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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously identified KT5720 and U-98017 as agents that had paclitaxel (taxol)-like activity in a Chinese hamster ovary (CHO) paclitaxel-dependent cell screen for paclitaxel mimetics. In vitro polymerization of purified brain tubulin is not affected substantially by these compounds, suggesting that, unlike paclitaxel, these agents do not directly affect tubulin. However, these compounds cause profound rearrangements of the cytoskeleton in intact cells, including an apparent alteration of microtubule length, overlapping of cells, and an increase in cell size. We show that KT5720 and U-98017 effectively inhibit mitogen-activated protein kinase (MAPK) activity in vitro. Staurosporine, a poor inhibitor of MAPK but a potent inhibitor of cAMP-dependent protein kinase A (PKA) activity, phospholipid/Ca++-dependent kinase (PKC), and cdc2, does not cause similar changes. In addition, paclitaxel-dependent cells grown in U-98017 have substantially decreased levels of stimulated MAPK. In correlation with these results, we have confirmed the presence of MAPK in isolated tubulin and microtubules in cells. We have examined the hypothesis that these compounds are working through inhibition of MAPK to alter microtubules by inhibiting the phosphorylation of microtubule-associated proteins. A MAPKK dominant negative mutation transfected in CHO cells inhibits activation of MAPK. Transfectants carrying this dominant mutant have impaired activation of MAPK and an altered cell morphology, similar in some respects to that seen with KT5720 and U-98017. These results support a role for MAPK family members in the control of microtubule dynamics and suggest that in intact cells U-98017 and KT5720 achieve their effects of altering cytoskeleton and supporting partial growth of paclitaxel-dependent cells through inhibition of kinases such as MAPK.
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PMID:KT5720 and U-98017 inhibit MAPK and alter the cytoskeleton and cell morphology. 969 5

The influence of osmolarity and compatible organic osmolytes on the phosphorylation of the MAP-kinases Erk-1 and Erk-2 and on the expression of taurine transporter (TAUT) and lipopolysaccharide (LPS)-induced nitric oxide synthetase (iNOS) was studied in RAW 264.7 mouse macrophages. Hypoosmolarity (205 mosmol/l) but not hyperosmolarity (405 mosmol/l) or challenge of the cells with betaine or taurine increased phosphorylation of Erk-1 and Erk-2. Hypoosmotic Erk-phosphorylation was blocked by the MEK-inhibitor PD098059 but was resistant to depletion of extracellular calcium and to inhibition of PLC, PKC, erbstatin-sensitive tyrosine kinases and elevation of intracellular cAMP. Hyperosmolarity stimulated Na+-dependent taurine uptake and led to an increase of TAUT mRNA levels, whereas hypoosmotic exposure diminished both and induced a rapid efflux of the osmolyte from taurine-preloaded cells. The hyperosmotic elevation of TAUT mRNA levels was antagonized upon addition of taurine but not of betaine or myo-inositol. Hyperosmolarity increased the LPS-induced iNOS expression at the mRNA and the protein level. This was suppressed by betaine but not by taurine or myo-inositol. The osmotic regulation of taurine transport and iNOS expression appeared independent of the MEK-Erk pathway and the p38MAPK.
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PMID:Compatible organic osmolytes and osmotic modulation of inducible nitric oxide synthetase in RAW 264.7 mouse macrophages. 970 50

We have previously shown that ethanol-induced injury to the gastric mucosa triggers increased expression of the angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and angiogenesis. To further investigate ethanol-induced angiogenesis, we used an in vitro angiogenesis model which employs the ability of an endothelial-derived cell line (EA hy926) to form tubelike structures resembling capillaries when plated on the matrix material, Matrigel. We report that serum-starved EA hy926 cells, incubated for as little as 5 minutes with ethanol concentrations of 1.0-2.5%, formed tubelike structures reflecting in vitro angiogenesis. Control cells, not incubated with ethanol, did not form tubelike structures. Incubation for 5 minutes with 2.5% ethanol resulted in increased activities of PKC and MAP kinase (ERK2) by 1.6-fold (p < 0.05) and 2.3-fold (P < 0.001), respectively. Furthermore, inhibitors of the MAPK kinase, MEK (PD98059) and PKC (GF 109203X) prevented the induction of in vitro angiogenesis by ethanol.
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PMID:Induction of in vitro angiogenesis in the endothelial-derived cell line, EA hy926, by ethanol is mediated through PKC and MAPK. 970 42

