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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

IkappaB proteins function as direct regulators of Rel/NF-kappaB transcription complexes. We show that the cell-death protease CPP32 (caspase-3) in vitro specifically cleaved chicken and human IkappaB-alpha at a conserved Asp-Ser sequence. This cleavage site appears to be identical to the site at which chicken IkappaB-alpha is cleaved in vivo in temperature-sensitive v-Rel-transformed chicken spleen cells undergoing apoptosis. Other caspases, namely interleukin-1beta-converting enzyme (caspase-1) and Ich-1 (caspase-2), did not cleave IkappaB-alpha. CPP32 also cleaved mammalian IkappaB-beta in vitro at the analogous Asp-Ser sequence. Cleavage of IkappaB-alpha by CPP32 was blocked by serine phosphorylation of IkappaB-alpha. Cleavage of IkappaB-alpha by a CPP32- like protease could generate a constitutive inhibitor of Rel transcription complexes. This report provides evidence for a direct biochemical interaction between the NF-kappaB signaling pathway and a cell-death protease signaling pathway.
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PMID:Phosphorylation of IkappaB-alpha inhibits its cleavage by caspase CPP32 in vitro. 936 96

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.
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PMID:Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy. 936 3

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.
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PMID:Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. 937 39

PC12 cells are a useful model system for studying neuronal apoptosis. Like neurons, they undergo apoptosis when deprived of trophic support. Involvement of caspases [interleukin 1beta-converting enzyme (ICE)-related proteases] has been implicated in apoptosis induced by various stimuli in many cell types, including neurons. In the present study we investigated the need for caspases participation in apoptosis induced by growth factor deprivation in naive and neuronal PC12 cells. For this purpose we generated PC12 cell lines that consistently express the viral caspases inhibitor genes p35 or crmA, and analyzed their susceptibility to trophic factor deprivation. We also examined the effects of cell-permeable peptide inhibitors of caspases. Our results showed that broad-spectrum inhibitors of the caspases, namely the baculovirus p35 gene and the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, effectively inhibit the death of both naive and neuronal PC12 cells. However, caspase-1 (ICE)-specific inhibitors, namely the peptides Ac-Try-Val-Ala-Asp-chloromethylketone and Ac-Try-Val-Ala-Asp-aldehyde, as well as crmA, were much less effective. These findings demonstrate that caspases, but not caspase-1, are needed for apoptosis induced by trophic factor deprivation in both naive and neuronal PC12 cells. Northern and Western blot analyses showed that PC12 cells express caspase-3. We therefore examined the involvement of caspase-3 in the death process of trophic factor-deprived PC12 cells. Our results showed that the pro-caspase-3 and its substrate poly-(ADP-ribose) polymerase are cleaved at similar rates in serum-deprived PC12 cells. Moreover, cell lysates prepared from these cells possess caspase-3-like activity, as determined by their ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. These findings strongly suggest that caspase-3 or caspase-3-like proteases are activated in trophic factor-deprived PC12 cells.
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PMID:Need for caspases in apoptosis of trophic factor-deprived PC12 cells. 937 95

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis, and here we report that NO prevents hepatocyte apoptosis initiated by the removal of growth factors or exposure to TNFalpha or anti-Fas antibody. We postulated that the mechanism of the inhibition of apoptosis by NO would include an effect on caspase-3-like protease activity. Caspase-3-like activity increased coincident with apoptosis due to all three stimuli, and treatment with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited both proteolytic activity and apoptosis. Endogenous or exogenous sources of NO prevented the increase in caspase-3-like activity in hepatocytes. Exposure of purified recombinant caspase-3 to an NO or NO+ donor inhibited proteolytic activity. Dithiothreitol (DTT), but not glutathione, reversed the inhibition of recombinant caspase-3 by NO. When lysates from cells stimulated to express inducible NO synthase or cells exposed to NO donors were incubated in DTT, caspase-3-like activity increased to about 55% of cells not exposed to a source of NO. Similarly, administration of an NO donor to rats treated with TNFalpha and D-galactosamine also prevented the increase in caspase-3-like activity as measured in liver homogenates. The effect of the NO donor was reversed by about 50% if the homogenate was incubated with DTT. TNFalpha-induced apoptosis and caspase-3-like activity were also reduced in cultured hepatocytes exposed to 8-bromo-cGMP, and both effects were inhibited by the cGMP-dependent kinase inhibitor KT5823. The suppression in caspase-3-like activity in hepatocytes exposed to an NO donor was partially blocked by an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one, (ODQ), while the incubation of these lysates in DTT almost completely restored caspase-3-like activity to the level of TNFalpha-treated controls. These data indicate that NO prevents apoptosis in hepatocytes by either directly or indirectly inhibiting caspase-3-like activation via a cGMP-dependent mechanism and by direct inhibition of caspase-3-like activity through protein S-nitrosylation.
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PMID:Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. 938 67

