Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously reported that gastrin (G17) inhibits apoptosis of AR4-2J pancreatic adenocarcinoma cells, through the activation of Akt. We dissected the mechanisms responsible for this effect. D2, a CCKB receptor antagonist, inhibited G17 induction of Akt phosphorylation, measured by Western blots with anti-phospho-Akt antibodies. The intracellular calcium chelator BAPTA-AM, but not the PKC inhibitor GF109203X, blocked G17 induction of Akt. G17 stimulated BAD phosphorylation, measured by both Western blots with anti-phospho-BAD antibodies and by in vitro Akt kinase assays using recombinant BAD as substrate. G17 also induced FOXO3 phosphorylation assessed by Western blots with anti-phospho-FOXO3 antibodies, and BAPTA-AM inhibited this effect. Gastrin inhibited luciferase activity in cells transfected with FOXO1 together with a vector containing insulin-responsive sequences upstream of the luciferase reporter gene. In conclusion, G17 induces Akt through activation of CCKB receptors and of intracellular calcium-dependent, PKC-independent, pathways. This effect leads to BAD phosphorylation and to forkhead transcription factors inactivation.
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PMID:Intracellular mechanisms mediating the anti-apoptotic action of gastrin. 1535 98

Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-alpha and -delta, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-delta siRNA, ROKalpha siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-delta and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-delta was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-delta downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-delta proteins, in stimulated gut peptide secretion.
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PMID:Myristoylated alanine-rich C kinase substrate-mediated neurotensin release via protein kinase C-delta downstream of the Rho/ROK pathway. 1562 35

Gastrointestinal peptides including mammalian bombesin-like peptides, cholecystokinin (CCK), gastrin, and neurotensin stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a number of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation. These agonists bind to G protein-coupled receptors (GPCRs) that promote Galpha q-mediated activation of beta isoforms of phospholipase C to produce two second messengers: Inositol (1,4,5) trisphosphate {Ins (1, 4, 5) P3} that mobilises Ca2+ from internal stores, and diacylglycerol that activates the classic and new isoforms of the protein kinase C (PKC) family. PKCs play a critical part in transducing bombesin/gastrin releasing peptide (GRP) receptor signals into activation of protein kinase cascades. Protein kinase D (PKD), a serine/threonine protein kinase with distinct structural and enzymological properties, is activated by phosphorylation in living cells through a new PKC-dependent signal transduction pathway. GPCR agonists including bombesin/GRP induce a rapid and striking activation of PKD by PKC. These results indicate that PKD functions downstream from PKCs and identify a new phosphorylation cascade that is activated by gastrointestinal peptide agonists. The bombesin/GRP GPCR also promotes rapid Rho-dependent assembly of focal adhesions, formation of actin stress fibres and tyrosine phosphorylation of multiple cellular proteins. We identified p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (CAS) and paxillin as prominent targets of gastrointestinal peptide-stimulated tyrosine phosphorylation and developed a model that envisages a G12/Rho-dependent pathway connecting GPCR activation to the tyrosine phosphorylation of these focal adhesion proteins. Separate pathways mediate gastrointestinal peptide stimulation of additional tyrosine kinase pathways including transactivation of Src and epidermal growth factor receptor (EGFR). Tyrosine phosphorylation has a critical role in gastrointestinal peptide-induced cellular migration and cooperates with Gq-stimulated events to promote mitogenesis. The growth-promoting effects of neuropeptides and the elucidation of the signalling pathways that mediate their effects assume an added importance because these agonists and their receptors are increasingly implicated in sustaining the proliferation of clinically aggressive solid tumours including those from lung, pancreas, and colon.
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PMID:Gastrointestinal peptide signalling in health and disease. 1614 98

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCK(B) receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.
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PMID:Gastrin regulates the TFF2 promoter through gastrin-responsive cis-acting elements and multiple signaling pathways. 1733 76

Our previous work revealed that gastrin regulates chromogranin A (CgA) transcription through enhanced binding of Sp1, CREB and Egr-1 to a proximal gastrin-responsive promoter element (Gas-RE). Here, we provide a detailed characterization of the signalling pathways transmitting the effect of gastrin on the CgA promoter. Gastrin treatment of gastric AGS-B cells potently stimulated MEK-1 as well as MAP kinases ERK-1/-2, JNK and p38 in a time-dependent manner. Interruption of ERK-1/-2/MEK-1 pathways abolished the transactivating effect of gastrin, whereas blockade of JNK or p38 activity was without effect. Functional promoter analysis revealed that the minimal element CgA-85/-64 was sufficient and necessary to confer MEK-1/ERK responsiveness. Analysis of proximal signalling pathways showed that activation of the MEK-1/ERK-1/2 module by gastrin does not require Ras, PI3-kinase or intracellular calcium signals, but depends on activation of kinases of the PKC family. This report demonstrates that a pathway comprising PKCs>Raf-1>MEK-1>ERK-1/-2 mediates the effect of gastrin on the CgA promoter, and strongly suggests that enhanced phosphorylation of Sp1 and CREB is crucial for CgA transactivation through the G protein-coupled CCK-B/gastrin receptor.
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PMID:Gastrin transactivates the chromogranin A gene through MEK-1/ERK- and PKC-dependent phosphorylation of Sp1 and CREB. 1788 8

The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter-reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter-reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-G(R) and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6-9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle alpha-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts.
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PMID:Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo. 1793 65