We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hyaluronan, can induce angiogenesis in vivo and, as such, o-HA is an important regulator of the neovascularization process. As a continuation of this work, we have studied the cytoplasmic signal transduction pathways responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 microg/ml) to bovine aortic endothelial cells induces tyrosine phosphorylation of multiple proteins within 1 minute and that the activity remains above basal levels for at least 24 hours. Increased phosphorylation of the CD44 receptor was also observed. Pretreatment of cells with an anti-CD44-receptor antibody (5 microg/ml) or the tyrosine kinase inhibitor genistein (10 microM) inhibited both o-HA-induced proliferation (p < 0.05) and protein tyrosine phosphorylation. In comparison, native hyaluronan had little effect on tyrosine phosphorylation across the same time period. Protein kinase C (PKC) activity was increased 2- to 3-fold in the membranes of cells treated with o-HA, and a pretreatment with phorbol 12,13-dibutyrate (PDBu) to down-regulate PKC significantly inhibited o-HA-induced cell proliferation (p < 0.05). Examination by Western blotting showed that only the betaI and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating the proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furthermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-regulated kinase [ERK-1]) by o-HA culminated in the nuclear translocation of ERK-1. This pathway is essentially linear, as shown by the ability of specific enzyme inhibitors (PDBu and PD98059) to prevent both activation of ERK-1- and o-HA-induced proliferation. We conclude that phosphorylation of the CD44 receptor results in an increase in tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed that o-HA-induced proliferation of endothelial cells is CD44-receptor-mediated and accompanied by early response gene activation.
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PMID:Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. 971 86

Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.
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PMID:Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway. 973 28

Protein kinase C (PKC) plays a key role in signal transduction and is an important mediator of events throughout development. However, no information exists regarding the effect of a specific PKC inhibitor on mammalian embryogenesis during neurulation. This investigation was undertaken to examine the effects of a specific inhibitor of PKC, as well as inhibitors of other important kinases, on cultured mouse embryos. CD-1 mouse embryos (3 to 6 somite stage) were exposed to bisindolylmaleimide I (a specific PKC inhibitor) as well as specific inhibitors of PKA, PKG, and MAP kinase kinase for 24 h. The PKC inhibitor was a potent embryotoxicant and elicited malformations at concentrations as low as 0.01 microM. Inhibitors of other kinases also produced malformations but at much higher concentrations than those required to produce similar defects with the PKC inhibitor. These data suggest that PKC plays an important role in mammalian neurulation. Further research is required to clarify the mechanism by which PKC inhibition at this developmental stage produces malformations and the potential effects of environmental toxicants with PKC inhibitory properties on this signal transduction pathway.
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PMID:Dysmorphogenic effects of a specific protein kinase C inhibitor during neurulation. 976 44

