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Originally published In Press as doi:10.1074/jbc.M412449200 on February 16, 2005

J. Biol. Chem., Vol. 280, Issue 17, 17371-17379, April 29, 2005
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Regulation of Monocyte Apoptosis by the Protein Kinase C{delta}-dependent Phosphorylation of Caspase-3*

Oliver H. Voss{ddagger}§, Sunghan Kim{ddagger}, Mark D. Wewers¶||, and Andrea I. Doseff§¶||**

From the ||Heart and Lung Research Institute and Division of Pulmonary and Critical Care, §Department of Molecular Genetics, the Ohio State University, Columbus, Ohio 43210

Received for publication, November 3, 2004 , and in revised form, February 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase C{delta} (PKC{delta}) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKC{delta}-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKC{delta} is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKC{delta} precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKC{delta} resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKC{delta}-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis plays an essential role in the daily maintenance and development of the immune system by regulating the number of the cells. Monocytes, key components of innate immunity, circulate in the bloodstream for 24–48 h and, in the absence of an appropriate stimulus, die spontaneously (1). Spontaneous monocyte apoptosis requires the activation of the apoptotic protease machinery (2). During inflammation, differentiation or malignant transformation and inflammatory and differentiation factors, including lipopolysaccharide and macrophage colony-stimulating factor, prolong monocyte survival (3–5). These survival signals block spontaneous apoptosis by inhibiting the activation of the caspases (2, 6, 7) by a mechanism yet to be elucidated.

Monocyte apoptosis requires the activation of caspase-3, a member of the evolutionarily conserved cysteine-aspartate-specific protease family (2, 8). The caspases are constitutively expressed as inactive precursors. In response to an apoptotic stimulus, the precursors are converted into active caspases by proteolytic processing (9). Caspase-3 is an "effector" caspase that requires two proteolytic cleavages to mature into an active enzyme (10). The first proteolytic step is dependent upon the activity of "initiator" caspases, such as caspase-8 or -9 that cleave caspase-3 between the long (p17) and the carboxyl-terminal (p12) domains (11, 12). Generation of the active caspase-3 also requires a second cleavage, occurring between the prodomain and the p17 domain, a cleavage that has been suggested to be autocatalytic (13).

Given the central importance of caspases in the regulation of cell fate in the immune system, the activation of caspases is controlled by several mechanisms, including protein-protein interactions and post-translational modifications (14). For example, caspase-3 activity is blocked by S-nitrosylation in human monocytes (15), and the denitrosylation of caspase-3 results in caspase-3 activation in B and T cell lines upon Fas-induced apoptosis (16). Phosphorylation is another well recognized mechanism of protein regulation that has been extensively implicated in apoptosis (reviewed in Ref. 17). However, the involvement of kinases as direct modulators of caspases is just starting to be recognized. For example, the activation of caspase-9 is inhibited by phosphorylation by the serine-threonine kinase protein kinase B/Akt or by the extracellular signal-regulated kinase (ERK) (18, 19). In this context, macrophage colony-stimulating factor promotes monocyte survival through the inhibition of caspase-9 activation due to phosphorylation by Akt in a phosphatidylinositol 3-kinase-dependent manner (6). Consistent with this, the reactivation of apoptosis in lipopolysaccharide-treated monocytes by the anti-inflammatory cytokine interleukin-4 depends, at least in part, on the suppression of ERK1 phosphorylation (20). Although the role of signal transduction cascades in the regulation of apoptosis and cell fate establishment in myeloid cells is broadly accepted, the specific signaling events that regulate monocyte apoptosis remain unknown.

Protein kinase C (PKC) constitutes a large family of serine/threonine protein kinases consisting of at least 10 different isoforms classified into three subgroups based on their cofactor requirements as follows: classical, novel, and atypical (21). The involvement of PKC in the regulation of apoptosis is well documented, functioning either as activators or inhibitors of apoptosis, depending on the particular cell type and the specific apoptotic stimulus (22, 23). PKC{delta}, a member of the novel subclass, is abundantly expressed in human monocytes (24) and one of the first substrates of caspase-3 to be identified (25). Activation of PKC{delta} during apoptosis was observed in several cell types in response to a variety of stimuli, and PKC{delta} promotes the apoptosis of neutrophils, myocytes, and etoposide-induced salivary gland acinar cells (26–30).

We show here that caspase-3 is phosphorylated in vivo in monocytic cells. We demonstrate that caspase-3 associates with and is phosphorylated in vitro by PKC{delta}. We found that activation of PKC{delta} precedes caspase-3 activity during spontaneous and induced monocyte apoptosis, and the phosphorylation enhances caspase-3 activity. In addition, we show that overexpression of PKC{delta} increases apoptosis, whereas down-regulation of PKC{delta} expression prolongs monocyte survival, inhibits caspase-3 activation, and decreases caspase-3 phosphorylation. Taken together, these studies suggest that the PKC{delta}-dependent phosphorylation of caspase-3 provides a novel pro-apoptotic mechanism involved in the regulation of the monocyte life span.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Monocyte Purification—Human monocytes were purified by clumping, as described previously (2). Briefly, fresh human monocytes were obtained from normal donors and diluted 1:1 with sterile saline solution. The solution was subsequently centrifuged through a Histopaque-1077 gradient column (Sigma) at 600 x g for 20 min at 4 °C. The mononuclear layer was removed, washed, and spun twice in RPMI 1640 (BioWhittaker, Walkersville, MD), and the cells were counted. The cells were resuspended in RPMI 1640, 10% fetal bovine serum (Hyclone, Logan, UT) at a concentration of 5 x 107 cells/ml. Cells were rotated at 70 rpm on a horizontal rotor for 1 h at 4 °C to induce clumping and then sedimented by gravity for 20 min through 100% fetal bovine serum at 4 °C. The sedimented cells were subsequently washed twice in RPMI 1640 and resuspended in RPMI at a final concentration of 1 x 106 cells/ml. The population of monocytes obtained was on average 70–80% pure as estimated by flow cytometry using an anti-CD14 marker (BD Biosciences). In all experiments monocytes were incubated at a concentration of 2 x 106 cells/ml in serum-free RPMI 1640 at 37 °C in 5% CO2. THP-1 premonocytic cells and HeLa cells were cultured in RPMI 1640 with 5% heat-inactivated fetal bovine serum in the presence of 50 µg/ml penicillin and streptomycin (Invitrogen) at 37 °C in 5% CO2. For the in vivo labeling experiments, cells were washed once with PBS and incubated for 1 h at 37 °C in RPMI 5% serum in the absence of sodium phosphate. 107 cells/ml were labeled with inorganic phosphate (1 mCi/ml, specific activity 9120 Ci/mmol, PerkinElmer Life Sciences) for 4 h in RPMI 1640 containing phosphate at 37 °C in 5% CO2.

