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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytosolic and nuclear Ca(2+) have been shown to differentially regulate transcription. However, the impact of spatially distinct Ca(2+) signals on mitogen-activated protein kinase-mediated gene expression remains unknown. Here we investigated the role of nuclear and cytosolic Ca(2+) signals in epidermal growth factor (EGF)-induced transactivation of the ternary complex factor Elk-1 using a GAL4-Elk-1 construct. EGF increased Ca(2+) in both the nucleus and cytosol of HepG2 or 293 cells. Pretreatment with the intracellular Ca(2+) chelator bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid significantly reduced EGF-induced transactivation of Elk-1, indicating that EGF-stimulated Elk-1 transcriptional activity is dependent on intracellular Ca(2+). To determine the relative contribution of nuclear and cytosolic Ca(2+) signals during EGF-mediated Elk-1 transactivation, Ca(2+) signals in either compartment were selectively impaired by targeted expression of the Ca(2+)-binding protein parvalbumin to either the nucleus or cytosol. Suppression of nuclear but not cytosolic Ca(2+) signals inhibited EGF-induced transactivation of Elk-1. However, suppression of nuclear Ca(2+) signals did not affect the ability of ERK either to become phosphorylated or to undergo translocation to the nucleus in response to EGF. Elk-1 phosphorylation and nuclear localization following EGF stimulation were also unaffected by suppressing nuclear Ca(2+) signals. These results suggest that nuclear Ca(2+) is required for EGF-mediated transcriptional activation of Elk-1 and that phosphorylation of Elk-1 alone is not sufficient to induce its transcriptional activation in response to EGF. Thus, subcellular targeting of parvalbumin reveals a distinct role for nuclear Ca(2+) signals in mitogen-activated protein kinase-mediated gene transcription.
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PMID:Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium. 1197 8

Phytochemicals bind to and regulate the human estrogen receptors (ERalpha and ERbeta), mimicking actions of the endogenous estrogen, 17beta-estradiol, and known antiestrogens such as ICI 182,780. Recently, however, some of these estrogenic phytochemicals have been shown to affect other signal transduction pathways, such as receptor tyrosine kinases and mitogen-activated protein kinases (MAPK). Previously, we found that certain phytochemicals, such as flavone, apigenin, kaempferide and chalcone, have potent antiestrogenic activity. However, the antiestrogenicity of these compounds does not correlate with their ER binding capacity, suggesting alternative signaling as a mechanism for their antagonistic effects. In this study, we examined the effects of these compounds on the transcription factor activator protein-1 (AP-1). Using AP-1-luciferase stable human endometrial adenocarcinoma Ishikawa and human embryonic kidney (HEK) 293 cells, chalcone, flavone and apigenin all stimulated AP-1 activity. Additionally, we determined the effects of the phytochemicals on transcription factors that are downstream targets of various MAPK pathways. To test this, we used HEK 293 cells stably cointegrated with GAL4 transcriptional activation systems of Elk-1, c-Jun or C/EBP homologous protein (CHOP). Chalcone was the only phytochemical that activated all three transcription factors [Elk-1, 2.7-fold (P < 0.001); c-Jun, 2.7-fold (P = 0.025); CHOP, 3.0-fold (P = 0.002)], whereas apigenin stimulated CHOP (3.9-fold; P < 0.001), but inhibited phorbol myristoyl acetate-induced c-Jun activity (71%;P = 0.006). This work suggests that phytochemicals affect multiple signaling pathways that converge at the level of transcriptional regulation. The ability of flavonoids to regulate MAPK-responsive pathways in a selective manner indicates a mechanism by which phytochemicals may influence human health and disease.
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PMID:Flavonoid phytochemicals regulate activator protein-1 signal transduction pathways in endometrial and kidney stable cell lines. 1209 58

