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:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.
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PMID:Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators. 1135 49

Endothelial cells (ECs) are constantly subjected to hemodynamic forces including cyclic pressure-induced strain. The role of protein kinase C (PKC) in cyclic strain-treated ECs was studied. PKC activities were induced as cyclic strain was initiated. Cyclic strain to ECs caused activation of PKC-alpha and -epsilon. The translocation of PKC-alpha and -epsilon but not PKC-beta from the cytosolic to membrane fraction was observed. An early transient activation of PKC-alpha versus a late but sustained activation of PKC-epsilon was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-alpha and -epsilon with the signaling molecule Raf-1 was shown. ECs treated with a PKC inhibitor (calphostin C) abolished the cyclic strain-induced Raf-1 activation. ECs under cyclic strain induced a sustained activation of extracellular signal-regulated protein kinases (ERK1/2), which was inhibited by treating ECs with calphostin C. ECs treated with a specific Ca(2+)-dependent PKC inhibitor (Go 6976) showed an inhibition in the early phase of ERK1/2 activation but not in the late and sustained phase. ECs transfected with the antisense to PKC-alpha, the antisense to PKC-epsilon, or the inhibition peptide to PKC-epsilon reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-alpha mediated mainly the early ERK1/2 activation, whereas PKC-epsilon was involved in the sustained ERK1/2 activation. Strained ECs increased transcriptional activity of Elk1 (an ERK1/2 substrate). ECs transfected with the antisense to each PKC isoform reduced Elk1 and monocyte chemotactic protein-1 promotor activity. Our findings conclude that a sequential activation of PKC isoform (alpha and epsilon) contribute to Raf/ERK1/2 activation, and PKC-epsilon appears to play a key role in endothelial adaptation to hemodynamic environment.
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PMID:Sequential activation of protein kinase C (PKC)-alpha and PKC-epsilon contributes to sustained Raf/ERK1/2 activation in endothelial cells under mechanical strain. 1139 52

ERK1b is an alternatively spliced form of ERK1, containing a 26-amino acid insertion between residues 340 and 341 of ERK1. Although under most circumstances the kinetics of ERK1b activation are similar to that of ERK1 and ERK2, we have previously found several conditions under which the activation of ERK1b by extracellular stimuli differs from that of other ERKs. We studied the molecular mechanisms that cause this differential regulation of ERK1b and found that ERK1b is altered in its ability to interact with MEK1 and this influenced its subcellular localization but not its kinetics of activation. ERK1b had a decreased ability to phosphorylate Elk1, but this did not change much the transcriptional activity of the latter. Importantly, the interaction of ERK1b with PTP-SL, which can act as a MAPK phosphatase, shortly after mitogenic stimulation, was significantly affected as well. Using mutants of ERK1b we found that the differential interaction of ERK1b with the three effectors is caused by the site of insertion that abrogates the cytosolic retention sequence/common docking motif of ERKs, and is not dependent on the actual sequence of the insert. Prolonged epidermal growth factor stimulation of Rat1 cells resulted in a differential inactivation and not activation of ERK1b as compared with ERK1 and ERK2. The reduced sensitivity to phosphatases without major differences in the kinetics of activation or activation of substrates, suggests that ERK1b plays a role in the transmission of extracellular signals under conditions of persistent stimulation, where ERK1b and MAPK phosphatases are induced, and the activity of ERK1 and ERK2 is suppressed.
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PMID:Altered regulation of ERK1b by MEK1 and PTP-SL and modified Elk1 phosphorylation by ERK1b are caused by abrogation of the regulatory C-terminal sequence of ERKs. 2855 Jan 38