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.
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PMID:p53-induced apoptosis in the human T-ALL cell line CCRF-CEM. 939 39

Type I and II keratins help maintain the structural integrity of epithelial cells. Since apoptosis involves progressive cell breakdown, we examined its effect on human keratin polypeptides 8, 18, and 19 (K8, K18, K19) that are expressed in simple-type epithelia as noncovalent type I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapid hyperphosphorylation of most known K8/18 phosphorylation sites and delayed formation of K18 and K19 stable fragments. In contrast, K8 is resistant to proteolysis and remains associated with the K18 fragments. Transfection of phosphorylation/glycosylation-mutant K8 and K18 does not alter fragment formation. The protein domains of the keratin fragments were determined using epitope-defined antibodies, and microsequencing indicated that K18 cleavage occurs at a conserved caspase-specific aspartic acid. The fragments are found preferentially within the detergent-insoluble pool and can be generated, in a phosphorylation-independent manner, by incubating keratins with caspase-3 or with detergent lysates of apoptotic cells but not with lysates of nonapoptotic cells. Our results indicate that type I keratins are targets of apoptosis-activated caspases, which is likely a general feature of keratins in most if not all epithelial cells undergoing apoptosis. Keratin hyperphosphorylation occurs early but does not render the keratins better substrates of the downstream caspases.
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PMID:Apoptosis generates stable fragments of human type I keratins. 940 8

Interleukin-16, a proinflammatory cytokine produced in CD8(+) lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a approximately 50-kDa form of pro-IL-16. Transfected COS cells released a approximately 20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal approximately 20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8(+) lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1beta-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8(+) lymphocytes and by inhibition of CD8(+) lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.
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PMID:Processing and activation of pro-interleukin-16 by caspase-3. 942 80

Upon treatment with NO-releasing compounds such as S-nitrosoglutathione or spermine NO, human myeloid leukemia U937 cells undergo apoptosis. Early NO-mediated signals comprise activation of a Z-A-DCB (benzoyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene)-sensit ive, caspase-3 like cysteine protease that cleaved poly (ADP-ribose) polymerase (PARP), U1 small nuclear ribonucleoprotein (U1 snRNP), and the fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin. In association with these early apoptotic alterations p21 (WAF1/Cip1) is upregulated, but NO affected cell proliferation and apoptosis at a similar dose. At later time points the classical antiapoptotic protein Bcl-2 is downregulated, indicating that decreased Bcl-2 expression is secondary and not a prerequisite for initiation of apoptosis. N-Acetylcysteine (1 mM) interfered with NO-mediated apoptotic signaling, blocking DNA fragmentation as well as PARP and U1 snRNP cleavage. In contrast Z-A-DCB suppressed DNA fragmentation and U1 snRNP cleavage, while PARP breakdown proceeded unaltered. Observing proteolytic PARP digestion without apoptotic alterations questions PARP cleavage as an apoptotic parameter. These results suggest that a Z-A-DCB-sensitive caspase that is distinct from the PARP-cleaving enzyme is activated during NO exposure. NO-mediated apoptotic signaling in U937 cells activates caspases, some of which are dispensable for propagating the death signal.
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PMID:U937 apoptotic cell death by nitric oxide: Bcl-2 downregulation and caspase activation. 945 54

Isothiocyanates exert strong anticarcinogenic effects in a number of animal models of cancer, presumably by modulation of xenobiotic-metabolizing enzymes, such as by inhibition of cytochrome P-450 and/or by induction of phase II detoxifying enzymes. Here, we report that phenethyl isothiocyanate and other structurally related isothiocyanates, phenylmethyl isothiocyanate, phenylbutyl isothiocyanate, and phenylhexyl isothiocyanate, but not phenyl isothiocyanate induced apoptosis in HeLa cells in a time- and dose-dependent manner. Treatment with apoptosis-inducing concentrations of isothiocyanates also caused rapid and transient induction of caspase-3/CPP32-like activity. Furthermore, these isothiocyanates, except phenyl isothiocyanate, stimulated proteolytic cleavage of poly(ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with a potent caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde inhibited isothiocyanate-induced caspase-3-like activity and apoptosis. These results suggest that isothiocyanates may induce apoptosis through a caspase-3-dependent mechanism. The induction of apoptosis by isothiocyanates may provide a distinct mechanism for their chemopreventive functions.
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PMID:Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity. 945 80


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