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)

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR-complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down-modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.
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PMID:Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. 751 43

Tumor growth is dependent upon angiogenesis. There is an intense search for pharmacological inhibitors of angiogenesis as a novel approach to treat angiogenic diseases, e.g., arthritis, diabetic retinopathy or cancer. A series of compounds, originally studied as potential protein kinase C inhibitors, included the diaminoanthraquinone NSC 639366 (1-[[3-(diethylamino)-2-hydroxypropyl]amino]-4-[(2,3- epoxypropyl)amino]-9,10-anthracenedione fumaric acid salt) (SPC-100097), was found to reversibly inhibit bovine endothelial cell growth with an IC50 that ranged between 1 and 4 nM. NSC 639366 reversibly inhibited endothelial cell migration, particularly endothelial cells stimulated by the potent angiogenic molecule, basic fibroblast growth factor. The activity of secreted urokinase-type plasminogen activator and active interstitial collagenase, but not gelatinase, was inhibited by NSC 639366. In vivo, angiogenesis was significantly inhibited by NSC 639366 by using the chick chorioallantoic membrane or the rat corneal bioassay. Two analogs of NSC 639366 did not inhibit endothelial cell growth. These experiments introduce a novel compound that could be clinically useful against angiogenic diseases and encourage further development of compounds that inhibit the plasminogen-plasmin system known to be a key regulator of angiogenesis.
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PMID:A diaminoantraquinone inhibitor of angiogenesis. 752 34

Butyrate is a potent differentiating agent present in high concentrations in colonic lumen as a result of metabolic breakdown of dietary fibre and, as such, may directly influence colonic cancer progression. We have investigated the effects of butyrate on an enzyme system important in colonic tumour progression, the plasminogen-activating system, in a poorly differentiated colon cancer cell. Butyrate was found to induce a rapid and transient increase in plasminogen activator inhibitor type 1 (PAI-1) mRNA while concomitantly suppressing the constitutive production of both urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) mRNA transcripts. We have investigated the mechanisms involved in mediating these effects by run-on transcription and RNA stability analyses. Our data show that PAI-1 mRNA induction occurs through both regulation of the stability of the alternately spliced 3.3 kb PAI-1 mRNA transcript and induction of the 2.4 kb PAI-1 mRNA transcript. Studies using modulators of signal transduction pathways demonstrate that induction of PAI-1 mRNA synthesis is independent of protein kinase C but dependent on the activation of protein kinase A. Suppression of uPA mRNA by butyrate was found to occur by down-regulation of gene transcription through a process independent of de novo protein synthesis. The transcription rate of the uPAR gene was not modulated by butyrate, but rapid turnover of the uPAR gene by butyrate was dependent on ongoing protein synthesis. Our results demonstrate that butyrate can effect rapid changes in the expression of genes of the plasminogen-activating system through several different mechanisms in a gene-specific manner.
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PMID:Butyrate regulates gene expression of the plasminogen activating system in colon cancer cells. 766 35

Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.
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PMID:Regulation of the endothelial cell urokinase-type plasminogen activator receptor. Evidence for cyclic AMP-dependent and protein kinase C-dependent pathways. 767 5

Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor-treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady-state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel-coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF-mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation.
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PMID:Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator. 768 25

The mouse urokinase-type plasminogen activator (uPA) gene was used as a model macrophage colony-stimulating factor 1 (CSF-1)-inducible gene to investigate CSF-1 signalling pathways. Nuclear run-on analysis showed that induction of uPA mRNA by CSF-1 and phorbol myristate acetate (PMA) was at the transcriptional level in bone marrow-derived macrophages. CSF-1 and PMA synergized strongly in the induction of uPA mRNA, showing that at least some components of CSF-1 action are mediated independently of protein kinase C. Promoter targets of CSF-1 signalling were investigated with NIH 3T3 cells expressing the human CSF-1 receptor (c-fms). uPA mRNA was induced in these cells by treatment with CSF-1, and a PEA3/AP-1 element at -2.4 kb in the uPA promoter was involved in this response. Ets transcription factors can act through PEA3 sequences, and the involvement of Ets factors in the induction of uPA was confirmed by use of a dominant negative Ets-2 factor. Expression of the DNA binding domain of Ets-2 fused to the lacZ gene product prevented CSF-1-mediated induction of uPA mRNA in NIH 3T3 cells expressing the CSF-1 receptor. Examination of ets-2 mRNA expression in macrophages showed that it was also induced synergistically by CSF-1 and PMA. In the macrophage cell line RAW264, the uPA PEA3/AP-1 element mediated a response to both PMA and cotransfected Ets-2. uPA promoter constructs were induced 60- to 130-fold by Ets-2 expression, and the recombinant Ets-2 DNA binding domain was able to bind to the uPA PEA3/AP-1 element. This work is consistent with a proposed pathway for CSF-1 signalling involving sequential activation of fms, ras, and Ets factors.
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PMID:Regulation of urokinase-type plasminogen activator gene transcription by macrophage colony-stimulating factor. 776 Aug 40