The gastric acid-secreting parietal cell exhibits profound morphological changes on stimulation. Studies in gastrin null (Gas-KO) mice indicate that maturation of parietal cell function depends on the hormone gastrin acting at the G-protein-coupled cholecystokinin 2 receptor. The relevant cellular mechanisms are unknown. The application of differential mRNA display to samples of the gastric corpus of wild-type (C57BL/6) and Gas-KO mice identified the cytoskeletal linker protein, ezrin, as a previously unsuspected target of gastrin. Gastrin administered in vivo or added to gastric glands in vitro increased ezrin abundance in Gas-KO parietal cells. In parietal cells of cultured gastric glands from wild-type mice treated with gastrin, histamine or carbachol, ezrin was localized to vesicular structures resembling secretory canaliculi. In contrast, in cultured parietal cells from Gas-KO mice, ezrin was typically distributed in the cytosol, and this did not change after incubation with gastrin, histamine or carbachol. However, priming with gastrin for approximately 24 h, either in vivo prior to cell culture or by addition to cultured gastric glands, induced the capacity for secretagogue-stimulated localization of ezrin to large vesicular structures in Gas-KO mice. Similarly, in a functional assay based on measurement of intracellular pH, cultured parietal cells from Gas-KO mice were refractory to gastrin unless primed. The priming effect of gastrin was not attributable to the paracrine mediator histamine, but was prevented by inhibitors of protein kinase C and transactivation of the epidermal growth factor receptor. We conclude that in gastrin null mice there is reduced ezrin expression and a defect in ezrin subcellular distribution in gastric parietal cells, and that both can be reversed by priming with gastrin.
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PMID:Identification of ezrin as a target of gastrin in immature mouse gastric parietal cells. 1856 1

Elevated serum concentrations of the hormone gastrin are associated with the development of gastric carcinoid tumors, but the mechanisms of tumor development are not fully understood. We hypothesized that the antiapoptotic effects of gastrin may be implicated and have therefore investigated the role of antiapoptotic members of the bcl-2 family of proteins. AGS-G(R) human gastric carcinoma cells stably transfected with the CCK-2 receptor were used to assess changes in the expression of bcl-2 family members following gastrin treatment and the function of mcl-1 during apoptosis was investigated by use of small-interfering RNA (siRNA). Treatment of AGS-G(R) cells with 10 nM gastrin for 6 h caused maximally increased mcl-1 protein abundance. Gastrin-induced mcl-1 expression was inhibited by the transcription inhibitor actinomycin D and by the protein synthesis inhibitor cycloheximide. Downstream signaling of mcl-1 expression occurred via the CCK-2 receptor, protein kinase C, and MAP kinase pathways, but not via PI 3-kinase. Transfection with mcl-1 siRNA significantly suppressed mcl-1 protein expression and abolished the antiapoptotic effects of gastrin on serum starvation-induced apoptosis. Mcl-1 protein expression was also specifically increased in the type I enterochromaffin-like cell carcinoid tumors of 10 patients with autoimmune atrophic gastritis and hypergastrinemia. Gastrin therefore signals via the CCK-2 receptor, protein kinase C, and MAP kinase to induce expression of antiapoptotic mcl-1 in AGS-G(R) cells, and mcl-1 expression is also increased in human hypergastrinemia-associated type I gastric carcinoid tumors. Gastrin-induced mcl-1 expression may therefore be an important mechanism contributing toward type I gastric carcinoid development.
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PMID:Gastrin increases mcl-1 expression in type I gastric carcinoid tumors and a gastric epithelial cell line that expresses the CCK-2 receptor. 1871 2

Several studies indicate that cholesterol esterification is deregulated in cancers. The present study aimed to characterize the role of cholesterol esterification in proliferation and invasion of two tumor cells expressing an activated cholecystokinin 2 receptor (CCK2R). A significant increase in cholesterol esterification and activity of Acyl-CoA:cholesterol acyltransferase (ACAT) was measured in tumor cells expressing a constitutively activated oncogenic mutant of the CCK2R (CCK2R-E151A cells) compared with nontumor cells expressing the wild-type CCK2R (CCK2R-WT cells). Inhibition of cholesteryl ester formation and ACAT activity by Sah58-035, an inhibitor of ACAT, decreased by 34% and 73% CCK2R-E151A cell growth and invasion. Sustained activation of CCK2R-WT cells by gastrin increased cholesteryl ester production while addition of cholesteryl oleate to the culture medium of CCK2R-WT cells increased cell proliferation and invasion to a level close to that of CCK2R-E151A cells. In U87 glioma cells, a model of autocrine growth stimulation of the CCK2R, inhibition of cholesterol esterification and ACAT activity by Sah58-035 and two selective antagonists of the CCK2R significantly reduced cell proliferation and invasion. In both models, cholesteryl ester formation was found dependent on protein kinase zeta/ extracellular signal-related kinase 1/2 (PKCzeta/ERK1/2) activation. These results show that signaling through ACAT/cholesterol esterification is a novel pathway for the CCK2R that contributes to tumor cell proliferation and invasion.
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PMID:Signaling through cholesterol esterification: a new pathway for the cholecystokinin 2 receptor involved in cell growth and invasion. 1950 90

Expression of gastrin and cholecystokinin 2 (CCK(2)) receptor splice variants (CCK(2)R and CCK(2i4sv)R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK(2) receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK(2i4sv)R or CCK(2)R. RT-PCR and intracellular Ca(2+) ([Ca(2+)](i)) imaging of RIE/CCK(2)R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK(2)R or CCK(2i4sv)R (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK(2) receptor selective antagonist, YM022, partially inhibited the growth of both receptor-expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor-expressing cells. Vector-NCM356 cells did not form tumors in nude mice, whereas, either CCK(2) receptor-expressing cells formed large tumors. Autocrine activation CCK(2) receptor variants are sufficient to increase in vitro growth and tumorigenicity of non-transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.
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PMID:CCK2 receptor expression transforms non-tumorigenic human NCM356 colonic epithelial cells into tumor forming cells. 1969 27


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