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)

HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with the lactogenic hormones prolactin, insulin, and glucocorticoids results in transcription of the beta-casein gene. Prolactin induces a signaling event which involves tyrosine phosphorylation of the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Here we show that HC11 cells express two Stat5 proteins, Stat5a and Stat5b. Phosphopeptide and phosphoamino acid analysis of Stat5a and Stat5b immunoprecipitated from phosphate-labeled HC11 cells revealed that both proteins were constitutively phosphorylated on serine. Lactogenic hormone treatment resulted in the appearance of a tyrosine-phosphorylated peptide in both Stat5 proteins. Consistent with this observation, a Western blot analysis of Stat5a and Stat5b showed that lactogenic hormones induced a rapid, transient increase in phosphotyrosine which paralleled the binding of Stat5 to its cognate recognition sequence in the beta-casein gene promoter. Lactogenic hormone treatment of the HC11 cells also led to a rapid activation of the mitogen-activated protein (MAP) kinase pathway. We examined the role of this pathway in beta-casein transcription using a specific MAP kinase kinase inhibitor, PD98059. Concentrations of PD98059 which completely abrogated lactogen-induced MAP kinase activation did not affect the phosphorylation state of Stat5, its DNA binding activity, or transcriptional activation of a beta-casein reporter construct. This indicates that the MAP kinase pathway does not contribute to lactogenic hormone induction of the beta-casein gene.
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PMID:Lactogenic hormone activation of Stat5 and transcription of the beta-casein gene in mammary epithelial cells is independent of p42 ERK2 mitogen-activated protein kinase activity. 894 29

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.
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PMID:Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility. 899 14

To investigate the molecular basis of the hypertrophic action of angiotensin II (AII) in vascular smooth muscle cells (SMC), we have examined the ability of the hormone to regulate the function of the translational repressor 4E-binding protein 1 (4E-BP1). Addition of AII to quiescent aortic SMC potently increased the phosphorylation of 4E-BP1 as revealed by a decreased electrophoretic mobility and an increased phosphate content of the protein. The stimulation of 4E-BP1 phosphorylation was maximal at 15 min and persisted up to 120 min. Results from affinity chromatography on m7GTP-agarose demonstrated that AII-induced phosphorylation of 4E-BP1 promotes its dissociation from eIF4E in target cells. Further characterization of 4E-BP1 phosphorylation by phosphoamino acid analysis and phosphopeptide mapping revealed that 4E-BP1 is phosphorylated on eight distinct peptides containing serine and threonine residues in AII-treated cells. The combination of results obtained from kinetics experiments, phosphopeptide analysis of in vitro and in vivo phosphorylated 4E-BP1, and pharmacological studies with the MAP kinase kinase inhibitor PD 98059 provided strong evidence that the MAP kinases ERK1/ERK2 are not involved in the regulation of 4E-BP1 phosphorylation in aortic SMC. Together, our results demonstrate that AII treatment of vascular SMC leads to hyperphosphorylation of the translational regulator 4E-BP1 and to its dissociation from eIF4E by a MAP kinase-independent mechanism.
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PMID:Angiotensin II stimulates phosphorylation of the translational repressor 4E-binding protein 1 by a mitogen-activated protein kinase-independent mechanism. 902 Jan 7

Point mutations in the Ras oncogene cause Ras to remain in its active GTP-bound state sending signals downstream continuously. Since 75 to 90% of all human pancreatic ductal adenocarcinomas harbor activating mutations at codon 12 of the K-ras oncogene it was our belief that Raf-1-MEK-MAPK will be activated in the majority of human pancreatic cancers. The aim of this study was to confirm activation of Raf-1 in K-ras mutant human pancreatic cancer. Additionally, we sought to determine if Raf-1 activation differed in K-ras mutant and nonmutant pancreatic cancer. Furthermore, we were interested in determining if Raf-1 activation in pancreatic cancer led to subsequent activation of downstream effectors such as MAP kinase. The presence of mutations in codon 12 of the K-ras oncogene in 14 human pancreatic adenocarcinoma cell lines was determined by use of mutant allele-specific PCR restriction fragment length polymorphism analysis. Raf-1 expression of quiescent cells was determined by immunoblotting using a rabbit anti-human polyclonal antibody and enhanced chemiluminescence. MAP kinase activity was determined by measuring the incorporation of phosphate into Myelin Basic Protein. Seven cell lines were noted to have mutations in codon 12 of K-ras while seven cell lines did not. There was no difference in expression of the 74 kDa-activated form of Raf-1 in K-ras mutant vs K-ras nonmutant cell lines. However, there was a significant increase in MAP kinase activity in the nonmutant cell lines compared to the cell lines with Ras mutations (P = 0.026). We conclude that Raf-1 is expressed in its active form in human pancreatic cancer regardless of K-ras status. However, signalling downstream of Raf-1 differs in cell lines with K-ras mutations compared to those cell lines without K-ras mutations.
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PMID:Activation of Raf-1 in human pancreatic adenocarcinoma. 920 70