The stress-activated protein kinase (SAPK) and mitogen-activated protein kinase (MAPK) cascades mediate cytotoxic and cytoprotective functions, respectively, in the regulation of leukemic cell survival. Involvement of these signaling systems in the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) and modulation of ara-C lethality by protein kinase C PKC inhibition/down-regulation was examined in HL-60 promyelocytic leukemia cells. Exposure to ara-C (10 microM) for 6 hr promoted extensive apoptotic DNA damage and cell death, as well as activation of PKC. This response was accompanied by downstream activation of the SAPK and MAPK cascades. PKC-dependent MAPK activity seemed to limit ara-C action in that the toxicity of ara-C was enhanced by pharmacological reductions of PKC, MAPK, or both. Thus, ara-C action was (1) partially attenuated by diradylglycerols, which stimulated PKC and MAPK, but (2) dramatically amplified by sphingoid bases, which inhibited PKC and MAPK. The cytotoxicity of ara-C also was substantially increased by pharmacological reductions of PKC, including down-regulation of PKC by chronic preexposure to the macrocyclic lactone bryostatin 1 or inhibition of PKC by acute coexposure to the dihydrosphingosine analog safingol. Significantly, both of these manipulations prevented activation of MAPK by ara-C. Moreover, acute disruption of the MAPK module by AMF, a selective inhibitor of MEK1, suppressed both basal and drug-stimulated MAPK activity and sharply increased the cytotoxicity of ara-C, suggesting the direct involvement of MAPK as a downstream antiapoptotic effector for PKC. None of these chemopotentiating agents enhanced ara-CTP formation. Ceramide-driven SAPK activity did not seem to mediate drug-induced apoptosis, given that (1) neutralization of endogenous tumor necrosis factor-alpha with monoclonal antibodies or soluble tumor necrosis factor receptor substantially reduced ceramide generation and SAPK activation by ara-C, whereas the induction of apoptosis was unaffected; (2) pharmacological inhibition of sphingomyelinase by 3-O-methoxysphingomyelin reduced ceramide generation and SAPK activation without limiting the drug's cytotoxicity; and (3) potentiation of ara-C action by bryostatin 1 or safingol was not associated with further stimulation of SAPK. These observations collectively suggest a primary role for decreased MAPK, rather than increased SAPK, in the potentiation of ara-C cytotoxicity by interference with PKC-dependent signaling.
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PMID:Evidence for involvement of mitogen-activated protein kinase, rather than stress-activated protein kinase, in potentiation of 1-beta-D-arabinofuranosylcytosine-induced apoptosis by interruption of protein kinase C signaling. 980 19

The current study focuses on the role of p38 MAP kinase in response to acute preconditioning stimuli and ischemia. Exposure of the rat myoblast cell line H9C2 to preconditioning stimuli, viz. brief duration of ischemia (metabolic inhibition) and adenosine, led to activation of p38 MAP kinase. The protective preconditioning effect of these stimuli against lethal ischemic insult was abolished in the presence or p38 MAP kinase inhibitor SB 203580 but not in the presence of MEK inhibitor PD 98509. Phorbol myristate acetate, PMA, which activates protein kinase C, PKC, activates p38 MAP kinase. and this activation is inhibited by PKC inhibitor G. 6850. The preconditioning effect of PMA was abolished by SB 203580 and also by protein kinase C inhibitor Go 6850. This indicates that the protective action of preconditioning by PKC is mediated via activation of p38 MAP kinase. Paradoxically, the presence of SB 203580 and Go 6850 during the lethal stress protected the cells against cell death. The mode of cell death in this study whether necrotic or apoptotic has not been established. Lethal ischemic stress activates p38 MAP kinase. Preconditioning the cells decreases the activation of p38 MAP kinase in response to the second lethal stress. These findings highlight the role of p38 MAP kinase in ischemic preconditioning v ischemia. Furthermore, our findings in an in vitro model using a proliferating cell line indicate that the duration and/or intensity of stimuli activating p38 kinase probably determines whether it would play a beneficial v deleterious role in cell survival in response to stress.
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PMID:Role of p38 MAP kinase in myocardial stress. 984 Dec 66

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.
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PMID:PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor. 988 95

Protein kinase C (PKC) is a multigene family of enzymes consisting of at least 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element (SRE) in the c-fos promoter is necessary and sufficient for induction of transcription of c-fos by serum, growth factors, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary complex factor (TCF) and with a dimer of the serum response factor (SRF). TCF is the target of several signal transduction pathways and SRF is the target of the rhoA pathway. In this study we generated dominant-negative and constitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to determine the roles of individual isoforms of PKC in activation of the SRE. Transient-transfection assays with NIH 3T3 cells, using an SRE-driven luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, but not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ERK2, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatment of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and constitutively active mutants of PKC-alpha and PKC-epsilon activated the transactivation domain of c-Jun, a major substrate of JNK. Constitutively active mutants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promoter which lacks the TCF binding site, and they also induce transactivation activity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked by dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, these findings indicate that PKC-alpha and PKC-epsilon can enhance the activities of at least three signaling pathways that converge on the SRE: c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of PKC may play a role in integrating networks of signal transduction pathways that control gene expression.
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PMID:Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. 989 Oct 65


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