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)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.
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PMID:Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells. 894 64

In rat aortic smooth muscle cells (RASMC), pretreatment with forskolin inhibited the activation of p42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to low concentrations of PDGF (10 ng/ml). This correlated with a strong inhibition of PDGF-stimulated MEK and C-Raf-1 kinase activity. However, the effect of forskolin could be surmounted by increasing the concentration of PDGF. Under such conditions forskolin was only effective against prolonged MAP kinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity was reversed by pretreatment of the cells with cycloheximide, suggesting the involvement of a protein synthesis step. This was not due to effects upstream of MAP kinase since PDGF-stimulated MEK activation was decreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulated the induction of the dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect was small relative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1 was abolished by the protein kinase A inhibitor H89 and this correlated with the reversal of forskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a role for intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both the inhibition of Raf-1 activation and the induction of MKP-1.
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PMID:Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1. 921 35

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.
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PMID:Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines. 938 11

In pituitary and other target tissues, estrogen acts through ERs, which are ligand-activated nuclear transcription factors. ERs can also be activated by intracellular signaling pathways in a ligand-independent manner in some cells. Because the pituitary is the target of several cAMP-activating factors, we examined the ability of cAMP to activate ERs in the alphaT3 gonadotrope cell line. Forskolin, 8-bromo-cAMP, and pituitary adenylate cyclase-activating polypeptide all enhanced ER-dependent promoter activity, which was inhibited by antiestrogen or a pituitary-specific inhibitory ER variant. Activation was PKA dependent and was blocked by the PKA inhibitor H89 or cotransfection of the inhibitor PKI. Although cAMP activated MAPK in alphaT3 cells, inhibition of MAPK with the MEK inhibitor PD98059 did not prevent forskolin-induced ER activation. Similarly, epidermal growth factor did not stimulate ER activity, although it increased MAPK activation. Forskolin-induced activation of ER was enhanced by cotransfection of steroid receptor coactivator-1 and was inhibited by the repressor of ER action, suggesting that cAMP does not alter the normal interactions between ER and cofactors. In contrast to results with estrogen, cAMP treatment did not decrease ER protein levels. These results demonstrate that in the pituitary, cAMP activates ER in a ligand-independent manner exclusively through PKA.
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PMID:Ligand-independent activation of pituitary ER: dependence on PKA-stimulated pathways. 1145 79

Cell migration requires precise coordination of many signaling pathways to achieve directed motility. We report here that NIH3T3 fibroblasts expressing a dominant negative PKA subunit (dnPKA) show diminished migration in response to serum or growth factors. This effect is not a general effect on cell motility, but rather a decreased capacity to enhance migration in response to stimuli. Control (neo) and dnPKA cells show very similar haptotactic migration toward fibronectin, but dnPKA cells show reduced stimulation of migration in response to EGF/PDGF or serum. These effects were not due to alterations in cell growth or adhesion to fibronectin. Forskolin, which elevates cyclic adenosine monophosphate (cAMP) levels, dramatically inhibited neo cell motility in a scrape migration assay, although dnPKA cell migration was unaffected. The MEK selective inhibitor U0126 and the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 inhibited migrating neo cells and were able to further inhibit residual dnPKA cell migration. Our data show that intermediate or well-controlled levels of PKA activity are required for optimal growth factor-stimulated migration in fibroblasts. PKA may play an important role in the signaling processes that lead to motility.
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PMID:Inhibition of PKA blocks fibroblast migration in response to growth factors. 1164 Aug 85