Extract Preparation and Immunoprecipitation—Cells were pelleted and washed once with KPM buffer (50 mM KCl, 50 mM PIPES, 10 mM EGTA, 1.92 mM MgCl2, pH 7, 1 mM DTT, 0.1 mM PMSF, 10 µg/ml cytochalasin B, and 2 µg/ml each of the protease inhibitors chymostatin, pepstatin, leupeptin, and antipain). Extracts were prepared by lysing the cells for 10 min on ice with Nonidet P-40 kinase extraction buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.5, 5 mM EDTA, pH 8, 10 mM sodium glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 100 µM PMSF, 2 µg/ml protease inhibitors). Extracts were then centrifuged for 10 min at 14,000 x g in a microcentrifuge. Supernatants were aliquoted and kept at –70 °C for future use. Immunoprecipitations were carried out by rocking extracts with 10 µl (50% slurry) of monoclonal antibodies coupled to protein A-SepharoseTM (Amersham Biosciences) for 3 h at 4 °C. In vivo labeled cell lysates, obtained as described above, were immunoprecipitated in the presence of 0.1% SDS after a 15-min incubation at 55 °C. Monocyte cell extract equivalent to 2 x 108 cells was used per each immunoprecipitation reaction for the caspase-3 and PKC{delta} coimmunoprecipitation experiments. For PKC{delta} immunoprecipitation followed by in vitro kinase reaction shown in Fig. 3B, less than 2 x 106 THP-1 cells were used in each reaction. Antibodies or IgG isotype control were cross-linked to protein A beads as follows: 225 µl of antibodies were rocked with 1 ml of wet protein A beads for 1 h at room temperature. The beads were washed with 30 ml of 0.1 M triethanolamine (Sigma), pH 8.3, and then incubated for 4 h at room temperature with 50 mM dimethylpimelimidate (Pierce). The beads were spun down and incubated for 2 h with 0.1 M ethanolamine (Sigma). The beads were washed in a column with 150 ml of PBS followed by 150 ml of KPM buffer. Immunoprecipitates were washed four times with RIPA kinase extraction buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 10 mM NaF, 10 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mM DTT, 100 µM PMSF, 2 µg/ml of protease inhibitors). Beads were then washed two times with the kinase assay buffer (25 mM HEPES, pH 7.3, 10 mM MnCl2, 1 mM MgCl2, 1 mM DTT).

In Vitro Kinase Assays—After immunoprecipitation, kinase assays were performed by incubating the beads for 1 h at 37 °Cinthe presence of 20 µl of kinase assay buffer containing 0.75 µCi of [{gamma}-32P]ATP (PerkinElmer Life Sciences), 0.5 mM ATP. To each reaction, 5 µg of histone H2B as exogenous substrate (Roche Applied Science) was added as exogenous substrate. Reactions were stopped by the addition of 10 µl of 5x Laemmli buffer. Samples were boiled for 5 min and loaded onto an SDS-polyacrylamide gel. The kinase inhibitors utilized were genistein, AG126, UO126, bisindolylmaleimide I, Ro 31-8425, LY294002, PD98059, H-89 (Calbiochem), and staurosporine (Sigma). CKI-7 (CKI-7, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide) was from Seikagaku America, Inc. (Falmouth, MA), and IC261 was from ICOS Corp. (Bothell, WA). Phosphorylation of human recombinant caspase-3 (rCasp-3) was achieved by using purified human PKC{delta} (Panvera, Invitrogen) as the kinase source. 250 ng of rCasp-3 expressed in Escherichia coli was added to 20 µl of kinase reaction mixture containing 25 mM HEPES, pH 7.4, 10 mM MnCl2, 1 mM MgCl2, 500 nM ATP and 5 µCi of [{gamma}-32P] ATP in the presence of 10 ng of human PKC{delta}. A mixture of phosphatidylserine (200 µg/ml final concentration) and diacylglycerol (20 µg/ml final concentration) was also added to the reaction as cofactors of PKC{delta}. The control reaction for Fig. 4 consists of the exactly same reaction set up except that PKC{delta} was boiled for 10 min before its addition onto the reaction mixture. Boiled PKC{delta} was unable to phosphorylate H2B or rCasp-3 in in vitro kinase assays (data not shown). The phosphorylation reaction was carried out at 37 °C for 1 h and was followed by SDS-PAGE. For the caspase-3 activation reaction to determine the effect of phosphorylation, first the phosphorylation was carried out as described above. Then the caspase-3 activation reaction was followed by addition of an equal volume of 2x caspase-3 activation buffer (100 mM HEPES, pH 7.5, 100 mM NaCl, 20% sucrose, 20 mM DTT and 2 mM PMSF) with 4 units/µl of recombinant caspase-9 (rCasp-9, Biomol, Plymouth Meeting, PA). In order to achieve an apparent stop of the kinase activity, the radioactive [{gamma}32P]ATP present in the kinase reaction was quenched by adding 500 µM cold ATP at the onset of the activation reaction. The activation reaction was allowed to proceed at 37 °C and was stopped by adding 5x Laemmli buffer at a desired time point. Reactions of phosphorylated and nonphosphorylated rCasp-3 were resolved by SDS-PAGE, and both the kinase activity and the caspase-3 activation kinetics were visualized by autoradiography and by Western blot analysis, respectively, after transferring the gel to a membrane.

