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)

Gastrin stimulates transcription of the human histidine decarboxylase (HDC) gene through binding to the G-protein-coupled cholecystokinin-B/gastrin receptor. We have explored the possibility that mitogen-activated protein kinase cascades play a role in mediating the effects of gastrin on transcription in a gastric cancer (AGS-B) cell line. Gastrin and phorbol 12-myristate 13-acetate (PMA) treatment of AGS-B cells was found to increase the phosphorylation of tyrosine residues of extracellular signal-regulated kinases (ERKs) 1 and 2 and increase ERK activity as determined by the in vitro phosphorylation of myelin basic protein. Reporter gene assays also demonstrated that gastrin and PMA stimulated Elk-1- and c-Myc-dependent transactivation, consistent with gastrin- and PMA-induced activation of ERKs. Overexpression of wild type ERK-1 and ERK-2 or activation of endogenous ERKs using activated MEK-1 (mitogen-activated protein kinase kinase or ERK kinase) overexpression stimulated HDC promoter activity in a dose-dependent fashion. Interruption of the ERK-related pathway using expression vectors for kinase-deficient ERKs or an ERK-specific phosphatase (PAC-1) blocked gastrin- and PMA-stimulated HDC promoter activity. In contrast, inhibition of the Jun kinase pathway using an interfering dominant negative SEK-1 (stress-activated protein kinase/ERK-1) mutant did not inhibit HDC promoter activity. Furthermore, whereas gastrin stimulated phosphorylation of Shc proteins and association with Grb2, activation of the HDC promoter was not influenced by expression of dominant negative Ras (N15 or N17) proteins. However, gastrin stimulated Raf-1 kinase activity, and activation of the HDC promoter was blocked by coexpression of a dominant negative Raf-1 construct. Overall, these data demonstrate that gastrin regulates HDC transcription in a Rafdependent, Ras-independent fashion predominantly through activation of the ERK-related pathway.
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PMID:Gastrin and phorbol 12-myristate 13-acetate regulate the human histidine decarboxylase promoter through Raf-dependent activation of extracellular signal-regulated kinase-related signaling pathways in gastric cancer cells. 934 Nov 40

We have previously observed that gastrin has a cholecystokinin B (CCK-B) receptor-mediated growth-promoting effect on the AR42J rat pancreatic acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose dependently induced luciferase activity in AR42J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element (SRE) of the c-fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the specific CCK-A receptor antagonist L-364,718 or by pertussis toxin, indicating that gastrin targets the SRE via specific CCK-B receptors through a mechanism independent of Gi. Inhibition of protein kinase C (PKC) either by prolonged (24 h) exposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (100 nM) or by incubation with the selective inhibitor GF-109203X (3.5 microM) resulted in an 80% reduction in luciferase activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 microM). We measured ERK2 activity in AR42J cells via in-gel kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2 enzyme activity in a dose-dependent manner. Addition of GF-109203X and PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin induction of ERK2 activity also resulted in a threefold increase in the transcriptional activity of Elk-1, a factor known to bind to the c-fos SRE and to be phosphorylated and activated by ERK2. PD-98059 blocked the growth-promoting effect of gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced c-fos gene expression via PKC-dependent and -independent pathways.
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PMID:Molecular mechanisms for the growth factor action of gastrin. 935 32

The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.
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PMID:Bombesin activates MAP kinase in non-small cell lung cancer cells. 1009 32

Ras mutations are common in lung adenocarcinomas and squamous-cell cancers, which are non-small-cell lung cancers (NSCLCs). However, small-cell lung cancers (SCLCs) rarely have ras mutations, suggesting that ras activation may not confer a growth advantage in these cells. In one SCLC cell line DMS53, activated ras expression induced increased neuroendocrine differentiation and decreased cell proliferation. We show here that DMS53 cells undergo differentiation and G1-specific growth arrest in response to ras/raf/ mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) pathway activation. To assess the consequences of activating the raf/MEK/MAPK pathway downstream of ras, we transfected a DMS53 cell line with DeltaRaf-1:ER, an activatable form of c-raf-1. DeltaRaf-1:ER activation suppressed cell proliferation and cloning on soft agar by 90% without evidence of apoptosis. Cell cycle analysis showed a reduced proportion of cells in S phase, and was associated with induction of the cyclin-dependent kinase (cdk) inhibitor p16(INK4). Expression of the cell cycle-specific proteins pRb, Rb2/p130, p107, cyclin A, cdc-2, and E2F-1 was decreased after DeltaRaf-1:ER activation in DMS53 cells. The activity cdk4 and cdk2 was also reduced, as consistent with cell cycle arrest in cells with activated DeltaRaf-1:ER cells. In addition, DeltaRaf-1:ER reduced the expression of neuroendocrine markers, gastrin releasing peptide, and ret gene in DMS53:DeltaRaf-1:ER cells. These results provide further evidence that activation of the raf/MEK/ MAPK signaling pathway, which is associated with transformation in many circumstances, can reduce the growth of SCLC cells, and suggest that activation of this pathway might be clinically efficacious in some settings.
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PMID:Raf-1 causes growth suppression and alteration of neuroendocrine markers in DMS53 human small-cell lung cancer cells. 1010 Sep 84

Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c-fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of 125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKB receptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c-fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.
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PMID:Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin. 1036 39

The human histidine decarboxylase gene is regulated by gastrin through a cis-acting element known as the gastrin response element (GAS-RE) that was initially localized to a site (+2 to +24) downstream of the transcriptional start site. Electrophoretic mobility shift assays using sequentially deleted DNA probes and nuclear extracts from AGS-B gastric cancer cells showed that the GAS-RE is actually composed of two overlapping binding sites (GAS-RE1, +1 to +19; and GAS-RE2, +11 to +27) that bind distinct nuclear factors. Reporter gene assays demonstrated that each element alone could confer gastrin responsiveness, but the presence of both elements was required for complete gastrin response. Stimulation of AGS-B cells with gastrin for 10-20 min resulted in a >2-fold increase in factor binding. The binding was inhibited by pretreatment of AGS-B cells with cycloheximide and the MEK1 inhibitor PD98059, indicating a requirement for protein synthesis and also indicating that activation occurs through the MEK/mitogen-activated protein kinase pathway. UV cross-linking and Southwestern blot analysis showed that GAS-RE1 bound a 52-kDa protein, whereas GAS-RE2 bound a 35-kDa protein. Hence, activation of histidine decarboxylase gene promoter activity by gastrin is most likely mediated by two separate nuclear factors.
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PMID:Activation of human histidine decarboxylase gene promoter activity by gastrin is mediated by two distinct nuclear factors. 1040 43

Helicobacter pylori infection of the gastric mucosa is accompanied by an activated histamine metabolism. Histamine plays a central role in the regulation of gastric acid secretion and is involved in the pathogenesis of gastroduodenal ulcerations. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production, and its activity is regulated through transcriptional mechanisms. The present study investigated the effect of H. pylori infection on the transcriptional activity of the human HDC (hHDC) promoter in a gastric epithelial cell line (AGS) and analyzed the underlying molecular mechanisms. Our studies demonstrate that H. pylori infection potently transactivated the hHDC promoter. The H. pylori-responsive element of the hHDC gene was mapped to the sequence +1 to +27 base pairs, which shows no homology to known cis-acting elements and also functions as a gastrin-responsive element. H. pylori regulates the activity of this element via a Raf-1/MEK/ERK pathway, which was activated in a Ras-independent manner. Furthermore, we found that H. pylori-induced transactivation of the hHDC promoter was independent of the cag pathogenicity island and the vacuolating cytotoxin A gene and therefore may be exerted through (a) new virulence factor(s). A better understanding of H. pylori-directed hHDC transcription can provide novel insights into the molecular mechanisms of H. pylori-dependent gene regulation in gastric epithelial cells and may lead to new therapeutic approaches.
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PMID:Helicobacter pylori activates the histidine decarboxylase promoter through a mitogen-activated protein kinase pathway independent of pathogenicity island-encoded virulence factors. 1065 59

Helicobacter pylori and proinflammatory cytokines have a direct stimulatory effect on gastrin release from isolated G cells, but little is known about the mechanism by which these factors regulate gastrin gene expression. We explored whether tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 directly regulate gastrin gene expression and, if so, by what mechanism. TNF-alpha and IL-1 significantly increased gastrin mRNA in canine G cells to 181 +/- 18% and 187 +/- 28% of control, respectively, after 24 h of treatment. TNF-alpha and IL-1 stimulated gastrin promoter activity to a maximal level of 285 +/- 12% and 415 +/- 26% of control. PD-98059 (a mitogen-activated protein kinase kinase inhibitor), SB-202190 (a p38 kinase inhibitor), and GF-109203 (a protein kinase C inhibitor) inhibited the stimulatory action of both cytokines on the gastrin promoter. In conclusion, both cytokines can directly regulate gastrin gene expression via a mitogen-activated protein kinase- and protein kinase C-dependent mechanism. These data suggest that TNF-alpha and IL-1 may play a direct role in Helicobacter pylori-induced hypergastrinemia.
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PMID:TNF-alpha and interleukin 1 activate gastrin gene expression via MAPK- and PKC-dependent mechanisms. 1170 45

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.
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PMID:CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells. 1174 87

MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase and cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [32P]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 microM PD 098059 (IC50 = 5 microM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton ("downregulation") of PKC by a long term (22 h) pretreatment with 1 microM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 microM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 microM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [3H]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genistein-sensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells.
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PMID:Role of protein kinase C, PI3-kinase and tyrosine kinase in activation of MAP kinase by glucose and agonists of G-protein coupled receptors in INS-1 cells. 1236 12


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