Phosphodiesterase 4D5 is the sole PDE4D cAMP phosphodiesterase isoform expressed in human aortic smooth muscle cells (HASMC). Phorbol 12-myristate 13-acetate (PMA) challenge of HASMC rapidly activated PDE4D5 through a process ablated by the mitogen-activated protein kinase kinase inhibitor PD98059. PMA elicited an inhibitory effect on PDE4D5 activity in HASMC treated with the cyclooxygenase (COX) inhibitor indomethacin, the COX-2 selective inhibitor NS-398, the phospholipase A(2) inhibitor quinacrine, and the cAMP-dependent protein kinase A (PKA) inhibitor H89. PMA challenge of COS-1 cells elicited the rapid inhibition and phosphorylation of both recombinant and endogenous PDE4D5 in a manner ablated by PD98059 and not seen in S651A mutant PDE4D5. PMA promoted the generation of PGE(2) in the medium of HASMC and caused activation of both extracellular signal-regulated kinase (ERK) and PKA through a process ablated by indomethacin, NS-398, quinacrine, and PD98059. Exogenous prostaglandin (PG) E(2) increased cAMP levels and activated PKA in HASMC. COX-2 was expressed in HASMC but not in COS-1 cells. Forskolin challenge of COS-1 cells activated PDE4D5 by causing the PKA-mediated phosphorylation of Ser126 as detected using a novel phosphospecific antiserum. PMA challenge of HASMC elicited phosphorylation of the stimulatory PKA-specific phosphorylation site, Ser126 in PDE4D5 in a manner ablated by PD98059, indomethacin, and H89. We propose that, in HASMC, PMA activates PDE4D5 through an ERK-controlled autocrine mechanism. This involves PGE(2) generation, which causes activation of adenylyl cyclase, allowing PKA to elicit net activation of PDE4D5 by phosphorylation at Ser126.
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PMID:Phorbol 12-myristate 13-acetate triggers the protein kinase A-mediated phosphorylation and activation of the PDE4D5 cAMP phosphodiesterase in human aortic smooth muscle cells through a route involving extracellular signal regulated kinase (ERK). 1164 39

Exposure of tilapia pituitary cells in culture to salmon gonadotropin-releasing hormone (sGnRH; 0.01-100 nM) elevated the phosphorylated extracellular signal-regulated kinase (pERK) levels. sGnRH also elevated the alpha, FSHbeta and LHbeta subunit mRNA levels. The phorbol ester, 1-O-tetradecanoyl phorbol-13-acetate (TPA; 12.5 nM) increased pERK levels, whereas protein kinase C (PKC) depletion or inhibition by GF109203X (GF; 0.01-10 microM) suppressed GnRH-activated ERKs. GF too abated the GnRH-induced alpha and LHbeta mRNA levels, but had no effect on those of FSHbeta. Forskolin (0.001-100 microM) activated ERK, while inhibition of protein kinase A (PKA) by H89 (0.01-10 microM) suppressed pERK levels and all GnRH-stimulated gonadotropin subunit transcripts. Exposure of cells to the mitogen-activated protein kinase kinase (MAPK kinase; MEK) inhibitor (PD98059; PD 10, 25 and 50 microM) completely blocked GnRH-induced increase in ERKs activation. Furthermore, PD suppressed the alpha and LHbeta mRNA responses to GnRH, but had no effect on FSHbeta mRNA levels. It is suggested that in tilapia the differential regulation of gonadotropin subunit gene expression by GnRH results from a divergent recruitment of signal transduction pathways, activated upon GnRH binding; PKC-ERK cascade is involved in elevating alpha and LHbeta mRNAs, whereas induction of FSHbeta transcript is ERK-independent and is under direct cAMP-PKA regulation or through other MAPK cascades.
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PMID:GnRH signaling pathways regulate differentially the tilapia gonadotropin subunit genes. 1203 71

We previously reported that erythropoietin (Epo) has a mitogenic effect on rat vascular smooth muscle cells (VSMC) and that activation of the mitogen-activated protein kinase (MAPK) cascade is an important mediator for Epo-induced mitogenesis. An increase in intracellular cAMP has an antiproliferative effect on VSMC. We therefore hypothesized that cAMP effectors inhibit Epo-induced MAPK activation in rat VSMC. When we exposed VSMC to recombinant human Epo (rHuEpo), DNA synthesis was increased. Forskolin (Fsk) or cilostazol (Cil) decreased the DNA synthesis stimulated by rHuEpo. Coincubation with Rp-cAMPS triethylamine canceled the suppression of DNA synthesis and MAPK activity by Fsk. Both rHuEpo and phorbol 12-myristate 13-acetate upregulated phosphorylations of MEK and MAPK. Pretreatment with Fsk inhibited these phosphorylations. Protein kinase C inhibitors also suppressed MEK and MAPK phosphorylations. Moreover, Fsk induced phosphorylation of Raf-1 at serine-259. These results indicated that cAMP inhibited Epo-induced MAPK activation and that this suppression might be regulated upstream or at Raf-1. The results also suggested that these agents, which could accumulate cAMP, might be protective for Epo-stimulated direct action.
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PMID:Modulation of the erythropoietin-induced proliferative pathway by cAMP in vascular smooth muscle cells. 1241 9