Preparation of Lysates and Detection of Caspase Activity—The presence of active caspase-3 was determined by using the 7-amino-4-trifluoromethyl coumarin assay (AFC) linked to the tetrapeptide DEVD as described previously (2). 250 ng of purified rCasp-3 were incubated with DEVD-AFC in a cyto-buffer (10% glycerol, 50 mM Pipes, pH 7.0, 1 mM EDTA, 1 mM DTT) containing 20 µM DEVD-AFC. Activity of active rCasp-9 was determined by using 80 units per reaction (488 ng) by the cleavage of LEHD-AFC in a buffer containing 100 mM MES, pH 6.5, 10% PEG (Mr 3350), 0.1% CHAPS, 10 mM DTT. DEVD and LEHD-AFC were obtained from Enzyme Systems Products (Livermore, CA). Release of free AFC was determined by using a Cytofluor 4000 fluorometer (Perceptive Co., Framingham, MA; filters: excitation, 400 nm; emission, 505 nm).

Immunoblots and Purification of Recombinant Proteins—Monoclonal antibodies (clone 3-11) that recognize and immunoprecipitate caspase-3 have been described previously (31). For Western blots, anti-caspase-3 antibody (clone 4-18) was used as described previously (2). PKC{delta} and PKC{alpha} polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). rCasp-3 carrying His6 tag at the amino terminus was generated by cloning full-length human caspase-3 into the expression vector pQE31 (Qiagen, Valencia, CA). Cultures were induced with 1 mM isopropyl 1-thio-{beta}-D-galactopyranoside at A550 0.5. After induction for 1 h at 20 °C, cells were lysed by sanitation in 50 mM sodium phosphate, pH 7.8, 300 mM NaCl, 5 mM {beta}-mercaptoethanol, 1% Tween 20, and protease inhibitors (2 µg/ml chymostatin, pepstatin, leupeptin, antipain, and 1 mM PMSF). Lysates were centrifuged for 10 min at 17,000 x g, and the supernatant was allowed to bind to the nickel-nitrilotriacetic acid-agarose (Qiagen) in the presence of RNase (5 µg/ml) for 2 h at 4 °C. After binding, the beads were washed with 16 ml of the buffer containing 50 mM HEPES, pH 7.4, 50 mM NaCl, 10% glycerol, 1% Tween 20, and 1 mM PMSF. The purification was made by batch elution with 1-ml aliquots of the step gradient imidazole dissolved in the wash buffer. Elution fractions containing rCasp-3 were identified by SDS-PAGE and further confirmed by caspase-3 activity assay and by Western blot analysis after dialysis against 50 mM HEPES, pH 7.4, 50 mM NaCl, 10% sucrose, 5 mM DTT.

Transient Transfection and siRNA—THP-1 cells were washed once in PBS and resuspended in the specified electroporation buffer (Amaxa, Cologne, Germany) to a final concentration of 2 x 106 cells/ml. 0.5 µgof empty vector (pcDNA3-HA), vector containing wild type full-length PKC{delta} (pcDNA3-PKC{delta}-wt-HA), PKC{delta}, or a dominant negative mutant pcDNA3-PKC{delta}-DN-HA (32) were mixed with 0.1 ml of cell suspension, transferred to a 2.0-mm cuvette, and nucleofected using the Amaxa NucleofectorTM apparatus according to the manufacturer's specifications. After transfection, the cells were immediately transferred into 3 ml of RPMI medium and cultured for 1 day. Twenty four hours after transfection, the cells were treated with 1 µM etoposide (Sigma) for 8 h to induce apoptosis. For silencing experiments, HeLa or THP-1 cells were transfected with 150 nM siRNA-PKC{delta} (the siRNA sequences for targeting PKC{delta} were sense, 5'-GGCUGAGUUCUGGCUGGACTT-3' on PKC{delta} (33) (Qiagen)), and the same duplex siRNA-PKC{delta} labeled with rhodamine (siRNA-PKC{delta}-R) or a random rhodamine-labeled-negative control (sense, 5'-UUCUCCGAACGUGUCACGUTT-3', Qiagen) was utilized. Forty hours after transfection, cells were treated with etoposide to induce apoptosis for 8 h as described above. Cells were then rinsed with PBS and fixed with 2% paraformaldehyde (Sigma) for 5 min at room temperature and treated with 0.2% Triton X-100 (Bio-Rad) followed by rinse with PBS. Cells were stained with 0.5 µg/ml DAPI (Sigma) for 15 min at 4 °C. Percentage of apoptosis was determined by nuclear fragmentation of cells. Nuclear morphology of 200 cells in each experiment was visualized using Olympus fluorescence microscope and Image ProPlus software (excitation at 300 nm; emission at 461 nm). In silencing experiments only red cells were counted to determine their apoptotic status.

Flow Cytometry—For flow cytometry studies, monocytes were purified by a CD14-positive selection and cultured for 16 h as described previously (20). CD14-positive monocytes were washed with PBS and stained for annexin V-FITC and propidium iodine using the annexin V-FITC apoptosis detection kit following the manufacturer's specification (Pharmingen). Flow cytometry analysis was performed using BD Biosciences FACSCalibur using Cellquest version 3.3 software.

Statistical Analysis—Results were expressed as means ± S.E. Data were analyzed using Student t test for Figs. 4, 6C, and 7B and by analysis of variance in Fig. 5C using the JMP-SAS statistical program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Caspase-3 Is a Phosphoprotein—We have shown previously that the activation of caspase-3 plays a central role in human spontaneous monocyte apoptosis (2, 6, 7). To assess whether caspase-3 is regulated by phosphorylation, we determined the phosphorylation status of endogenous caspase-3. For this purpose, 32P metabolically labeled THP-1 whole-cell extracts were immunoprecipitated with monoclonal anti-caspase-3 antibodies (Fig. 1). Two bands, one of 35 kDa and a second of 80 kDa (p35 and p80, Fig. 1A), were specifically visualized by autoradiography in the immunoprecipitates with the anti-caspase-3 antibodies (Fig. 1A, Casp-3) and were absent in the isotype control immunoprecipitation (Fig. 1A, Control). When the same blot was probed with the anti-caspase-3 monoclonal antibody, only one band co-migrating with p35 was observed (Fig. 1B). These results suggest that caspase-3 interacts with phosphorylated proteins in vivo. Because the p35 protein of 35 kDa corresponds to the size of the unprocessed precursor caspase-3 (34), which is specifically recognized in the immunoprecipitation and the subsequent Western blot, these results also suggest that caspase-3 is phosphorylated in vivo in monocytes.

