Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Sphingosine, sphinganine, and phytosphingosine are structural analogs of sphingolipids and are classified as long-chain sphingoid bases. Sphingosine and sphinganine are known to play important roles in apoptosis. In the present study, we examined the phytosphingosine-induced apoptosis mechanism, focusing on mitochondria in human T-cell lymphoma Jurkat cells. Phytosphingosine significantly induced chromatin DNA fragmentation, which is a hallmark of apoptosis. Enzymatic activity measurements of caspases revealed that caspase-3 and caspase-9 are activated in phytosphingosine-induced apoptosis, but there is little activation of caspase-8 suggesting that phytosphingosine influences mitochondrial functions. In agreement with this hypothesis, a decrease in DeltaPsi(m) and the release of cytochrome c to the cytosol were observed upon phytosphingosine treatment. Furthermore, overexpression of mitochondria-localized anti-apoptotic protein Bcl-2 prevented phytosphingosine apoptotic stimuli. Western blot assays revealed that phytosphingosine decreases phosphorylated Akt and p70S6k. Dephosphorylation of Akt was partially inhibited by protein phosphatase inhibitor OA and OA attenuated phytosphingosine-induced apoptosis. Moreover, using a cell-free system, phytosphingosine directly reduced DeltaPsi(m). These results indicate that phytosphingosine perturbs mitochondria both directly and indirectly to induce apoptosis.
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PMID:Phytosphingosine induced mitochondria-involved apoptosis. 1572 52

Recent evidence has implicated the protein phosphatase PP5 in a variety of signaling pathways. Whereas several proteins have been identified that interact with PP5 and regulate its activity, a possibility of its regulation by second messengers remains speculative. Activation of PP5 in vitro by polyunsaturated fatty acids (e.g. arachidonic acid) and fatty acyl-CoA esters (e.g. arachidonoyl-CoA) has been reported. We report here that PP5 is strongly inhibited by micromolar concentrations of a natural polyamine spermine. This inhibition was observed both in assays with a low molecular weight substrate p-nitrophenyl phosphate as well as phosphocasein and apoptosis signal-regulating kinase 1 (ASK1), thought to be a physiological substrate of PP5. Furthermore, a decrease in polyamine levels in COS-7 cells induced by alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, led to accelerated dephosphorylation of oxidative stress-activated ASK1. This effect was suppressed by okadaic acid and by siRNA-mediated PP5 depletion, indicating that the effect of polyamine levels on ASK1 dephosphorylation was mediated by PP5. In line with the decreased ASK1 activation, polyamine depletion in COS-7 cells abrogated oxidative stress-induced activation of caspase-3, which executes ASK1-induced apoptosis, as well as caspase-3 activation induced by ASK1 overexpression, but had no effect on basal caspase-3 activity. These results implicate polyamines, emerging intracellular signaling molecules, as potential physiological regulators of PP5. Our findings also suggest a novel mechanism of the anti-apoptotic action of a decrease in polyamine levels via de-inhibition of PP5 and accelerated dephosphorylation and deactivation of ASK1.
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PMID:Regulation of apoptosis signal-regulating kinase 1 (ASK1) by polyamine levels via protein phosphatase 5. 1589 Jun 60

Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein-protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition.
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PMID:Geldanamycin activates Hsp70 response and attenuates okadaic acid-induced cytotoxicity in human retinal pigment epithelial cells. 1595 Jul 70

Calcineurin (CaN), a Ca2+-calmodulin (CaM)-dependent protein phosphatase, is important for Ca2+-mediated signal transduction. The main objective of this study was to examine the potential role of CaN in epileptic brain and its involvement in neuronal apoptosis. We investigated CaN expression and its interaction with various signaling molecules in normal, carrier and epileptic brain tissues of chicken. Our results revealed higher Ca2+-CaM-dependent phosphatase activity of CaN and a correspondingly strong immunoreactive band of CaN A in epileptic and carrier brain samples compared with normal brain. Furthermore, immunohistochemical analysis showed a higher level of expression of CaN in epileptic brain tissue. However, the intensity of immunoreactivity was less in carrier than epileptic brain. We observed that the interaction of CaN with m-calpain and micro-calpain was strong in carrier and epileptic chickens compared with that in normal birds. In addition, the interaction of CaN with Bcl-2, caspase-3 and p53 was greater in carrier and epileptic fowl than in normal chickens. The greater interaction of CaN with various apoptotic factors in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis.
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PMID:Expression of calcineurin and its interacting proteins in epileptic fowl. 1633 33