After dissemination from a primary tumor, cancer cells may resume growth, leading to overt metastasis, or enter a state of protracted dormancy. However, mechanisms that determine their fate, or markers that predict it, are mostly unavailable. We previously showed that in HEp3 human head and neck carcinoma, the extracellular signal-regulated kinase (ERK)(MAPK)/p38(SAPK) activity ratio predicts whether the cells will proliferate or enter a state of dormancy in vivo. The proliferative balance of high ERK/p38 ratio was induced by high urokinase (uPA) receptor (uPAR) expression, which activated alpha5beta1-integrin and epidermal growth factor receptor. This signaling pathway was additionally enhanced by uPA binding to uPAR and fibronectin binding to alpha5beta1-integrin. We tested whether the ERK/p38 balance is predictive of in vivo behavior in other cancer cell types and whether altering the balance will shift their phenotype between proliferation and dormancy. ERK and p38 activities were determined using either phospho-specific monoclonal antibodies or a trans-reporting system where GAL4-Elk and GAL4-CHOP trans-activation of luciferase gene served as reporters for ERK and p38 activities, respectively. We show that in breast, prostate, melanoma, and fibrosarcoma cell lines, the level of active phospho-ERK and the ERK/p38 activity ratio predict for the in vivo behavior in approximately 90% of the cell lines tested. Modulation of ERK/p38 activity ratio by multiple pharmacological and genetic interventions confirms that high ERK/p38 ratio favors tumor growth, whereas high p38/ERK ratio induces tumor growth arrest (dormancy) in vivo and that ERK is negatively regulated by p38. A melanoma cell line appeared to have developed an escape mechanism to avoid the growth inhibitory effect of high p38 activity. Mechanistic analysis implicated high uPAR expression and its interaction with and activation of alpha5beta1-integrin as determinants of the in vivo growth promoting high ERK/p38 ratio in several cell lines. The small GTPase, Cdc42, was implicated in activation of p38 and growth arrest. These results suggest that even cells that originate in advanced cancers retain a degree of dependence on surface receptors and matrix for their proliferative signals in vivo and provide a therapeutic opportunity to change their phenotype from tumorigenic to dormant.
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PMID:ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK). 1267 Sep 23

The neu (c- erbB-2 or HER2 ) proto-oncogene which encodes a receptor protein homologous to the epidermal growth factor receptor is overexpressed in 20%-30% of human breast and ovarian cancers. Oncogenic activation of Neu can also occur through multiple molecular mechanisms, including a point mutation in the transmembrane domain, deletion of the extracellular domain and short in-frame deletions of 7-12 amino acids in the extracellular region proximal to the transmembrane domain. Because of the highly vascularized phenotype of breast and ovarian cancers and the contribution of the Neu receptor to the development and progression of these tumors, we investigated the effect of Neu on the expression of the tumor angiogenesis factor VEGF. Expression of various activated Neu receptors but not wild-type Neu in Rat-1 cells, leads to increased VEGF expression on mRNA as well as on protein level. This effect is mediated by transcriptional activation of the VEGF promoter via a cluster of Sp 1 binding sites. Molecular analysis of the activation mechanism of Sp 1 revealed that neither the VEGF promoter binding activity of Sp 1 nor the expression of Sp 1 is affected by Neu transformation of the cells. Instead, functional Neu-induced transactivation of Sp 1 was observed by using a GAL4-based transactivation assay. These results demonstrate that functional changes of the transcription factor Sp 1 mediates a Neu-signaling cascade leading to VEGF promoter activation.
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PMID:Activated Neu/ErbB-2 induces expression of the vascular endothelial growth factor gene by functional activation of the transcription factor Sp 1. 1530 97

The DNA replication-related element binding factor (DREF) has been suggested as being involved in regulation of DNA replication- and proliferation-related genes in Drosophila. Recently, by searching the Drosophila genome database, we also found DRE-like sequences in the 5'-flanking regions of many genes with other functions. In addition, immunostaining of polytene chromosomes with an anti-DREF monoclonal antibody revealed that DREF can bind to a hundred regions of polytene chromosomes, suggesting regulation of multiple genes and multiple roles in vivo. When we over-expressed DREF protein or inverted repeat RNA of the DREF gene in wing imaginal discs using the GAL4-UAS targeted expression system in Drosophila, the results were veins of increased width and a loss of veins, respectively. With DREF over-expression, Rolled, a Drosophila MAPK homologue, was ectopically activated. Furthermore, half reduction of the D-raf gene dose suppressed this DREF-induced vein of increased width phenotype. In addition, when DREF transcripts were reduced by introducing double-stranded RNA of the DREF gene into S2 cells, the D-raf gene promoter activity was diminished to 4%. These data indicate that DREF is involved in regulation of vein formation through the activation of EGFR signalling in the Drosophila wing imaginal discs.
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PMID:DREF is required for EGFR signalling during Drosophila wing vein development. 1546 64

