Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3

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 Cdelta (PKCdelta) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCdelta-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCdelta is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCdelta precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCdelta resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCdelta-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.     

Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase-mediated apoptosis of HeLa cells

We show that caspase-3 cleaves Cdc6 at D(290)/S and D(442)/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-alpha-induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-alpha-induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-alpha- or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53-Bax-mediated apoptosis.     

Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis

The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).     

The protein kinase C-dependent phosphorylation of serine 166 is controlled by the phospholipid species bound to the phosphatidylinositol transfer protein alpha

The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.     

Regulation of transferrin receptor cycling by protein kinase C is independent of receptor phosphorylation at serine 24 in Swiss 3T3 fibroblasts

Treatment of Swiss 3T3 fibroblasts with tumor-promoting phorbol diester or with platelet-derived growth factor caused the phosphorylation of the transferrin receptor by protein kinase C (Ca2+/phospholipid-dependent enzyme) at serine 24 and increased the cell surface expression of the transferrin receptor. The hypothesis that the regulation of transferrin receptor cycling by protein kinase C is causally related to the phosphorylation of the receptor at serine 24 was critically tested. Site-directed mutagenesis of the human transferrin receptor cDNA was used to substitute serine 24 with threonine or alanine residues in order to create phosphorylation defective receptors. Wild-type and mutated transferrin receptors were expressed in Swiss 3T3 fibroblasts using the retrovirus vector pZipNeoSV (X). These receptors were functionally active and caused the receptor-mediated endocytosis of diferric transferrin. Incubation of the fibroblasts with phorbol diester caused the phosphorylation of the wild-type (Ser-24) human transferrin receptor, but this treatment did not result in the phosphorylation of the mutated (Ala-24 and Thr-24) receptors. The cycling of the phosphorylation defective receptors was regulated by phorbol diester and platelet-derived growth factor in a manner similar to that observed for the wild-type receptor. We conclude that the regulation of transferrin receptor cycling by protein kinase C is independent of receptor phosphorylation at serine 24 in Swiss 3T3 fibroblasts.     

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3–activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.     

The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.     

Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.     

Endothelin-1 induces serine phosphorylation of the adaptor protein p66Shc and its association with 14-3-3 protein in glomerular mesangial cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC). In the current study, it is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66(Shc)). ET-1-induced serine phosphorylation of p66(Shc) requires activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling module and is efficiently inhibited by both a MAPK/ERK kinase (MEK)-selective inhibitor and adenovirus-mediated transfer of a dominant interfering MEK1 mutant. Furthermore, adenovirus-mediated transfer of a constitutively active MEK1 mutant was found to markedly increase p66(Shc) serine phosphorylation. Adenoviruses encoding constitutively active mutants of MAPK kinases 3 and 6 (upstream kinases of p38(MAPK)) and 7 (upstream kinase of c-Jun NH(2)-terminal kinase) failed to induce serine phosphorylation of this adaptor protein. Serine phosphorylation of p66(Shc) resulted in its association with the serine binding motif-containing protein 14-3-3. ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66(Shc) was confirmed in experiments employing anti-phospho-14-3-3 binding motif antibodies. These studies are the first to demonstrate that G protein-coupled receptors stimulate serine phosphorylation of p66(Shc) and the first to report the formation of a signaling complex between p66(Shc) and 14-3-3.     

Histone H2B deacetylation at lysine 11 is required for yeast apoptosis induced by phosphorylation of H2B at serine 10

Chromatin alterations, induced by covalent histone modifications, mediate a wide range of DNA-templated processes, including apoptosis. Apoptotic chromatin condensation has been causally linked to the phosphorylation of histone H2B (serine 14 in human; serine 10 in yeast, H2BS10ph) in human and yeast cells. Here, we extend these studies by demonstrating a unidirectional, crosstalk pathway between H2BS10 phosphorylation and lysine 11 acetylation (H2BK11ac) in yeast. We demonstrate that the H2BK11 acetyl mark, which exists in growing yeast, is removed upon H(2)O(2) treatment but before H2BS10ph occurs, in a unidirectional fashion. H2B K11Q mutants are resistant to cell death elicited by H(2)O(2), while H2B K11R mutants that mimic deacetylation promote cell death. Our results suggest that Hos3 HDAC deacetylates H2BK11ac, which in turn mediates H2BS10ph by Ste20 kinase. Together, these studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death.     