Cannabinoids activate several members of the mitogen-activated protein kinase superfamily including p44 and p42 extracellular signal-regulated kinase (ERK). We used N1E-115 neuroblastoma cells and the cannabinoid receptor agonist WIN 55,212-2 (WIN) to examine the signal transduction pathways leading to the activation of ERK. ERK phosphorylation (activation) was measured by Western blot. The EC50 for stimulation of ERK phosphorylation was 10 nm, and this effect was blocked by pertussis toxin and the CB1 (cannabinoid) receptor antagonist SR141716A. The MEK inhibitors PD 98059 and U0126 blocked ERK phosphorylation, as did the adenylate cyclase activator forskolin. The phosphatidylinositol (PI) 3-kinase inhibitor LY 294002 and the Src kinase inhibitor PP2 partially occluded the response but also decreased basal levels of phospho-ERK. The PI 3-kinase and Src pathways are known to promote cell survival in many systems; therefore, MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) conversion was used to examine the effects of these inhibitors on cellular viability. LY 294002 decreased the number of viable cells after 18 h of treatment; therefore, the inhibition of ERK by this inhibitor is probably because of cytotoxicity. Forskolin blocked ERK phosphorylation with an EC50 of <3 microm, and the protein kinase A (PKA) inhibitor H-89 enhanced ERK phosphorylation. c-Raf phosphorylation at an inhibitory PKA-regulated site (Ser259) was also reduced by WIN. This is probably due to constitutive phosphatase activity because WIN did not directly stimulate PP1 or PP2A activity when measured using 6,8-difluoro-4-methylumbelliferyl phosphate as a fluorogenic substrate. These data implicate the inhibition of PKA as the predominant pathway for ERK activation by CB1 receptors in N1E-115 cells. PI 3-kinase and Src appear to contribute to ERK activation by maintaining activation of kinases, which prime the pathway and maintain cellular viability.
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PMID:A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells. 1451 12

Prostanoids can suppress vascular smooth muscle cell (VSMC) proliferation, but the mechanism through which this is mediated has not been identified. In this study, we show rat aortic VSMCs to express the EP1, EP2, EP3, EP4, and IP receptors. The EP4 receptor-specific agonist, 11-deoxy-PGE1, induced a time-dependent phosphorylation of protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 in serum-depleted (0.1%) VSMCs, whereas the EP2 receptor agonist, butaprost, was without effect. PGI2 or iloprost at the IP receptor inhibited basal ERK phosphorylation with IC50 values of 10 nmol/L. Iloprost also attenuated the sustained activation of ERK induced by endothelin-1 or basic fibroblast growth factor (bFGF). Endothelin-1 or bFGF significantly increased the number of VSMCs counted 24 hours later compared with basal, and both responses were blocked by the MEK inhibitor, U0126, or iloprost. Under basal conditions, U0126 or iloprost reduced the number of viable cells and increased caspase-3 activity, which could be reversed by coapplication with endothelin-1, bFGF, or the adenylate cyclase inhibitor, SQ22536. Endothelin-1, bFGF, or SQ22536 prevented the depression to below basal levels of ERK phosphorylation induced by iloprost. Forskolin activated caspase-3 and attenuated basal ERK phosphorylation, which were prevented by SQ22536, endothelin-1, or bFGF. These data suggest that iloprost induces apoptosis via a cAMP-mediated suppression of ERK activity. In turn, this apoptotic response can be blocked by a mitogenic stimulus that re-establishes ERK activity back to basal levels, but at the expense of any concomitant proliferative activity. However, ERK stimulation by a selective EP4 receptor agonist, suggests that prostanoids may have diverse and complex roles in VSMC physiology.
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PMID:Prostacyclin induces apoptosis of vascular smooth muscle cells by a cAMP-mediated inhibition of extracellular signal-regulated kinase activity and can counteract the mitogenic activity of endothelin-1 or basic fibroblast growth factor. 1496 6


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