In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.
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PMID:Dual inhibition of protein phosphatase-1/2A and calpain rescues nerve growth factor-differentiated PC12 cells from oxygen-glucose deprivation-induced cell death. 1638 61

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.
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PMID:Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: involvement of Ser/Thr phosphatase. 1701 55

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.
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PMID:Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells. 1702 93

Testicular germ cell tumours (TGCT) represent the most common malignancy in young males. We reported previously that two prototype members of the mitogen-activated protein kinase (MAPK) family, the MAPK ERK kinase (MEK) and extracellular signal-regulated kinase (ERK), are inactive in malignant testicular germ cells and become active after drug stimulation, leading to apoptosis of tumour cells. In this study, we asked whether the protein phosphatase PP2A, a known inhibitor of the MEK-ERK pathway, participates in the proliferation and/or apoptosis of primary TGCT (n = 48) as well as two TGCT cell lines (NTERA and NCCIT). Quantitative RT-PCR, immunohistochemistry, western blot analyses and phosphatase assay indicate that primary TGCT as well as TGCT cell lines express PP2A and that PP2A is active in TGCT cell lines. The inhibition of PP2A by application of two PP2A inhibitors, cantharidic acid (CA) and okadaic acid (OA), results in a significant increase in caspase-3-mediated apoptosis of TGCT cell lines. Thereby, PP2A inhibition was accompanied by phosphorylation and activation of MEK and ERK. Functional assays using the MEK inhibitor PD98059 demonstrated that the phosphorylation of MEK and ERK was required for the induction of caspase-3-mediated apoptosis of malignant germ cells. Thus, our data suggest that inhibition of PP2A mediates its apoptosis-inducing effect on TGCT through activation of the MEK-ERK signalling pathway that leads to caspase-3-mediated apoptosis of tumour cells. In addition our results support previous observations that PP2A exerts an anti-apoptotic effect on malignant tumour cells.
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PMID:Expression and function of protein phosphatase PP2A in malignant testicular germ cell tumours. 1759 Aug 61

A-kinase-anchoring protein 149 (AKAP149) is a member of a structurally diverse, though functionally similar anchoring protein family and is localized to the outer membrane of mitochondria and in the endoplasmic reticulum-nuclear envelope network. AKAP149 plays an important role in controlling the subcellular localization and temporal specificity of protein phosphorylation and mRNA metabolism by tethering kinases and phosphatases, such as protein kinase A and type I protein phosphatase, through its N-terminal protein-binding motifs and mRNAs via its C-terminal RNA-binding motifs. It is well recognized that caspases play a central role in transducing and amplifying the intracellular death signal and that apoptosis is executed as a consequence of caspase-mediated cleavage of multiple cellular substrates. The identification of novel death substrates and elucidation of the consequences of their proteolytic cleavages by caspases are therefore crucial for our understanding of cell death and other biological processes. Herein, we demonstrated that AKAP149 is a direct substrate of active caspase-3, -8 -and -10 in vitro and in vivo. 35S-labeled full-length AKAP149 was completely cleaved in vitro by active caspase-3, -8 and -10 into two fragments of approximately 105 and 45 kDa, while caspase-2 cleaved it partially and caspase-1 did not cleave it at all. AKAP149 was also cleaved by caspases during Fas- and staurosporine-induced apoptosis in Jurkat T and HeLa cells, which were blocked by specific inhibitors of caspase-3 and -8. The specific cleavage site for these caspases was mapped in vitro and in vivo to Asp582 at AKAP149, which is located between the protein kinase A regulatory subunit anchoring and KH RNA-binding domains. In addition, HeLa cells transiently overexpressing AKAP149 D582E mutant were resistant to staurosporine-induced HeLa cell apoptosis. Taken together, these data suggest that AKAP149 activity may be deregulated by caspase-dependent proteolysis during apoptotic cell death and may provide useful information for elucidating the apoptosis signaling pathways in detail.
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PMID:Specific proteolysis of the A-kinase-anchoring protein 149 at the Asp582 residue by caspases during apoptosis. 1849 68

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cgamma1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cgamma1 activity and expression. Depletion of PP1cgamma1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cgamma1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cgamma1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cgamma1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.
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PMID:Identification of a protective role for protein phosphatase 1cgamma1 against oxidative stress-induced vascular smooth muscle cell apoptosis. 1854 44


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