Caspase-3 Associates with a Kinase Activity—To investigate whether caspase-3 associates with a kinase activity, the endogenous caspase-3 from freshly isolated monocytes or THP-1 cells was immunoprecipitated with the anti-caspase-3 antibody and subjected to in vitro kinase assays in the presence of [{gamma}-32P]ATP, using H2B as a substrate. Endogenous caspase-3 from monocytes coprecipitated with a kinase activity capable of phosphorylating H2B (Fig. 2A, lane 2). In addition to the phosphorylated H2B, a 35-kDa phosphoprotein was recognized in the caspase-3 immunoprecipitated extracts (Fig. 2A, lane 2). This kinase activity was absent in immunoprecipitates performed with the isotype control (Fig. 2A, lane 1). When the same membrane was re-probed with the anti-caspase-3 antibody, only the 35-kDa phosphoprotein corresponding to caspase-3 was recognized in immunoprecipitates with the caspase-3 antibodies (Fig. 2A, lane 2), absent in the isotype control (Fig. 2A, lane 1). Similar results were obtained when caspase-3 was immunoprecipitated from extracts obtained from THP-1 cells and assayed for the associated kinase using H2B as a substrate (Fig. 2B). These results show that caspase-3 associates with a kinase activity in human primary monocytes and in THP-1 monocytic cells.



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  		FIG. 1.
Caspase-3 is phosphorylated in vivo. THP-1 premonocytic cells were radiolabeled with inorganic phosphate, and extracts were immunoprecipitated (IP) with anti-caspase-3 antibody (Casp-3) or an isotype control (Control). A, immunoprecipitated proteins were resolved by SDS-PAGE and transferred to a membrane. Phospholabeled proteins were visualized by autoradiography. B, the same membrane shown in A was immunoblotted with anti-caspase-3 antibodies.

 
PKC Inhibitors Block the Caspase-3-associated Kinase Activity—To identify the kinase that associates with caspase-3, the effect of different kinase inhibitors was tested on the phosphorylation of H2B in extracts immunoprecipitated with the anti-caspase-3 antibody. Extracts from freshly isolated monocytes were immunoprecipitated with the anti-caspase-3 antibody (Fig. 2C, Casp-3) or an isotype control (Fig. 2C, Control). Following the immunoprecipitation, in vitro kinase assays were performed in the presence (each inhibitor indicated) or absence (–) of protein kinase inhibitors and using H2B as the substrate. The phosphorylation products were separated by SDS-PAGE and transferred to membranes. The relative levels of kinase activity in each treatment were assessed by quantifying the phosphorylation of H2B. Next, the same membranes were re-probed with the anti-caspase-3 antibody to ensure comparable immunoprecipitation efficiency (Fig. 2C). Most of the inhibitors tested by the method described above had no effect on the phosphorylation of H2B by the caspase-3-associated kinase activity (Table I). However, the treatment with very low doses of the potent PKC inhibitor, staurosporine, effectively blocked the caspase-3-associated kinase activity in a dose-response manner (Fig. 2C, lanes 3–5). In addition, other inhibitors considered selective for PKC, such as bisindolylmaleimide (35) and Ro 31-8425 (36), blocked the kinase activity at concentrations lower than 10 nM (Fig. 2C, lanes 8–13; Table I). These findings indicated that a member of the PKC family is likely to be the kinase associated with caspase-3.


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  		TABLE I
Effect of different inhibitors on the activity of caspase-3-associated kinase activity

The caspase-3-associated kinase was immunoprecipitated with anti-caspase-3 antibody from monocytes and subjected to in vitro kinase assays in the presence of various kinase inhibitors.–indicates no inhibitory effect; + indicates inhibition, and +/–indicates some minor inhibition. PKA, cAMP-dependent protein kinase.

 
PKC{delta} Associates with Caspase-3 in Monocytes—Based on the above-described results and because PKC{delta} is abundantly expressed in monocytes (24), we next investigated whether PKC{delta} is a PKC isoform that associates with caspase-3. Toward this goal, freshly isolated human monocytes extracts were immunoprecipitated with the monoclonal anti-caspase-3, an isotype control, or anti-PKC{delta} antibodies. Western blot analysis of the immunoprecipitates obtained with the anti-PKC{delta} antibody revealed that a fraction of PKC{delta} was associated with caspase-3 in human monocytes (Fig. 2D, lane 4). Immunoprecipitations using isotype controls (Control A, rabbit IgG, and Control B mouse IgG) did not reveal any caspase-3, indicating the specificity of this interaction (Fig. 2D, Control A and Control B). In contrast, when similar immunoprecipitation experiments were carried out with commercial antibodies against PKC{alpha}, no association with caspase-3 was revealed (data not shown). Thus, these results show that PKC{delta} associates with caspase-3 in monocytes.



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  		FIG. 2.
Caspase-3 associates with PKC{delta} in human monocytes. A and B, extracts from monocytes or from THP-1 cells were immunoprecipitated (IP) with anti-caspase-3 antibodies (Casp-3, lanes 2 and 4) or an isotype control (Control, lanes 1 and 3). Immunoprecipitations were subjected to in vitro kinase assays in the presence of H2B and [{gamma}-32P]ATP. Kinase reaction products were resolved by SDS-PAGE and transferred to a membrane. Phospholabeled proteins were visualized by autoradiography. The same membrane was immunoblotted with anti-caspase-3 antibody. C, extracts from fresh primary human monocytes were immunoprecipitated with the anti-caspase-3 antibodies (Casp-3) or an isotype control (Control). Subsequently, immunoprecipitations were subjected to in vitro kinase assays in the presence of H2B, [{gamma}-32P]ATP, and different kinase inhibitors. Kinase products were resolved by SDS-PAGE and transferred to a membrane. The activity of the kinase that immunoprecipitates with caspase-3 was compared in the presence or absence (–) of inhibitors based on the level of H2B phosphorylation as visualized by autoradiography. The same membranes were immunoblotted with anti-caspase-3 antibody (lower panels). D, extracts from freshly isolated monocytes (lane 1) were immunoprecipitated with anti-caspase-3 (Casp-3, lane 4) or with anti-PKC{delta} antibodies (lane 3) or with isotype controls (Control A: polyclonal, Control B: monoclonal, lanes 2 and 5). Immunoprecipitations were resolved by SDS-PAGE and transferred to a membrane and immunoblotted with the anti-PKC{delta} antibodies.