Cleavage of the Bloom's syndrome gene product during apoptosis by caspase-3 results in an impaired interaction with topoisomerase IIIalpha

In higher eukaryotes, the integration of signals triggered in response to certain types of stress can result in programmed cell death. Central to these events is the sequential activation of a cascade of proteinases known as caspases. The final activated effector caspases of this cascade digest a number of cellular proteins, in some cases increasing their enzymatic activity, in others destroying their function. Of the proteins shown to be targets for caspase-mediated proteolysis, a surprisingly large proportion are proteins involved in the signalling or repair of DNA damage. Here we investigate whether BLM, the product of the gene mutated in Bloom’s syndrome, a human autosomal disease characterised by cancer predisposition and sunlight sensitivity, is cleaved during apoptosis. BLM interacts with topoisomerase IIIα and has been proposed to play an important role in maintaining genomic integrity through its roles in DNA repair and replication. We show that BLM is cleaved during apoptosis by caspase-3 and reveal that the main cleavage site is located at the junction between the N-terminal and central helicase domains of BLM. Proteolytic cleavage by caspase-3 produces a 120 kDa fragment, which contains the intact helicase domain and three smaller fragments, the relative amounts of which depend on time of incubation with caspase-3. The 120 kDa fragment retains the helicase activity of the intact BLM protein. However, its interaction with topoisomerase IIIα is severely impaired. Since the BLM–topoisomerase interaction is believed to be necessary for many of the replication and recombination functions of BLM, we suggest that caspase-3 cleavage of BLM could alter the localisation and/or function of BLM and that these changes may be important in the process of apoptosis.     

The small heat shock protein alpha B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation

Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.     

Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.     

Different effects on activity caused by phosphorylation of tyrosine hydroxylase at serine 40 by three multifunctional protein kinases.

Tyrosine hydroxylase was maximally phosphorylated by protein kinase C, with a stoichiometry of 0.43 mol of phosphate/mol of tyrosine hydroxylase subunit at Ser40, and by calmodulin-dependent protein kinase II, with stoichiometries of 0.43 mol/mol at Ser40 and 0.76 mol/mol at Ser19, respectively, without undergoing any significant direct activation. In contrast, the enzyme was maximally phosphorylated with a stoichiometry of 0.78 mol of phosphate/mol of subunit at Ser40 by cAMP-dependent protein kinase, which resulted in a large activation of the enzyme (about 3-fold activation under the assay conditions). Incubation of the enzyme, which had previously been maximally phosphorylated by calmodulin-dependent protein kinase II, with protein kinase C under phosphorylating conditions resulted in no additional incorporation of phosphate into the enzyme, suggesting that both protein kinases phosphorylated Ser40 of the same subunits of the enzyme. Since tyrosine hydroxylase is thought to be composed of four identical subunits, the results may indicate that calmodulin-dependent protein kinase II or protein kinase C phosphorylates only two of the four subunits of the enzyme at Ser40 without affecting the enzyme activity and that cAMP-dependent protein kinase phosphorylates Ser40 of all four subunits of the enzyme molecule, causing a marked activation. Based on a linear relationship between phosphorylation and the resulting activation of the enzyme by cAMP-dependent protein kinase, possible mechanisms for the activation of the enzyme by the protein kinase are discussed.     

Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis

Protein kinase C (PKC) family members play pivotal roles in cellular signal transduction and nPKCdelta and theta are known to be subjected to restrictive proteolysis during apoptosis. Here we show that nPKCepsilon was specifically cleaved and generates 43-kDa and 36-kDa C-terminal fragments during chemotherapeutic drug-induced apoptosis. The proteolytic cleavage of nPKCdelta and epsilon was completely inhibited by pretreatment with Ac-DEVD-cho, a specific inhibitor of caspase-3 family enzymes. Furthermore, nPKCepsilon in non-treated U937 cell lysates was cleaved by purified recombinant caspase-3 to generate the 43-kDa fragment, identical in size to the fragment observed in vivo. This cleavage was prevented by the addition of Ac-DEVD-cho. These results suggest that caspase-3 specifically cleaves nPKCepsilon. These findings suggest the possibility that nPKC subfamily members are generally involved in the execution of apoptosis but they are regulated diversely depending on the different apoptotic stimuli.