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)

Treatment of human platelets with phorbol 12-myristate 13-acetate (PMA) and arginine vasopressin (AVP) increase the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Electrophoretic retardation of MAPK mobility on SDS-polyacrylamide gels was used for determination of MAPK phosphorylation. The activity of MAPK was tested in myelin basic protein (MBP)-containing polyacrylamide gels. In this study we compared the PMA and AVP signal transduction pathways leading to the activation of MAPKs and Na+/H+ exchanger (NHE). Both agonists stimulate MAPK and NHE activities in a similar time frame and concentration dependence. The MAPK and NHE activities induced by PMA were inhibited by staurosporine, a potent inhibitor for protein kinase C (PKC), and by MAPK kinase (MEK) inhibitor, PD98059, but were not affected by the tyrosine kinase inhibitor genistein. In contrast, both AVP-induced MAPK and NHE activities were inhibited by genistein and MEK inhibitor but were not affected by staurosporine. Immunoprecipitation studies demonstrate that PMA, but not AVP, enhances the basal phosphorylation of the NHE-1. In this study, MAPKs are suggested to be a part of converging signaling leading to NHE activation by PKC-dependent and AVP-tyrosine kinase-dependent pathways. We propose that the MAPK activation of the NHE-1 does not involve phosphorylation of this exchanger protein. On the other hand, PKC can lead to phosphorylation and to additional activation of the NHE-1 through a MAPK-independent pathway.
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PMID:Stimulation of mitogen-activated protein kinase and Na+/H+ exchanger in human platelets. Differential effect of phorbol ester and vasopressin. 866

The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.
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PMID:Cellular stresses and cytokines activate multiple mitogen-activated-protein kinase kinase homologues in PC12 and KB cells. 866 97

The duration of extracellular signal-regulated protein kinase (ERK) activation is critical for cell signaling decisions and probably determines whether a stimulus elicits proliferation or differentiation. We studied the intracellular signals regulating sustained ERK-2 activity in glomerular mesangial cells (GMC), utilizing combination of GMC mitogens of different potency. Incubation of GMC with both endothelin-1 (ET-1) and platelet-derived growth factor BB (PDGF-BB) led to a long-lasting, monophasic increase in ERK-2 activity. In contrast, when ET-1 was administered together with epidermal growth factor (EGF), a less pronounced and shorter activation occurred. Long-term stimulation of ERK-2 was accompanied by an increase in p45 MEK activity, which again was more pronounced when ET-1 was administered together with PDGF-BB compared with EGF. In the presence of actinomycin D (Act D), an inhibitor of RNA synthesis, ERK-2 activity induced by ET-1 and PDGF-BB but not by ET-1 and EGF remained elevated more than sixfold throughout the whole incubation period of 6 h. The effect of Act D on ET-1- and PDGF-BB-induced ERK-2 activation was mimicked by the protein phosphatase inhibitor sodium orthovanadate. In addition, vanadate also unmarked an ET-1- and EGF-induced ERK-2 activity after 6 h. The serine/threonine phosphatase inhibitor okadaic acid (OA) did neither alter agonist-induced ERK-2 activity after 6 h (0.5 nM OA) nor after 10 min or 1 h (250 nM). Together these results suggest that, in GMC, long-term activation of the mitogen-activated protein kinase ERK-2 is differentially regulated, depending on the combination of agonists administered. ET-1- and PDGF-BB-induced long-term activation of ERK-2 is regulated by a vanadate-sensitive protein phosphatase(s) and by a transcriptionally regulated protein(s). In contrast, ET-1- and EGF-induced sustained ERK-2 stimulation is regulated by a vanadate-sensitive protein phosphatase(s) but not by a transcriptionally regulated protein. Agonist-specific and time-dependent stimulation of ERK-2-regulating protein phosphatases may be critical for the length of ERK-2 activation in GMC and could thus be of pathophysiological significance in glomerular diseases associated with alterations in cell proliferation or cell differentiation.
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PMID:Sustained ERK-2 activation in rat glomerular mesangial cells: differential regulation by protein phosphatases. 877 Jan 75