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.
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PMID:Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway. 922 71

We previously showed that sphingosine 1-phosphate (SPP) acts as a second messenger for tumor necrosis factor alpha-induced interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism of IL-6 synthesis induced by SPP in MC3T3-E1 cells. SPP significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity. PD98059, an inhibitor of MAP kinase kinase, suppressed SPP-induced IL-6 synthesis as well as SPP-induced MAP kinase activation. The patterns of both inhibitions were similar. TMB-8, an inhibitor of Ca2+ mobilization from intracellular Ca2+ stores, significantly suppressed the SPP-induced IL-6 synthesis. These results strongly suggest that SPP-induced IL-6 synthesis is mediated via p42/p44 MAP kinase activation in osteoblast-like cells and that the SPP-induced IL-6 synthesis is dependent on intracellular Ca2+ mobilization.
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PMID:Activation of mitogen-activated protein kinase is involved in sphingosine 1-phosphate-stimulated interleukin-6 synthesis in osteoblasts. 941 15

Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.
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PMID:Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs. 941 3

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.
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PMID:Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages. 955 2

Glucose-6-phosphate dehydrogenase (G6PDH) controls the flow of carbon through the pentose phosphate pathway and also produces NADPH needed for maintenance of reduced glutathione and reductive biosynthesis. Hepatic expression of G6PDH is known to respond to several dietary and hormonal factors, but the mechanism behind regulation of this expression has not been characterized. We show that insulin similarly induces expression of endogenous hepatic G6PDH and a reporter construct containing 935 base pairs of the G6PDH promoter linked to luciferase in transient transfection assays. Using well tested and structurally distinct inhibitors of Ras farnesylation, lovastatin and B581, and a specific inhibitor of mitogen-activated protein kinase kinase activation, PD 98059, we show that the Ras/Raf/mitogen-activated protein kinase pathway is not utilized for the insulin-induced stimulation of G6PDH gene expression in primary rat hepatocytes. Similarly, using well characterized inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002, we show that PI 3-kinase activity is necessary for the induction of G6PDH expression by insulin. Rapamycin, an inhibitor of FRAP protein, which is involved in the activation of pp70 S6 kinase, blocks the insulin induction of G6PDH, suggesting that S6 kinase is also necessary for the insulin induction of G6PDH expression.
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PMID:Insulin regulation of glucose-6-phosphate dehydrogenase gene expression is rapamycin-sensitive and requires phosphatidylinositol 3-kinase. 961 3

Protein phosphorylation in bovine alveolar macrophages (BAM) activated by quartz dusts and metal oxide-coated silica particles was investigated by means of two-dimensional electrophoresis (2D-PAGE) and densitometric 32[P]-phosphate image analysis. BAM activity was monitored by determining generated superoxide anions and hydrogen peroxide. In vitro stimulation of BAM with cadmium oxide-coated silica particles (LiC-CdO) resulted in characteristic time-dependent changes in 2D-PAGE spot patterns, that were similar to the effects induced by 4ss-phorbol-12-myristate-13-acetate (PMA). Phosphorylation of two proteins with apparent molecular masses of 29 and 42 kDa appeared as main signals in both LiC-CdO and in PMA treated BAM but with different kinetics. Phosphoprotein spot pp29 was identified as an isoelectric form of Hsp27 by microsequence and Western blot analysis. In contrast to PMA stimulation, LiC-CdO-induced Hsp27 and p42 phosphorylation did not correlate with the amount of generated reactive oxygen intermediates. Other potent BAM activators like quartz dust SIKRON F600 or VO-coated silica particles did not show Hsp27 and p42 phosphorylation. LiC-CdO-mediated Hsp27 phosphorylation was inhibited by SB 203580 indicating that p38 MAP kinase is the upstream mediator of the activated signaling pathway(s), while MEK inhibitor PD 98059 had no effect.
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PMID:Hsp27 phosphorylation is induced in alveolar macrophages exposed to CdO-coated silica particles. 967 16


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