Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Caenorhabditis elegans, fem-1, fem-2, and fem-3 play pivotal roles in sex determination. Recently, a mammalian homologue of the C. elegans sex-determining protein FEM-1, F1Aalpha, has been described. Although there is little evidence to link F1Aalpha to sex determination, F1Aalpha and FEM-1 both promote apoptosis in mammalian cells. Here we report the identification and characterization of a human homologue of the C. elegans sex-determining protein FEM-2, hFEM-2. Similar to FEM-2, hFEM-2 exhibited PP2C phosphatase activity and associated with FEM-3. hFEM-2 shows striking similarity (79% amino acid identity) to rat Ca(2+)/calmodulin (CaM)-dependent protein kinase phosphatase (rCaMKPase). hFEM-2 and FEM-2, but not PP2Calpha, were demonstrated to dephosphorylate CaM kinase II efficiently in vitro, suggesting that hFEM-2 and FEM-2 are specific phosphatases for CaM kinase. Furthermore, hFEM-2 and FEM-2 associated with F1Aalpha and FEM-1 respectively. Overexpression of hFEM-2, FEM-2, or rCaMKPase all mediated apoptosis in mammalian cells. The catalytically active, but not the inactive, forms of hFEM-2 induced caspase-dependent apoptosis, which was blocked by Bcl-XL or a dominant negative mutant of caspase-9. Taken together, our data suggest that hFEM-2 and rCaMKPase are mammalian homologues of FEM-2 and they are evolutionarily conserved CaM kinase phosphatases that may have a role in apoptosis signaling.
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PMID:The Caenorhabditis elegans sex-determining protein FEM-2 and its human homologue, hFEM-2, are Ca2+/calmodulin-dependent protein kinase phosphatases that promote apoptosis. 1155 3

MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.
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PMID:Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells. 1167 38

Heat-shock protein (Hsp) 70 is an inhibitor of apoptosis and has been shown to protect against nitric oxide-mediated toxicity. To gain mechanistic insights into the actions of Hsp70, we stably transfected RAW 264.7 mouse macrophages with the human Hsp70 gene and investigated critical steps in the progression towards cell demise. Incubation of control and Hsp70-transfected macrophages with S-nitrosoglutathione induced accumulation of the tumour suppressor p53, expression of p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1) and G(1) cell-cycle arrest. However, cytochrome c translocation to the cytosol and activation of caspase 9 and caspase 3 were markedly reduced in Hsp70-overexpressing cells. In addition, changes in nuclear morphology, as determined by Hoechst staining, and the appearance of cells in the sub-G(1) phase were diminished in Hsp70-overexpressing cells compared with controls. We conclude that, in macrophages, Hsp70 interferes with cytochrome c release from mitochondria and, thereby, prevents nitric oxide-induced apoptosis, but leaves p53 accumulation and interference in the cell cycle intact.
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PMID:Heat-shock protein 70 attenuates nitric oxide-induced apoptosis in RAW macrophages by preventing cytochrome c release. 1187 90

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5--15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCdelta, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCdelta activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCdelta cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCdelta. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCdelta fragment. Attenuation of ROS generation, caspase-3 activation, and PKCdelta activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCdelta(K376R) (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.
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PMID:Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. 1188 May 3

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.
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PMID:Role of the CC chemokine receptor 9/TECK interaction in apoptosis. 1199 71

Trefoil factor (TFF)1 is synthesized and secreted by the normal stomach mucosa and by the gastrointestinal cells of injured tissues. The link between mouse TFF1 inactivation and the fully penetrant antropyloric tumor phenotype prompted the classification of TFF1 as a gastric tumor suppressor gene. Accordingly, altered expression, deletion, and/or mutations of the TFF1 gene are frequently observed in human gastric carcinomas. The present study was undertaken to address the nature of the cellular and molecular mechanisms targeted by TFF1 signalling. TFF1 effects were investigated in IEC18, HCT116, and AGS gastrointestinal cells treated with recombinant human TFF1, and in stably transfected HCT116 cells synthesizing constitutive or doxycycline-induced human TFF1. We observed that TFF1 triggers two types of cellular responses. On one hand, TFF1 lowers cell proliferation by delaying G1-S cell phase transition. This results from a TFF1-mediated increase in the levels of cyclin-dependent kinase inhibitors of both the INK4 and CIP subfamilies, leading to lower E2F transcriptional activity. On the other hand, TFF1 protects cells from chemical-, anchorage-free-, or Bad-induced apoptosis. In this process, TFF1 signalling targets the active form of caspase-9. Together, these results provide the first evidence of a dual antiproliferative and antiapoptotic role for TFF1. Similar paradoxical functions have been reported for tumor suppressor genes involved in cell differentiation, a function consistent with TFF1.
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PMID:The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1-S phase transition and reducing apoptosis. 1203 70