 
PKC{delta} Phosphorylates Caspase-3—Based on our findings that caspase-3 is a phosphoprotein and associates with PKC{delta}, we next evaluated whether caspase-3 is a substrate for PKC{delta}. For this purpose, we performed in vitro kinase assays with rCasp-3 and purified PKC{delta} in the presence of [{gamma}-32P]ATP. After separation of the phosphorylation products on SDS-polyacrylamide gels followed by transferring to the membrane and autoradiography, it was evident that purified PKC{delta} phosphorylates caspase-3 (Fig. 3A). The result also revealed an additional phosphoprotein of a higher molecular weight (Fig. 3A), which Western blot analysis using anti-PKC{delta} antibody identified as PKC{delta} (data not shown). The probing of the same membrane with the anti-caspase-3 antibody showed equal loading of rCasp-3 (Fig. 3A, lower panel).

We next analyzed whether endogenous PKC{delta} from monocytes was able to phosphorylate rCasp-3. For this purpose, PKC{delta} was immunoprecipitated from THP-1 cell extracts using PKC{delta}-specific antibodies and subsequently subjected to an in vitro kinase assay in the presence of [{gamma}-32P]ATP using both H2B and the rCasp-3 as substrates. Each immunoprecipitation was performed with 2 x 106 cells, the amount in which the endogenous caspase-3 is undetectable. Phosphorylation of the rCasp-3 was only observed in the in vitro kinase reaction performed with immunoprecipitates carried out with the PKC{delta}-specific antibodies (Fig. 3B, lane 3). Neither the anti-PKC{alpha} nor the isotype control immunoprecipitates were able to phosphorylate rCasp-3 (Fig. 3B, lanes 1 and 5). Similar negative results were obtained with PKC{beta} (data not shown). However, immunoprecipitates of both PKC{delta} and PKC{alpha} retained similar activities for the phosphorylation of H2B (Fig. 3B, lanes 2–5). The same membrane was re-probed successively with antibodies against caspase-3, PKC{delta}, and PKC{alpha} to ensure equivalent loading of rCasp-3 as well as the specificity of the immunoprecipitations (Fig. 3B). Together, these results demonstrate that caspase-3 is a specific substrate for PKC{delta}.



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  		FIG. 3.
PKC{delta} phosphorylates caspase-3. Human purified recombinant caspase-3 (rCasp-3) was subjected to in vitro kinase assays. A, kinase reaction using rCasp-3 was carried out in the presence (lane 1) or absence (lane 2) of purified human PKC{delta}, and the kinase products were resolved by SDS-PAGE and transferred to a membrane. Phosphorylated rCasp-3 (P-rCasp-3) was visualized by autoradiography. The same membrane was immunoblotted with anti-caspase-3 antibody. B, THP-1 cells extracts were immunoprecipitated (IP) with anti-PKC{delta} (lane 2 and 3), anti-PKC{alpha} (lane 4 and 5) antibodies or an isotype control (Control, lane 1) followed by in vitro kinase assay in the presence of [{gamma}-32P]ATP and both rCasp-3 and H2B as substrates. Phosphorylated kinase products were resolved by SDS-PAGE and transferred to a membrane. Phospholabeled proteins were visualized by autoradiography. The same membrane was immunoblotted with anti-caspase-3, anti-PKC{delta}, and anti-PKC{alpha} antibodies. C, THP-1 lysates (lane 4) were immunoprecipitated with anti-PKC{delta} antibody (lanes 2 and 3) or an isotype control (lane 1). Depleted lysates were analyzed by immunoblotting with anti-PKC{delta} and anti-{beta}-tubulin antibodies. D, immunodepleted lysates obtained in C were immunoprecipitated with anti-caspase-3 antibody (lanes 1–3) or an isotype control (lane 4) and subjected to in vitro kinase assays in the presence of H2B as exogenous substrate and [{gamma}-32P]ATP. Phospholabeled proteins were visualized by autoradiography (upper panel). The same membrane was immunoblotted with anti-caspase-3 antibody (lower panel).

 
Next, we investigated whether PKC{delta} was required for caspase-3 phosphorylation. For this purpose, THP-1 lysates (Fig. 3C, lane 4) were immunoprecipitated using different amounts of anti-PKC{delta} antibody (4 or 8 µl Fig. 3C, lanes 2 and 3, respectively) to deplete endogenous PKC{delta} or an isotype used as mock immunoprecipitation control (Fig. 3C, lane 1). Supernatants from the immunoprecipitations were analyzed by immunoblotting using anti-PKC{delta} antibodies. Lysates from which PKC{delta} was immunoprecipitated showed a reduced level of endogenous PKC{delta} (Fig. 3C, lanes 2 and 3), when compared with the immunoprecipitates using an isotype control (Fig. 3C, lane 1). The same membrane was re-probed with anti-PKC{alpha} and anti-{beta}-tubulin to ensure specificity and equal loading (Fig. 3C, lower panels).By using these lysates, we next investigated the status of caspase-3 phosphorylation. For this purpose, depleted PKC{delta} lysates (shown in Fig. 3C, lanes 2 and 3) were immunoprecipitated with the anti-caspase-3 antibody (Fig. 3D, lanes 2 and 3, respectively) or an isotype control (Fig. 3D, lane 4). Caspase-3 was also immunoprecipitated from mock lysate controls that contained PKC{delta}, as shown in Fig. 3C, lane 1. These immunoprecipitates were subjected to in vitro kinase assays in the presence of H2B. Lysates lacking PKC{delta} had a reduced ability to phosphorylate caspase-3 (Fig. 3D, lanes 2 and 3) than mock depletion lysates containing PKC{delta} (Fig. 3D, lane 1, lysates obtained as in Fig. 3C, lane 1). Similarly, phosphorylation of H2B was also reduced in lysates depleted of PKC{delta}. Caspase-3 was absent in the isotype control immunoprecipitates (Fig. 3D, lane 4, upper and lower panel). The same membrane was re-probed with antibodies against caspase-3 to ensure the specificity of the immunoprecipitations (Fig. 3D, lower panel). These results taken together show that PKC{delta} is required for caspase-3 phosphorylation.