CD4 T-lymphocytes, which orchestrate immune responses, receive a cognitive signal when clonally distributed receptors are occupied by MHC class II bound peptides on antigen-presenting cells. The latter provide costimulatory or accessory signals through macromolecules such as B7.1 and B7.2 which interact with coreceptors on T-cells to regulate outcomes in terms of T-cell activation or specific non-responsiveness. Complementary studies at the chemical level have implicated Schiff base formation between specialised carbonyls and amines, constitutively expressed on antigen-presenting cell and T-cell surfaces, as an essential element in specific T-cell activation. The small xenobiotic Schiff base forming molecule tucaresol, which substitutes for the physiological donor of carbonyl groups to provide a costimulatory signal to CD4 T-helper lymphocytes (Th-cells), has been developed for testing as an immunopotentiatory drug. Tucaresol, which is orally bioavailable and systemically active, enhances CD4 Th-cell and CD8 cytotoxic T-cell responses in vivo and selectively favours a Th1-type profile of cytokine production. In murine models of virus infection and syngeneic tumour growth it has substantial therapeutic activity. Schiff base formation by tucaresol on T-cell surface amines provides a costimulatory signal to the T-cell through a mechanism that activates clofilium-sensitive K+ and Na+ transport. The signalling pathway utilised by tucaresol converges with T-cell receptor signalling at the level of MAP kinase, promoting the tyrosyl phosphorylation of ERK2 by MEK (mitogen-activated protein kinase kinase). The Schiff base forming class of immunopotentiatory drug provides the first orally active, mechanism-based immunopotentiatory agents for therapeutic testing. Tucaresol is currently undergoing pilot phase I/II clinical trials as an immunopotentiator in chronic hepatitis B virus infection, HIV infection and malignant melanoma.
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PMID:Schiff base forming drugs: mechanisms of immune potentiation and therapeutic potential. 889 54

We have previously shown that osmotic stress activates both the mitogen-activated protein kinase (MAPK) cascade and the stress-activated protein kinase (SAPK, also known as JNK) cascade in rat fibroblastic 3Y1 cells and rat PC12 cells. Here, we show that treatment of these cells with sodium arsenite, a chemical compound that mimics the effects of heat shock, or anisomycin, a protein synthesis inhibitor, induces activation of SAPKs potently. These chemical compounds also stimulated the activity of SEK1/MKK4/JNKK, SAPK activator, and the activity of MEKK, SEK1 activator. Expression of a dominant negative mutant of Ras blocked the anisomycin-induced activation of SAPK and SEK1, but did not affect markedly the arsenite-induced or heat shock-induced activation in PC12 cells. The osmotic-stress-induced activation of SAPK was insensitive to the expression of a dominant negative Ras, but was partly sensitive to down-regulation of protein kinase C. These results suggest the existence of Ras-dependent and Ras-independent activation pathways for the SAPK cascade triggered by environmental stresses including chemical stress in PC12 cells. Cell staining with a specific anti-SAPK serum showed that SAPKs were present in both the cytoplasm and the nucleus under normal conditions, and became located mainly in the nucleus after osmotic stress or ultraviolet treatment, suggesting the nuclear translocation of SAPKs.
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PMID:Ras-dependent and Ras-independent activation pathways for the stress-activated-protein-kinase cascade. 891 25

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.
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PMID:Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes. 906 20

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.
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PMID:Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells. 914 52

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix degradation in part by accelerating plasmin formation at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitro invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-activated protein kinases (MAPKs) regulate u-PAR expression in two cultured colon cancer cell lines. An in-gel kinase assay showed that ERK1 activity was considerably higher in RKO cells, which display > or = 10(5) receptors/cell, than the GEO cells which have approximately 10(4) urokinase receptors per cell. The expression of either an ERK-inactivating phosphatase (CL100), or a kinase-defective ERK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constitutive ERK1 activity in RKO cells was largely a result of an activated MEK1. Further, treatment of RKO cells with a specific inhibitor (PD 098059) of MEK1 activation, which diminished ERK1 activity, reduced the amount of urokinase specifically bound to the cell surface and this was associated with reduced laminin degradation. The expression of a dominant negative c-Raf-1 also reduced u-PAR promoter activity suggesting that MEK1 activation involved an activator at, or upstream, of this serine-threonine kinase. Transfection of the u-PAR-deficient GEO cells with a constitutively activated MEK1 expression construct up-regulated u-PAR promoter activity. Similarly treatment of GEO cells with a phosphatase inhibitor (sodium vanadate) caused a dose-dependent increase in ERK1 activity which paralleled increased cell surface binding of urokinase. Taken together, these data suggest that elevated u-PAR expression, in at least a sub-population of colon cancer, is partly a consequence of a constitutively activated ERK-1-dependent signaling cascade.
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PMID:Elevated urokinase-type plasminogen activator receptor expression in a colon cancer cell line is due to a constitutively activated extracellular signal-regulated kinase-1-dependent signaling cascade. 919 Oct 56