We describe a novel approach that allows detection of primary and metastatic cells in vivo in which either the extracellular signal-regulated kinase (ERK) or the p38 pathway is activated. Our recent findings showed that ERK and p38 kinases regulate, respectively, programs dictating cell proliferation (high ERK-to-p38 ratio) or growth arrest and dormancy (low ERK-to-p38 ratio) in vivo. Thus, we were able to use green fluorescent protein (GFP) to reflect ERK and p38 activities and, consequently, the proliferative state of cancer cells. This was accomplished by transfecting tumorigenic T-HEp3 and HT1080 cells, and dormant D-HEp3 cells, with plasmids coding for Elk-GAL4 or CHOP-GAL4 fusion proteins that, when phosphorylated by either ERK or p38, respectively, transactivated a GFP-reporter gene. The fate of these cells was examined in culture, in primary sites, and in spontaneous metastasis in chick embryos and nude mice. In culture GFP level was directly proportional to the previously established levels of ERK or p38 activation. In contrast, during the first 24 hours of in vivo inoculation, both the tumorigenic and the dormant cells strongly activated the p38 pathway. However, in the tumorigenic cells, p38 activity was rapidly silenced, correcting the ERK/p38 imbalance and contributing to high ERK activity throughout the entire period of tumor growth. In contrast, in the small nodules formed by dormant cells, the level of ERK activity was dramatically reduced, whereas p38 activity remained high. Strong activation of ERK was evident in metastatic sites, whereas p38 activation was silenced in this anatomic location as well. These results show that it is possible to directly measure cancer cell response to microenvironment with this reporter system and that only proliferation-competent cells have the ability to rapidly adapt ERK and p38 signaling for proliferative success. This approach allows isolation and further characterization of metastatic cells with specific signaling signatures indicative of their phenotypes.
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PMID:Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth. 1549 54

The ubiquitous heat shock protein 90 (hsp90) has been shown to participate directly in the function of a wide variety of cellular signal transduction components, including steroid receptors (SRs). However, there is still no direct evidence for an in vivo association of SRs with hsp90. This study utilizes the mammalian two-hybrid system to study the ability of hsp90 to interact with various (non)liganded nuclear receptors (NRs) in vivo in mammalian cells. As bait, we used ligand-binding domain (LBD) of various NRs fused with the GAL4-DBD. hsp90/Receptor interactions were monitored in COS cells. When NR-LBDs were co-transfected along with hsp90/VP16, none (RxR(2)-LBD) or only minimal (SR-LBDs) transcription inductions were observed (1.9-4.7-fold) in the absence of ligand. Addition of ligand further abolished the observed minimal induction. As a positive control for interaction we used TIF-2, which interacts with SRs in a ligand inducible manner. When co-transfected with NR-LBDs in the absence of ligand TIF-2/VP16 induced minimal activation of transcription (1.6-4.5-fold) that was comparable to the activation induced by the NR-LBDs. In contrast, in the presence of the ligand, the activation ranged between 62- and 134-fold depending on the receptor. The results suggest that the interaction of SRs with the hsp90 is minimal when compared to a bona fide type of interaction with the co-factors.
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PMID:Interaction of nuclear receptors with hsp90 in living cells. 1590 99

We developed a bigenic reporter system composed of a hybrid transcription factor that combines the regulatory and activation domains of either Elk-1 or cyclic AMP-responsive element binding protein (CREB) with the DNA binding, dimerization, and regulatory domains from a synthetic variant of the bacterial Tet repressor (TetR). The novel hybrid transcription factor TetR-Elk-1 was regulated by MAPK ERK kinase 1 (MEK-1) overexpression, and TetR-CREB was regulated by protein kinase A (PKA) overexpression or elevation of cyclic AMP levels. These hybrid transcription factors could be useful reporters of cell signaling pathways because, unlike previous GAL4 hybrid reporters, TetR hybrid transcription factors are inhibited by the administration of doxycycline. We validated this system in cell culture transfection experiments utilizing luciferase assays to monitor reporter gene expression and Western blot analysis to monitor transcription factor expression and phosphorylation levels. This system may be useful in creating temporally restricted windows of response to cell signaling and may be of value in the advancement of methods used to study signal transduction.
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PMID:TetR hybrid transcription factors report cell signaling and are inhibited by doxycycline. 1623 65

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established that csw(N308D)'s ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.
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PMID:Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations. 1639 95

The majority of colon cancers develop from pre-existing adenomas. We analyzed the expression profiles in the sequence of normal colon crypts, adenomas and early-stage carcinomas using microdissected cells from tubular adenomas with foci of malignant transformation. Differentially expressed genes were detected between normal-adenoma and adenoma-carcinoma, and were grouped according to the patterns of expression changes in the sequence. Down-regulated genes in the sequence included PLA2G2A, TSPAN1, PDCD4, FCGBP, AATK, EPLIN, FABP1, AGR2, MTUS1, TSC1, galectin 4 and MT1F. PLA2G2A has been shown to suppress colon tumorigenesis in mice, but the pathobiological role in humans has been controversial. Our data showed continuous down-regulation of PLA2G2A in the sequence supporting an implication in human colon cancer. Tumor suppressor and/ or proapoptotic activities have also been reported in other genes. Up-regulated genes included ribosomal proteins, IER3 and TPR. TGF-beta2 and matrix metalloproteinase 23B were up-regulated in carcinoma but not in adenoma, supporting the pathobiological roles in malignant transformation. Differentially expressed genes partly coincided with those in the adenoma-carcinoma sequence of the stomach, which was published previously, suggesting a partial overlap between the adenoma-carcinoma sequences of the colon and stomach.
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PMID:Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon. 1696 89


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