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide which shares structural domains with enzymes of the blood clotting cascade. HGF/SF is secreted by cells of mesodermal origin and has powerful mitogenic, motogenic and morphogenic activity on epithelial and endothelial cells. HGF/SF is produced as a biologically inactive single-chain precursor (pro-HGF/SF) most of which is sequestered on the cell surface or bound to the extracellular matrix. Maturation into the active alpha beta heterodimer results from proteolytic cleavage by a urokinase-type protease, which acts as a pro-HGF/SF convertase. The primary determinant for receptor binding appears to be located within the alpha-chain. The interaction of the alpha-chain with the receptor is sufficient for the activation of the signal cascade involved in the motility response. However, the complete HGF/SF protein seems to be required to elicit a mitogenic response. HGF/SF binds with high affinity to a transmembrane receptor, p190MET, encoded by the MET proto-oncogene. p190MET is the prototype of a distinct subfamily of heterodimeric tyrosine kinases, including the putative receptors Ron and Sea. The mature form of p190MET is a heterodimer of two disulfide-linked subunits (alpha and beta). The alpha-subunit is extracellular and heavily glycosylated. The beta-subunit consists of an extracellular portion involved in ligand binding, a membrane spanning segment, and a cytoplasmic tyrosine kinase domain. Both subunits derive from glycosylation and proteolytic cleavage of a common precursor of 170 kDa. In polarized epithelial cells the HGF/SF receptor is selectively exposed in the basolateral plasmalemma, where it is associated with detergent-insoluble components. Two Met isoforms, carrying an intact ligand binding domain but lacking the kinase domain due to truncation of the beta-subunit, arise from alternative post-transcriptional processing of the mature form. One truncated form is soluble and released from the cells. HGF/SF binding triggers tyrosine autophosphorylation of the receptor beta-subunit. Autophosphorylation on the major phosphorylation site Y1235 upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. Negative regulation of the kinase activity occurs through phosphorylation of a unique serine residue (S985) located in the juxtamembrane domain of the receptor. This phosphorylation is triggered by two distinct pathways involving either protein kinase C activation or increase in intracellular Ca2+ concentration. Upon ligand binding, the HGF/SF receptor recruits and activates several cytoplasmic effectors, including phosphatidylinositol 3-kinase (PI 3-K), phospholipase C-gamma (PLC-gamma), pp60c-Src, a tyrosine phosphatase, and a Ras-guanine nucleotide exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering. 776 52

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.
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PMID:Multiple instability-regulating sites in the 3' untranslated region of the urokinase-type plasminogen activator mRNA. 800 88

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.
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PMID:Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor. 805 May 1

1,2-Diglycerides with long-chain fatty acid residues related to nutritional fat (LCDGs) specifically affect growth and urokinase secretion in human colonic tumor cells, but not in normal mucosa. This allows them to advance and enhance carcinogenesis in the colon and rectum. SW480 colon carcinoma cells are LCDG sensitive in the same way as primary colonic tumor cells and have therefore been used as a model system to study the mechanism of LCDG action and to search for inhibitors of tumor development in the colon. Using this model system, we have shown that the effects of LCDGs are transmitted by protein kinase C and abolished by downregulation of the enzyme. Retinol, retinoic acid, and beta-carotene in nanomolar concentrations inhibit LCDG-induced growth and urokinase secretion and block stimulation of protein kinase C. Although retinol and retinoic acid at higher concentrations also display stimulatory activity, beta-carotene does not. At 100 nM, a concentration that can easily be reached in the plasma of humans, beta-carotene reduces LCDG-induced urokinase secretion about 50%. Inasmuch as beta-carotene does not have side effects due to intrinsic activities and storage effects, beta-carotene and foods rich in carotenes could be useful in the prevention of colorectal cancer.
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PMID:Retinoids inhibit protein kinase C-dependent transduction of 1,2-diglyceride signals in human colonic tumor cells. 805 26


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