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces HL-60 cells to differentiate along the monocyte/macrophage pathway and stimulates expression of the extracellular adhesion protein osteopontin (OPN). In this study, the mechanism of TPA-mediated OPN mRNA expression and its relationship to differentiation were investigated. The induction of OPN mRNA by TPA was dose dependently inhibited by staurosporine (0.4-10.0 nM) and chelerythrine (0.1-5.0 microM), indicating that OPN expression requires PKC activation. Furthermore, the mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD 098059 (1.0-10.0 microM), inhibited the effect of TPA in a dose-dependent fashion. Cycloheximide (10 microg/ml) ablated the induction of OPN mRNA by TPA. To determine if OPN mRNA expression was associated with a particular differentiational pathway, HL-60 cells were treated with RA, 9-cis-RA, calcitriol, or sodium butyrate. None of these agents stimulated OPN mRNA. Treatment with TPA subsequent to a 120-h pretreatment with retinoic acid (RA), 9-cis-RA, or calcitriol resulted in a potentiation of the induction of OPN mRNA. These results support a role for protein kinase C (PKC) in promoting OPN expression because each of these agents increased PKC levels. An hOPN promoter/reporter construct was responsive to TPA, indicating that this effect is at the level of transcription. Thus, TPA-stimulated transcription of the OPN gene apparently occurs via a PKC/MAPK-dependent mechanism that is independent of that associated with differentiation and is not dependent on the maturational state of these cells.
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PMID:Stimulation of osteopontin mRNA expression in HL-60 cells is independent of differentiation. 922 25

Schiff base formation on specialized T cell surface amines provides a costimulatory signal to T cells through a mechanism that activates Na+ and K+ transport, substantially enhancing TCR-dependent IL-2 production. Schiff base-forming molecules that mimic the natural carbonyl donor potently enhance immune responses and provide the first mechanism-based, orally active immunopotentiatory agents. In the present study, costimulation by the Schiff base-forming molecule tucaresol was investigated at the level of mitogen-activated protein kinase (MAPK) in T cell lines. Both TCR-directed stimulation by anti-CD3 and Schiff base stimulation by tucaresol produced a distinct mobility shift in MAPK, characterized by direct immunoblotting of cell lysate proteins subjected to SDS-PAGE, that corresponded with increased phosphorylation. Combined TCR-CD3 and tucaresol stimulation substantially enhanced and prolonged the MAPK response, providing a biochemical basis for the costimulatory nature of the pathway utilized by Schiff base signaling. The MAPK affected was identified by immunoprecipitation as ERK2. Both the direct effects and the TCR signal-enhancing effects of tucaresol on MAPK activation were also demonstrated in a functional MAPK assay measuring substrate phosphorylation. Borohydride reduction of tucaresol's Schiff base-forming carbonyl group abolished both enhancement of MAPK phosphorylation and IL-2 production, as did a selective inhibitor of the MAPKK, MEK1. Tucaresol had no effect on TCR-mediated rises in intracellular free Ca2+ or inositol 1,4,5-triphosphate generation, while tucaresol signaling occurred normally in the lck-deficient J.CaM1.6 T cell line, consistent with convergence of tucaresol- and TCR-induced signals downstream of early TCR-mediated events.
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PMID:Convergence of Schiff base costimulatory signaling and TCR signaling at the level of mitogen-activated protein kinase ERK2. 927 16


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