Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood. We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner. Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases. To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases. Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo. The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase. Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif. Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells.
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PMID:Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase. 763 60

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.
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PMID:Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases. 897 82

In order to establish what role members of the activating protein-1 (AP-1) gene families, i.e., c-fos, c-jun, junB, and junD, play in thymic apoptosis, we have analyzed changes in their expression in response to three different agents: a glucocorticoid analog dexamethasone, an inhibitor of topoisomerase II teniposide VM26, and gamma radiation. All three agents induced thymic death at a similar rate and with the same morphological and biochemical features. There was a rapid and transient increase in the steady-state mRNA level of junB and c-fos genes in all treatments, including control cultures, reminiscent rather of cellular stress response to the environmental changes than to the apoptotic stimuli. On the other hand, treatments with the DNA-damaging agents, VM26 and gamma radiation, resulted in superinduction of the c-jun mRNA and in the activation of the stress response signaling pathway of c-Jun N-terminal kinase. Gene transcription ceased completely in cells with fragmented DNA and the down-regulation of genes such as junD and tubulin was reflective of the thymocytes' commitment to apoptosis. The DNA-binding activities of the serum response factors, cyclic AMP response element binding proteins, and AP-1 factors, indicative of their transcriptional competence, were compromised shortly after induction of apoptosis regardless of the agent employed, consistent with previously reported enhancement in cellular proteolysis which is an essential component of the apoptotic cell death.
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PMID:Jun and JNK kinase are activated in thymocytes in response to VM26 and radiation but not glucocorticoids. 902 81

The exact mechanisms for the selective toxicity of chemotherapeutic drugs against tumor cells are not fully understood. We designed a series of experiments to test the possibility that the positive proliferative signal initiated by oncogenes might change the sensitivity for apoptosis induction by the anticancer drug etoposide (VP16), an inhibitor of topoisomerase II (Topo II). Treatment with VP16 induced significantly increased apoptosis in NIH3T3 cells transformed by oncogenic src, ras or raf, compared with the normal 3T3 cells. Apopototic changes involved nuclear DNA fragmentation, morphological alterations and decreased viability. Furthermore it was shown that stress-activated protein kinase (SAPK) was activated much more strongly in all three transformed lines compared to untransformed cells by VP16 treatment, while slight activation of extracellular signal-regulated kinase (ERK1) was observed in all four cell lines. In addition, the transformed cells displayed arrest in mid-S-phase following the treatment, whereas NIH3T3 cells were primarily arrested in late S and G2/M phase. Finally, the cyclin-dependent kinase inhibitor p21 WAF1 was induced in all four cell lines, although induction of p53 was not detected in any of these cell lines. Taken together our results demonstrated that oncogenic transformation can sensitize the cells to apoptosis induction, stress kinase activation and cell cycle arrest in response to VP16 treatment. These results may have important implications for understanding the selective toxicity of anti-cancer drugs in tumor cells.
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PMID:Oncogenic transformation potentiates apoptosis, S-phase arrest and stress-kinase activation by etoposide. 934 97

Human monocytic leukemia U937 cells undergo apoptosis when treated with antitumor drugs, such as etoposide, camptothecin and mitomycin C. The molecular mechanism of the drug-induced apoptosis is not well understood. In this study, we found that 2-deoxyglucose (2DG), an analog of D-glucose and an inducer of glucose-regulated stress, inhibited anticancer drug-induced but not tumor necrosis factor-alpha-induced apoptosis of U937 cells. 2DG did not reduce initial cellular damage caused by etoposide, an inhibitor of topoisomerase II, suggesting that 2DG affected subsequent cellular responses involved in apoptosis. 2DG inhibited the etoposide-induced activation of c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and the subsequent activation of CPP32, both of which are positive regulators for etoposide-induced apoptosis of U937 cells. Our results indicate that 2DG inhibits apoptosis by blocking the signals from cellular DNA damage for JNK1/SAPK activation.
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PMID:2-Deoxyglucose inhibits chemotherapeutic drug-induced apoptosis in human monocytic leukemia U937 cells with inhibition of c-Jun N-terminal kinase 1/stress-activated protein kinase activation. 953 66

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermium radix, inhibited the growth of various lines of cancer cells derived from human solid tumors at low concentrations between 10(-8) and 10(-6) M. When HL-60 cells were treated with 10(-6) M beta-HIVS for 3 h, characteristic features of apoptosis, such as DNA fragmentation, nuclear fragmentation, and activation of caspase-3-like activity, were observed. The most characteristic features of the effect of beta-HIVS were the remarkable morphological changes induced upon treatment of HL-60 cells with beta-HIVS, as visualized on the staining of actin filaments with phalloidin labeled with tetramethylrhodamine B isothiocyanate. Moreover, activation of MAP kinases, such as ERK2, JNK and p38, was detected after treatment with 10(-6) M beta-HIVS preceding the appearance of the characteristics of apoptosis, and the features of the activation of these MAP kinases were quite different from those of Fas and anticancer drug-induced apoptosis. The activation of JNK by beta-HIVS was not inhibited by inhibitors of caspases, suggesting that JNK is located either upstream or independent of the caspase signaling pathway. beta-HIVS did not inhibit the activity of topoisomerase II. These results indicate that beta-HIVS induces apoptosis in HL-60 cells through a mechanism unlike those reported for anti-Fas antibodies and etoposide.
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PMID:beta-hydroxyisovalerylshikonin inhibits the cell growth of various cancer cell lines and induces apoptosis in leukemia HL-60 cells through a mechanism different from those of Fas and etoposide. 988 Jul 90