The serine/threonine protein kinase C (PKC) has been implicated in the regulation of drug resistance and cell survival in many types of cancer cells. However, the one or more precise mechanisms remain elusive. In this study, we have identified and determined the mechanism by which PKC-epsilon, a novel PKC isoform, modulates drug resistance in lung cancer cells. Western blot analysis demonstrates that expression of PKC-epsilon, but not other PKC isoforms, is associated with the chemo-resistant phenotype of non-small cell lung cancer (NSCLC) cell lines. Northern blotting and nuclear run-on transcription analysis further reveals that the failure of expression of PKC-epsilon in the chemo-sensitive phenotype of small cell lung cancer (SCLC) cells results from transcriptional inactivation of the gene. Importantly, forced expression of PKC-epsilon in NCI-H82 human SCLC cells confers a significant resistance to the chemotherapeutic drugs, etoposide and doxorubicin. Resistance is characterized by a significant reduction in apoptosis in PKC-epsilon-expressing cells. Treatment of NCI-H82 cells with etoposide induces a series of time-dependent events, including the release of cytochrome c from the mitochondria to the cytosol, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP). All of these events are blocked by PKC-epsilon expression. Furthermore, caspase-specific inhibitors, z-VAD-fmk and z-DEVD-fmk, significantly attenuate the accumulation of sub-G(1) population and block the PARP cleavage in response to etoposide. These results suggest that PKC-epsilon prevents cells from undergoing apoptosis through inhibition of the mitochondrial-dependent caspase activation, thereby leading to cell survival. Finally, down-regulation of PKC-epsilon expression by the antisense cDNA in NSCLC cells results in increased sensitivity to etoposide. Taken together, our findings suggest an important role for PKC-epsilon in regulating survival of lung cancer cells.
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PMID:Protein kinase C-epsilon promotes survival of lung cancer cells by suppressing apoptosis through dysregulation of the mitochondrial caspase pathway. 1212 73

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.
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PMID:Sensitization of tumor cells to Apo2 ligand/TRAIL-induced apoptosis by inhibition of casein kinase II. 1215 14

Recent studies have shown that inhibition of stress-induced signaling via the mitogen activated protein kinase (MAPK) pathway can potentiate the toxic effects of chemotherapeutic drugs and ionizing radiation. Because of these observations, we have further investigated the impact upon growth and survival of mammary (MDA-MB-231, MCF7, T47D), prostate (DU145, LNCaP, PC3) and squamous (A431) carcinoma cells following irradiation and combined long-term exposure to MEK1/2 inhibitors. Exposure of carcinoma cells to ionizing radiation resulted in MAPK pathway activation initially (0-4 h) and modestly enhanced MAPK activity at later times (24 h-96 h). Inhibition of radiation-induced MAPK activation using MEK1/2 inhibitors potentiated radiation-induced apoptosis in two waves, at 21-30 h and 96-144 h after exposure. The potentiation of apoptosis was not observed in MCF7, LNCaP, or PC3 cells. At 24 h, the potentiation of apoptosis was independent of radiation dose whereas at 108 h, apoptosis correlated with increasing dose. Removal of the MEK1/2 inhibitor either 6 h or 12 h after exposure abolished the potentiation of apoptosis at 24 h. At this time, the potentiation of apoptosis correlated with cleavage of pro-caspases -8, -9 and -3, and with release of cytochrome c into the cytosol. Inhibition of caspase function using a pan-caspase inhibitor ZVAD blocked the enhanced apoptotic response at 24 h. Selective inhibition of caspase 9 with LEHD or caspase 8 with IETD partially blunted the apoptotic response in MDA-MB-231, DU145 and A431 cells, whereas inhibition of both caspases reduced the response by > 90%. Removal of the MEK1/2 inhibitor either 24 h or 48 h after exposure abolished the potentiation of apoptosis at 108 h. Incubation of cells with ZVAD for 108 h also abolished the potentiation of apoptosis. In general agreement with the finding that prolonged inhibition of MEK1/2 was required to enhance radiation-induced apoptosis at 108 h, omission of MEK1/2 inhibitor from the culture media during assessment of clonogenic survival resulted in either little or no significant alteration in radiosensitivity. Collectively, our data show that combined exposure to radiation and MEK1/2 inhibitors can reduce survival in some, but not all, tumor cell types. Prolonged blunting of MAPK pathway function following radiation exposure is required for MEK1/2 inhibitors to have any effect on carcinoma cell radiosensitivity.
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PMID:Pharmocologic inhibitors of the mitogen activated protein kinase cascade have the potential to interact with ionizing radiation exposure to induce cell death in carcinoma cells by multiple mechanisms. 1217 Jul 77

We have previously reported that combretastatin-A4 prodrug (CA4P), anantitubulin/antiangiogenic agent isolated from the South African willow tree Combretum caffrum, induced cell death primarily through mitotic catastrophe in a panel of human B-lymphoid tumors. In this study, we investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. For this we studied the effect of CA4P on selected markers of apoptosis [caspases 9 and 3, poly(ADP-ribose) polymerase (PARP), bcl-2, and bax] and G2-M protein regulators (p53, MDM2, 14-3-3sigma, GADD45, cdc2, cdc25, chk1, wee1, p21, and cyclin B1). The chronic lymphocytic leukemia cell line WSU-CLL was used for this purpose. Western blot analysis showed that 24 h of CA4P (5 nM) exposure induces caspase 9 activation and PARP cleavage. However, the addition of Z-Val-Ala-Asp-fluoromethylketone (a general caspase inhibitor) or Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (a caspase 9 inhibitor) before CA4P treatment did not block cell death. No change in bcl-2 or bax protein expression was observed. Exposure of WSU-CLL cells to 4 and 5 nM CA4P was associated with overproduction of total p53 and no dramatic change in MDM2, 14-3-3sigma, GADD45, the cyclin-dependent kinase cdc2, its inhibitory phosphorylation, the cdc2-inhibitory kinase (wee1), chk1, or cdc25 hyperphosphorylation. The overaccumulation of p21 and cyclin B1 protein was obvious at 24 h. Furthermore, CA4P treatment showed an increase in the expression of a marker of mitosis (mitotic protein monoclonal-2 antibody) and an overaccumulation of the cyclin B in the nucleus. Our findings suggest that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of caspase 9 and PARP cleavage. The two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other in our model.
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PMID:Combretastatin-A4 prodrug induces mitotic catastrophe in chronic lymphocytic leukemia cell line independent of caspase activation and poly(ADP-ribose) polymerase cleavage. 1217 7


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