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC mu/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC mu/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC mu partially colocalizes with PKC eta in different cell types. Colocalization depends on the presence of the PKC mu pleckstrin homology domain. Coexpression of constitutively active PKC eta with PKC mu leads to a significant enhancement of the PKC mu substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser(738/742) of PKC mu, whereas Ser(910) autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC eta directly phosphorylates PKC mu on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC eta-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC mu/PKC eta affecting different arms of the p38/ERK/JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC mu for interaction with and activation by PKC eta as pleckstrin homology domain and activation loop.
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PMID:Protein kinase C (PKC)eta-mediated PKC mu activation modulates ERK and JNK signal pathways. 1174 79

beta-Arrestins are cytosolic proteins that mediate homologous desensitization of G protein-coupled receptors (GPCRs) by binding to agonist-occupied receptors and by uncoupling them from heterotrimeric G proteins. The recent finding that beta-arrestins bind to some mitogen-activated protein (MAP) kinases has suggested that they might also function as scaffolds for GPCR-stimulated MAP kinase activation. To define the role of beta-arrestins in the regulation of ERK MAP kinases, we examined the effect of beta-arrestin overexpression on ERK1/2 activation and nuclear signaling in COS-7 cells expressing angiotensin II type 1a receptors (AT1aRs). Expression of either beta-arrestin1 or beta-arrestin2 reduced angiotensin-stimulated phosphatidylinositol hydrolysis but paradoxically increased angiotensin-stimulated ERK1/2 phosphorylation. The increase in ERK1/2 phosphorylation in beta-arrestin-expressing cells correlated with activation of a beta-arrestin-bound pool of ERK2. The beta-arrestin-dependent increase in ERK1/2 phosphorylation was accompanied by a significant reduction in ERK1/2-mediated, Elk1-driven transcription of a luciferase reporter. Analysis of the cellular distribution of phospho-ERK1/2 by confocal immunofluorescence microscopy and cellular fractionation revealed that overexpression of beta-arrestin resulted in a significant increase in the cytosolic pool of phospho-ERK1/2 and a corresponding decrease in the nuclear pool of phospho-ERK1/2 following angiotensin stimulation. beta-Arrestin overexpression resulted in formation of a cytoplasmic pool of beta-arrestin-bound phospho-ERK, decreased nuclear translocation of phospho-ERK1/2, and inhibition of Elk1-driven luciferase transcription even when ERK1/2 was activated by overexpression of cRaf-1 in the absence of AT1aR stimulation. These data demonstrate that beta-arrestins facilitate GPCR-mediated ERK activation but inhibit ERK-dependent transcription by binding to phospho-ERK1/2, leading to its retention in the cytosol.
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PMID:beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. 1177 2

In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl(+) myeloblasts fail to undergo terminal maturation. The extracellular signal-regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been shown to mediate terminal differentiation of myeloid cells. Interestingly, Bcr-Abl(+) CML cell lines established from blast crisis were found to have low Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking protein in regulation of the Erk MAP kinase in Bcr-Abl(+) K562 human CML cells. Overexpression of Gab2 in K562 cells resulted in transcriptional activation of the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2, Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2, and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOP trans-reporting assays indicated that overexpression of Gab2 selectively activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of elevating the Gab2 level in K562 cells, stable cell lines for doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe expression, led to Erk activation, growth arrest, cell spreading, and enlargement; expression of megakaryocyte/platelet lineage-specific integrins alphaIIb/beta3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet proteins. All of these changes are characteristics of megakaryocytic differentiation. Together, these results reveal Gab2 as a limiting signaling component for Erk MAP kinase activation and terminal differentiation of K562 CML cells.
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PMID:Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl(+) K562 leukemic cells by Gab2. 1183 Apr 91

Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. AtT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.
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PMID:Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways. 1208 57