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.
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PMID:Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation. 1020 78

We recently demonstrated that physiological induction of apoptosis by cytotoxic sphingolipid messengers proceeds via activating protein-1 (AP1)-dependent and AP1-independent mechanisms in U937 human monoblastic leukemia cells. Here we examine involvement of the stress-activated protein kinase (SAPK) cascade and AP1 in the initiation of apoptosis in U937 cells by podophyllotoxin-derived inhibitors of topoisomerase II. Induction of apoptotic cell death and DNA damage by treatment of U937 cells with etoposide (100 microM) was associated with phosphorylation and activation of the c-Jun NH(2)-terminal kinase (JNK1) SAPK enzymes p46 and p54-JNK2 and transient increases in expression of the transcription factor c-Jun, a primary JNK substrate. These responses were accompanied by a modest, but sustained, recruitment of the mitogen-activated protein kinases p42-extracellular signal receptor-activated kinase (ERK)1 and p44-extracellular signal receptor-activated kinase 2. The capacity of etoposide to promote double-stranded DNA degradation and cell death was unaffected by manipulations that interfere with SAPK signaling outflow through c-Jun/AP1, including: 1) pharmacological inhibition of AP1 activity by diferuloylmethane and 2) molecular ablation of normal c-Jun function by the Jun dominant-negative mutant TAM-67. Cytotoxicity of the structurally related compound teniposide was similarly unaffected. In parallel trials, the lethal actions of ceramide (but not of sphingosine) were markedly diminished by pretreatment with diferuloylmethane or expression of TAM-67, confirming the effectiveness of these interventions in suppression of SAPK/AP1-dependent apoptosis. The involvement of AP1 in the proapoptotic actions of other inhibitors of topoisomerase II activity was also evaluated. Induction of cell death by the anthracyclines daunorubicin, daunorubicin, and idarubicin was found to be insensitive to pretreatment with diferuloylmethane or expression of TAM-67. Collectively, the present data indicate that induction of apoptosis by etoposide and related inhibitors of topoisomerase II is mediated through a cell death pathway that does not require SAPK-dependent recruitment of AP1. These findings additionally suggest that activation of the SAPK represents a consequence, rather than an underlying cause, of etoposide-induced apoptosis in myeloid leukemia cells.
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PMID:Evidence that the apoptotic actions of etoposide are independent of c-Jun/activating protein-1-mediated transregulation. 1045 18

Several sets of non-receptor protein tyrosine kinases (PTK) play important roles in apoptosis induced by various extracellular stresses. Anti-cancer drugs induce cellular DNA damage and cytotoxic events, leading to apoptotic cell death. We utilized the established chicken B cell line, DT40 cells and their derived mutants, lacking the respective PTK [DT40/Syk(-), DT40/Lyn(-) and DT40/Btk(-)], to examine a role of these PTK in apoptotic processes induced by anti-cancer drugs. All anti-cancer drugs examined induced apoptosis of wild-type DT40 cells. Interestingly,DT40/Lyn(-), but not DT40/Syk(-) and DT40/Btk(-) cells, become resistant to apoptosis induced by adriamycin and etoposide, topoisomerase II (Topo II) inhibitory agents, compared to wild-type DT40 cells, as assessed by DNA fragmentation and TUNEL analyses. Ectopic expression of Fyn, another Src family member, in DT40/Lyn(-) cells restores largely the susceptibility of the cells against Topo II inhibitor-induced apoptosis. Furthermore, it was found that Topo II inhibitors activate c-Jun N-terminal kinase (JNK) slightly in both wild-type and DT40/Lyn(-) cells to similar extents. Collectively, these results suggest that Lyn is involved in Topo II inhibitor-induced apoptotic signaling in DT40 cells independent of JNK.
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PMID:Protein tyrosine kinase Lyn mediates apoptosis induced by topoisomerase II inhibitors in DT40 cells. 1046 58

Using combined immunofluorescence and fluorescence in situ hybridization (FISH) analysis we have extensively characterized the proteins associating with two different homologue human neocentromeres at interphase and prometaphase/metaphase, and compared these directly with those found with normal human centromeres. Antisera to CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immuno-fluorescence experiments in conjunction with FISH employing specific DNA probes to clearly identify neocentromeric DNA. We found that except for the total absence of CENP-B binding, neocentromeres are indistinguishable from their alpha satellite-containing counterparts in terms of protein composition and distribution. This suggests that the DNA base of a potential centromeric locus is of minimal importance in determining the overall structure of a functional kinetochore and that, once seeded, the events leading to functional kinetochore formation occur independently of primary DNA sequence.
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PMID:Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins. 1060 28


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