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)

The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary for the EGF response. Both the EGF-induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant-negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid-independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.
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PMID:Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. 864 Dec 83

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
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PMID:Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells. 866 40

The PDGF beta-receptor in which the active-site lysine in the kinase domain has been mutated to arginine (K634R) tacks intrinsic kinase activity. When expressed in HepG2 cells, the kinase-inactive PDGF beta-receptor was tyrosine phosphorylated in response to PDGF-BB. Previously, HepG2 cells were thought to be devoid of PDGF alpha-receptor primarily due to lack of specific antibody which precluded detection of the PDGF alpha-receptor. In fact, these cells express low levels of PDGF alpha-receptor. In HepG2 cells that express the kinase-inactive PDGF beta-receptor, PDGF-BB activates the PDGF alpha-receptors to trans phosphorylate the kinase-inactive PDGF beta-receptor in an intermolecular fashion. As a result, stimulation of HepG2 cells that express the kinase-inactive receptor leads to activation of serine/threonine kinases of the MAP kinase cascade which include RAF-1, MEK-1 and p42 MAP kinase. In contrast, the kinase-inactive receptor does not activate any signaling pathways when it is expressed in PC12 cells which do not express the endogenous PDGF alpha-receptor. Thus, the kinase-inactive K634R PDGF beta-receptor is able to enhance PDGF-BB signaling in HepG2 cells that express the PDGF alpha-receptor.
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PMID:The kinase-inactive PDGF beta-receptor mediates activation of the MAP kinase cascade via the endogenous PDGF alpha-receptor in HepG2 cells. 870 May 41

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues. Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated alpha-, beta-, and gamma-PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive delta-, epsilon-, theta-, eta-, and mu-PKCs. The kinases that belong to both of these groups display two cysteine-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include zeta, lambda, and tau-PKCs that lack both the C2 and one cysteine-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart, evidence that multiple PKC isoforms exist was first provided by Kosaka et at. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates. This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells.
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PMID:Signalling by protein kinase C isoforms in the heart. 873 30

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

Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine specific, proline directed, protein kinases which are activated by a wide spectrum of extracellular stimuli. MAPK activation is achieved through kinase cascades, which include a MAPK kinase (MAPKK or MEK) and a MAPKK/MEK kinase (MAPKKK/MEKK). These cascades serve as information relays, connecting cell-surface receptors to specific transcription factors and other regulatory proteins, thus allowing extracellular signals to regulate the expression of specific genes. Genetic and biochemical analyses have revealed many tiers in the regulation of the activities of MAPKs, as well as different routes that lead to the activation of an individual MAPK. An emerging topic of great interest is the basis for specificity in the activation of individual MAPKs and their ability to recognize their substrates.
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PMID:Mitogen-activated protein kinase cascades and regulation of gene expression. 879 94

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

Results of this study document a biphasic activation of protein kinases of the MAP kinase cascade-MEK and MAP kinases-upon interleukin-1 stimulation in human HeLa cells. The specific activities of both MEK and MAP kinases were increased within 1 min, declined rapidly to control levels and increased again after 15 min of interleukin-1 stimulation. Inhibition by okadaic acid of serine/threonine specific phosphatases resulted in a marked increase in interleukin-1 stimulated MEK and MAP kinase activities. Elevation by interleukin-1 of the specific activities of MEK and MAP kinases correlated with suppression of serine/threonine phosphatases in the late phase of stimulation. The data indicate, that enhanced phosphorylation of cellular proteins by enzymes of the MAP kinase cascade might represent a fine balance between activated protein kinases and repressed phosphoprotein phosphatase 2A in interleukin-1 stimulated HeLa cells.
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PMID:Interleukin-1 induced signalling: biphasic activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinases in HeLa cells. Involvement of phosphoprotein phosphatases. 901 Jun 81

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


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