Mapping inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to alcohol intoxication. Herein, we characterize the pharmacological and signal transduction mechanisms related to alcohol-induced c-Fos expression in Edinger-Westphal neurons. Using immunohistochemistry, we show that pretreatment with gamma-aminobutyric acid (GABA)-ergic antagonists (4 mg/kg bicuculline and 45 mg/kg pentylenetetrazole) attenuates induction of c-Fos expression by alcohol (2.4 g/kg, intraperitoneal). In addition, 10 mg/kg 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)4,5-dihydro-1H-imidazole (RX 821002), an alpha(2A/D)-adrenoceptor antagonist, and 20 mg/kg haloperidol, a dopamine antagonist, also block alcohol-induced c-Fos expression in Edinger-Westphal neurons. No effects were seen in alcohol-induced c-Fos after the pretreatment of 20 mg/kg propranolol (beta-adrenoceptor antagonist), 10 mg/kg 2-(2-(4-(2-methoxyphenyl)piperazin-1-yl) ethy)-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC 239) (alpha(2B/C)-adrenoceptor antagonist), or 30 mg/kg naltrexone (opioid antagonist). Although positive modulators for the GABA(A) receptor (20 mg/kg 3alpha-hydroxy-5alpha-pregnan-20-one and 10-30 mg/kg chlordiazepoxide) and opioid receptor (10 mg/kg morphine) produced significant elevations, agonists for alpha(2)-adrenoceptors (clonidine) and dopamine receptors (apomorphine) had no effect on Edinger-Westphal c-Fos expression. These findings suggest that alcohol-induced c-Fos expression in Edinger-Westphal results from direct interactions with GABA(A) receptors, which are modified by alpha(2A/D)-adrenoceptors and dopamine receptors. Also using immunohistochemistry to identify potential intracellular mechanisms associated with alcohol-induced c-Fos expression in Edinger-Westphal, we show time-dependent increases in serine 727 phospho-signal transducer and activator of transcription 3 (Stat3) but no changes in phospho-cAMP response element-binding protein and phospho-Elk1. Time-dependent increases in phospho-extracellular signal-regulated kinase (ERK) 1/2 were found to occur simultaneously with increases in serine 727 phospho-Stat3. Finally, blockade of ERK 1/2 phosphorylation with the mitogen-activated protein kinase (MEK) 1/2 inhibitor SL327 blocked alcohol-induced c-Fos expression, suggesting that alcohol induces c-Fos in Edinger-Westphal neurons through activation of the MEK1/2-ERK1/2-Stat3 pathway.
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PMID:Alcohol-induced c-Fos expression in the Edinger-Westphal nucleus: pharmacological and signal transduction mechanisms. 1213 Jul 10

Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.
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PMID:A new ERK2 binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling. 1222 May 2

The main function of K vitamins is to act as co-factors for gamma-glutamyl carboxylase. However, they have also recently been shown to inhibit cell growth. We have chemically synthesized a series of K vitamin analogs with various side chains at the 2 or 3 position of the core naphthoquinone structure. The analogs with short thio-ethanol side chains are found to be more potent growth inhibitors in vitro of various tumor cell lines. Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone] is one of the most potent. The anti-proliferation activity of these compounds is antagonized by exogenous thiols but not by non-thiol antioxidants. This suggests that the growth inhibition is mediated by sulfhydryl arylation of cellular glutathione and cysteine-containing proteins and not by oxidative stress. The protein tyrosine phosphatases (PTP) are an important group of proteins that contain cysteine at their catalytic site. PTPs regulate mitogenic signal transduction and cell cycle progression. PTP inhibition by Cpd 5 results in prolonged tyrosine phosphorylation and activation of several kinases and transcription factors including EGFR, ERK1/2, and Elk1. Cpd 5 could activate ERK1/2 either by signaling from an activated EGFR, which is upstream in the signaling cascade, or by direct inhibition of ERK1/2 phosphatase(s). Prolonged ERK1/2 phosphorylation strongly correlates with Cpd 5-mediated growth inhibition. Cpd 5 can also bind to and inhibit the Cdc25 family of dual specific phosphatases. As a result, several Cdc25 substrates (Cdk1, Cdk2, Cdk4) involved in cell cycle progression are tyrosine phosphorylated and thereby inhibited by its action. Cpd 5 could also inhibit both normal liver regeneration and hepatoma growth in vivo. DNA synthesis during rat liver regeneration following partial hepatectomy, transplantable rat hepatoma cell growth, and glutathione-S-transferase-pi expressing hepatocytes after administration of the chemical carcinogen diethylnitrosamine, are all inhibited by Cpd 5 administration. The growth inhibitory effect during liver regeneration and transplantable tumor growth is also correlated with ERK1/2 phosphorylation induced by Cpd 5. Thus, Cpd 5-mediated inhibition of PTPs, such as Cdc25 leads to cell growth arrest due to altered activity of key cellular kinases involved in signal transduction and cell cycle progression. This prototype K vitamin analog represents a novel class of growth inhibitor based upon its action as a selective PTP antagonist. It is clearly associated with prolonged ERK1/2 phosphorylation, which is in contrast with the transient ERK1/2 phosphorylation induced by growth stimulatory mitogens.
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PMID:K vitamins, PTP antagonism, and cell growth arrest. 1238 79


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