PKC{delta} Increases Caspase-3 Activation—According to the current model, the proteolytic activation of caspase-3 involves two steps (9). The first step is the cleavage between the p17 and the p12 domains, mediated by initiator caspases such as caspase-9. The second step involves the autocatalytic cleavage between the prodomain and the p17 domain (12, 37). By having established that caspase-3 is phosphorylated by PKC{delta}, we next examined the effect of phosphorylation on the activity of caspase-3. For this purpose, full-length rCasp-3 was first phosphorylated in vitro by PKC{delta}. In the experimental conditions used, we routinely obtain 1 molar eq of phosphate incorporated per 4 molar eq of rCasp-3 (see "Materials and Methods"). The control constituting nonphosphorylated caspase-3 was composed by the same reaction mixture except that PKC{delta} was boiled prior to the addition to the mix (see "Materials and Methods").

After completion of the kinase reaction for 1 h at 37 °C, rCasp-9 was added to the reaction. After 0, 2, or 4 h of incubation with rCasp-9, caspase-3 activity was measured using the DEVD-AFC substrate (see "Materials and Methods"). Based on the amount of fluorescent product formed (Fig. 4A), the enzymatic activity of caspase-3 was compared in the phosphorylated and nonphosphorylated samples. The results show a statistically significant increase of rCasp-3 activity after phosphorylation (Fig. 4B). For example, after 2 h there is 50% more caspase-3 activity in phosphorylated caspase-3 than in the nonphosphorylated caspase-3 reaction. At 4 h, ~30% more caspase activity was found in the phosphorylated caspase-3, compared with the nonphosphorylated counterpart. Unlike caspase-3, no activation of caspase-9 was observed when it was added to the kinase reaction mixture containing PKC{delta}, as determined using LEHD-AFC, a substrate specific for caspase-9 (data not shown). Taken together, these results indicate that caspase-3 can be specifically activated by PKC{delta}-mediated phosphorylation and suggest that PKC{delta} may function as a pro-apoptotic regulator by directly modulating the activity of caspase-3 in the cell.



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  		FIG. 4.
Phosphorylation increases the activity of caspase-3. Human full-length recombinant caspase-3 (rCasp-3) was subjected to in vitro kinase assays in the presence of [{gamma}-32P]ATP and PKC{delta} or boiled inactivated-PKC{delta}. Products of the kinase reaction were mixed with active rCasp-9. After incubating the reaction for 2 and 4 h, the reaction samples were assayed for caspase-3 activity by the DEVD-AFC cleavage assay. A, cleavage of DEVD-AFC was monitored as released fluorescent AFC (fluorescence units) over time for phosphorylated (black symbols) and unphosphorylated (white symbols) rCasp-3 incubated for 2 (triangles) or 4 h (circles) with rCasp-9. Phosphorylated rCasp-3 samples not subjected to rCasp-9 activation (white square) were included as a control. A typical example of several similar results is shown. B, the slopes from A were used to calculate and compare the caspase-3 activity of phosphorylated and nonphosphorylated samples. Values are means ± S.E. of phosphorylated samples (black bars) and unphosphorylated control (gray bars) at 2-h (n = 3; *, p < 0.01) and 4-h time points (n = 3, **, p < 0.05, Student's t test).

 
PKC{delta} Activation during Spontaneous Apoptosis of Primary Human Monocytes—To assess the function of PKC{delta} in monocytic cells, we next examined the activity of PKC{delta} during monocyte apoptosis. For this purpose, first we used human monocytes undergoing spontaneous apoptosis by culturing them for different lengths of time, as described previously (2). To determine the activity of PKC{delta}, monocytes lysates were immunoprecipitated with the anti-PKC{delta} antibody and subjected to in vitro kinase assay using H2B as a substrate. The activity of PKC{delta} rapidly increased at least four times during the 1st h of incubation and was followed by a second increase around 4 h (Fig. 5A). Caspase-3 activation was apparent 2 h after the start of monocyte incubation (Fig. 5B). These results show that there is detectable PKC{delta} activity that precedes caspase-3 activation, which is followed by a second increase of PKC{delta} activity occurring after the activation of caspase-3.

We next examined the effect of the PKC inhibitor staurosporine on human spontaneous monocytes apoptosis. For this purpose, freshly isolated human monocytes were cultured for 16 h in the presence of staurosporine or were left untreated (apoptotic). We found that caspase-3 activity was inhibited by the treatment with 10 nM staurosporine (Fig. 5C), whereas the ERK inhibitor, PD098059, failed to reduce the activation of caspase-3 (Fig. 5C). Consistent with this result, we found that the number of apoptotic monocytes was reduced in the presence of 10 nM staurosporine when compared with the monocytes left untreated, as determined by the number of cell stained with annexin V (Fig. 5D). Taken together, these results demonstrate that PKC{delta} was acting as an activator of cell death in monocytic cells supporting its role in the activation of caspase-3.

PKC{delta} Acts as a Pro-apoptotic Kinase in Monocyte Apoptosis—To investigate further the role of PKC{delta} on apoptosis, we determined the activity of PKC{delta} in cells induced to undergo apoptosis. For this purpose, lysates from THP-1 cells treated with 1 µM etoposide for different lengths of time to induce apoptosis were used to determine the activity of PKC{delta}. THP-1 lysates were immunoprecipitated with the anti-PKC{delta} antibody and subjected to in vitro kinase assay using H2B as a substrate. The activity of PKC{delta} rapidly increased during the 1st h of incubation with etoposide (Fig. 6A). The caspase-3 activity in the same lysates was determined, showing that the activation of caspase-3 occurred approximately after 3 h of etoposide treatment (Fig. 6B). The increase of PKC{delta} activity occurred prior to the activation of caspase-3 in etoposide-induced apoptosis. Similar results were obtained when taxol was used to induce apoptosis (data not shown). Thus, these results show that PKC{delta} activation occurs prior caspase-3 activation in etoposide-induced apoptosis.

