kinase mRNA is increased prior to cell death resulting from transient forebrain ischemia [8]. The apoptotic function of the ΔCaM-DAP kinase mutant is blocked by overexpression of natural caspase inhibitors, i.e. CrmA and p35, but not by overexpression of a dominant negative caspase-8 mutant, indicating that DAP kinase functions downstream of caspase-8 and upstream of some members of the caspase family other than caspase-8 [6]. A recent study by Raveh et al. [41] demonstrated DAP kinase activates p53 via p19ARF and suppresses oncogenic transformation. Taking together these results and those of the present study, it is thus conceivable that DAP kinase participates in a novel cascade involved not only in C2-ceramide-induced apoptosis, but also in physiologically induced apoptosis, as a result of caspase activation, and that DAP kinase activity is a key element leading to apoptosis.

Acknowledgements

We thank Dr Adi Kimchi (Weizmann Institute of Science, Israel) for providing helpful advice and DAP kinase expression plasmids, Dr␣Xiaofen Sun and Mr Christopher Booth for assistance in cell viability assay.

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