To establish further the participation of PKC{delta} in monocyte apoptosis, cells were transfected with a full-length PKC{delta}-wt-HA construct driven by the cytomegalovirus promoter, a dominant negative mutant (PKC{delta}-DN-HA), or by the empty vector control (see "Materials and Methods"). Twenty four hours after transfection, cells were induced to undergo apoptosis with 1 µM etoposide for 8 h. Cells overexpressing PKC{delta} and treated with etoposide showed an increased number of cells undergoing apoptosis, as determined by the number of cells with DNA fragmentation compared with cells transfected with vector alone or with the dominant negative mutant (Fig. 6C). Similarly, PKC{delta}-overexpressing cells showed an increase of apoptotic cells when treated with taxol (data not shown). These experiments indicate that overexpression of PKC{delta} increases DNA fragmentation, an apoptotic change that requires caspase-3 activity.

We next examined the effect of down-regulation of PKC{delta} expression on apoptosis. For this purpose, we determine first the ability of siRNA duplexes that target PKC{delta} (siRNA-PKC{delta} (33)) to silence PKC{delta} expression in highly transfectable HeLa cells. Cells were transfected with siRNA-PKC{delta}, with the same duplexes labeled with rhodamine (siRNA-PKC{delta}-R), and with a rhodamine-labeled random duplex control (siRNA-Control-R and see "Materials and Methods"). Immunoblotting analysis with anti-PKC{delta} and PKC{alpha} antibodies showed the specific down-regulation of PKC{delta} expression in cells transfected with the siRNA-PKC{delta} or the rhodamine-labeled duplex counterpart (Fig. 7A, lanes 3 and 4, respectively). Lysates from cells transfected with the control showed levels of PKC{delta} similar to nontransfected cells (Fig. 7A, lanes 2 and 1, respectively). The same membrane was re-probed with anti-PKC{alpha} antibodies to ensure the specificity of the silencing and with {beta}-tubulin to ensure equivalent loading (Fig. 7A). Next, we investigated the effect of silencing PKC{delta} on DNA fragmentation, a process that requires caspase-3 activity and is considered a hallmark of apoptosis (38, 39). For this purpose, THP-1 cells were transfected with siRNA-PKC{delta}-R or siRNA-Control-R (Fig. 7B, see red cells). Forty hours after transfection, cells were left untreated or induced to undergo apoptosis for 8 h with 1 µM etoposide. After this period, nuclei were stained with DAPI, and the number of apoptotic cells was determined as the percentage of cells with fragmented DNA (only nuclei from red transfected cells were considered). We found that silencing PKC{delta} reduced the number of apoptotic cells as determined by the reduction on the number of cells with DNA fragmentation (Fig. 7B, g and h). Cells transfected with the siRNA-Control underwent DNA fragmentation at higher percentage (Fig. 7B, e and f, see arrows). On the contrary, in cells transfected with siRNA-PKC{delta}-R (Fig. 7B, g and h, see arrows), shown in red, DNA fragmentation is absent, whereas cells in the same panels that were not transfected (cells are not red) still showed DNA fragmentation. These results taken together suggest that PKC{delta} induces DNA fragmentation, an apoptotic change dependent on caspase-3 activity.



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  		FIG. 5.
PKC{delta} activity precedes caspase-3 activity in monocyte apoptosis. A, extracts from human monocytes freshly isolated or cultured for various lengths of time were immunoprecipitated (IP) with anti-PKC{delta} antibodies and subjected to in vitro kinase assay using H2B as substrate in the presence of [{gamma}-32P]ATP. The kinase reaction products were resolved by SDS-PAGE and transferred to a membrane, and phosphorylated H2B was visualized by autoradiography. The kinase activity shown at the top was measured by PhosphorImager, and absolute quantitation is shown at the bottom. The same membrane was immunoblotted with anti-PKC{delta} antibody to ensure equal loading of the samples (lower panel). B, caspase-3 activity from monocytes extracts cultured as in A was measured by DEVD-AFC assay. C, freshly isolated human monocytes were cultured for 16 h in the presence of varying concentrations of staurosporine or 10 µM PD098059, and caspase-3 activity in the extracts was measured by the DEVD-AFC assay. Values are means ± S.E. (n = 3, p < 0.001, analysis of variance). D, the same monocyte cells used in C were labeled with annexin V-FITC and propidium iodide-phycoerythrin (PE) to determine the number of apoptotic cells and analyzed by flow cytometry.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of caspase-3 is a key process in the execution of monocyte apoptosis (2). In the present study we describe a novel mechanism involved in the regulation of caspase-3. The results from in vivo labeling experiments of monocytic cells suggest that caspase-3 is a phosphoprotein in vivo (Fig. 1). Consistent with these findings, we established that endogenous caspase-3 from human monocytes or THP-1 cells associates with a kinase activity capable of phosphorylating caspase-3 (Fig. 2, A and B). By using a number of different kinase inhibitors, our results indicated a participation of PKC in the phosphorylation of caspase-3 (Table I and Fig. 2C). Because PKC{delta} is abundantly expressed in monocytes (24) and is a substrate of caspase-3 (25, 40, 41), we examined whether PKC{delta} might associate with caspase-3. We found that PKC{delta} associates with caspase-3 in monocytes (Fig. 2D). We also demonstrated that purified PKC{delta} phosphorylates caspase-3 and that the immunoprecipitated endogenous PKC{delta} from THP-1 cells phosphorylates caspase-3 (Fig. 3, A and B). We cannot rule out at this point the possibility that caspase-3 is a substrate for other kinases in addition to PKC{delta}; however, experiments performed under similar conditions failed to detect an association with PKC{alpha} or PKC{beta}, both isoforms also abundantly expressed in monocytes (24). Moreover, PKC{alpha} immunoprecipitates failed to phosphorylate caspase-3 (Fig. 3B). Hence, these results strongly suggest that caspase-3 is associated and phosphorylated by PKC{delta}.

The role played by PKCs as either pro- or anti-apoptotic regulators appear to depend on the specific isoform, the cell type in study, and the particular apoptotic stimulus (29). Recent studies revealed that mice lacking PKC{delta} have increased proliferation of B cells and develop autoimmune diseases (42, 43). Consistent with these findings, our studies show an increase of 4-fold in PKC{delta} activity during the 1st h of spontaneous monocyte apoptosis, prior to caspase-3 activation (Fig. 5A). Similarly, PKC{delta} activity preceded caspase-3 activation when apoptosis was induced with etoposide or taxol (Fig. 6 and data not shown). A second increase in PKC{delta} activity was observed after the activation of caspase-3 (Fig. 5A), reflecting the reported activation of PKC{delta} by caspase-3 (44). An increase of PKC{delta} activity prior to caspase-3 activation has also been reported on tetrandrine-induced apoptosis of U937 monocytic cells (30). In addition, we found that PKC{delta} overexpression increased apoptosis in monocytic cells treated with etoposide or taxol (Fig. 6, and data not shown). In support of a pro-apoptotic role, treatment with a PKC inhibitor prevented spontaneous monocyte apoptosis and inhibited caspase-3 activity (Fig. 5). Consistent with these findings, attenuation of PKC{delta} decreased caspase-3 phosphorylation (Fig. 3D), and silencing of PKC{delta} expression inhibited etoposide-induced-DNA fragmentation (Fig. 7B), a process in which caspase-3 activity is essential (38, 39). Hence, one possible mechanism for the pro-apoptotic function of PKC{delta} in the cell might be achieved through the phosphorylation and induction of the activity of caspase-3. In this context, we found a 30% increase in caspase-3 activity when caspase-3 is phosphorylated by PKC{delta} (Fig. 4B). Although our results showed that phosphorylation of caspase-3 increases its overall activity, future experiments will be required to elucidate whether the phosphorylation affects the activity per se or acts by increasing the activation of caspase-3.



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  		FIG. 6.
PKC{delta} acts as a pro-apoptotic kinase in monocyte apoptosis. A, extracts from THP-1 cells cultured for various lengths of time with 1 µM etoposide to induce apoptosis were immunoprecipitated (IP) with anti-PKC{delta} antibodies and subjected to in vitro kinase assay using H2B as substrate in the presence of [{gamma}-32P]ATP. The kinase reaction products were resolved by SDS-PAGE and transferred to a membrane, and phosphorylated H2B was visualized by autoradiography (upper panel). The same membrane was immunoblotted with anti-PKC{delta} antibody to ensure equal loading of the samples (lower panel). The kinase activity shown at the top was measured by PhosphorImager and normalized by PKC{delta} density shown at the bottom. B, caspase-3 activity from the same lysates used in A was measured by DEVD-AFC assay. C, THP-1 cells transfected with plasmid DNA carrying PKC{delta}-wt-HA full-length, a dominant negative mutant PKC{delta}-DN-HA, or a vector-HA control were left untreated (black bars) or induced to undergo apoptosis for 8 h with 1 µM etoposide (white bars). Percentage of apoptotic cells was determined by DNA fragmentation and visualized by DAPI staining. Values are means ± S.E. (n = 3; *, p < 0.01, Student's t test, after etoposide treatment and represents the measurements of PKC{delta}-wt-HA compared with vector control; #, p < 0.05, PKC{delta}-DN-HA to vector control).

 



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  		FIG. 7.
Silencing of PKC{delta} inhibits monocyte apoptosis. A, immunoblots using anti-PKC{delta}, anti-PKC{alpha}, or anti-{beta}-tubulin antibodies from lysates of HeLa cells (mock, lane 1) or cells transfected with siRNA-Control-R (lane 2), siRNA-PKC{delta} (lane 3), and siRNA-PKC{delta}-R (lane 4). B, THP-1 cells transfected (red) with siRNA-Control-R (a, b, e, and f) or siRNA-PKC{delta}-R (c, d, g, and h) were left untreated (a, b, e, and f) or induced to undergo apoptosis for 8 h with 1 µM etoposide (c, d, g, and h) were stained with DAPI. Values represent means ± S.E. of DAPI-stained cells that have DNA fragmentation over total cells (n = 3; *, p < 0.01). DNA morphology of only red cells was taken into consideration.

 
So far, only caspase-9, another member of the caspase family, was showed to be phosphorylated by Akt or ERK (18, 19). Despite these previous findings, we observed no effect of the phosphatidylinositol 3-kinase inhibitor LY294002 nor the PD098059 and UO126 (mitogen-activated protein kinase inhibitors) on the caspase-3-associated kinase activity (Table I). In agreement with previous reports, we failed to find an association of caspase-3 with ERK or Akt (18, 19) in monocytic cells. Moreover, PKC{delta} was not able to stimulate caspase-9 activity (data not shown). Our findings that PKC{delta} associates and phosphorylates caspase-3 support a pro-apoptotic role in which phosphorylation of caspase-3 promotes its activity during monocyte apoptosis. Taken together these observations suggest an emerging model in which a network of different kinases regulates particular caspases, with the ultimate results of determining cell fate.


   FOOTNOTES
 
* This work was supported by American Cancer Society Grant ACS/IRG98-278-01 (to A. I. D.) and by American Lung Association Grant RG-044-N (to A. I. D.). 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. Back

{ddagger} Both authors contributed equally to this work.1 Back

** To whom correspondence should be addressed: 201 Heart and Lung Research Institute, the Ohio State University, 473 West 12th Ave., Columbus, OH 43210. Tel.: 614-292-9507; Fax: 614-292-7778; E-mail: doseff-1{at}medctr.osu.edu.

1 The abbreviations used are: ERK, extracellular signal-regulated kinase; PKC, protein kinase C; DAPI, 4,6-diamidino-2-phenylindole; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; FITC, fluorescein isothiocyanate; AFC, 7-amino-4-trifluoromethyl coumarin assay; siRNA, small interfering RNA; MES, 4-morpholineethanesulfonic acid; rCasp-3, recombinant caspase-3; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Back


   ACKNOWLEDGMENTS
 
We thank Dr. Grotewold for suggestions and critical reading of the manuscript. We thank Dr. Kuret for the gift of the CKI inhibitors and Martha Monick for the gift of PKC antibodies.



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