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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.
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PMID:Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization. 681 52

Serine esterase inhibitors (phenylmethanesulfonyl fluoride, 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) fluoride, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, or p-nitrophenyl anthranilate) blocked the production of malonyldialdehyde by platelets induced with a variety of stimuli (including thrombin, trypsin, collagen, and A23187). These inhibitors did not block malonyldialdehyde production by platelets from exogenous arachidonic acid. Those inhibitors studied in greater detail (phenylmethanesulfonyl fluoride and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) were shown to inhibit the release of [1-14C]arachidonic acid from phosphatidylinositol and phosphatidylcholine in intact platelets but not the conversion of arachidonic acid to thromboxanes, prostaglandins, or hydroxyfatty acids. These inhibitors also blocked the stimulus-induced production of [32P]phosphatidic acid in intact platelets. Both arachidonic acid release from phosphatidylinositol and phosphatidic acid production have been reported to depend on the production of diglyceride by the action on the phosphatidylinositol-specific phospholipase C.f a phosphatidylinositol-specific phospholipase C. That enzyme in the soluble fraction from disrupted platelets was inhibited at concentrations of serine esterase inhibitors which block arachidonic acid release in intact platelets. These results indicate that serine esterase inhibitors block the stimulus-induced mobilization of arachidonic acid in platelets at least in part by their action o
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PMID:Serine esterase inhibitors block stimulus-induced mobilization of arachidonic acid and phosphatidylinositide-specific phospholipase C activity in platelets. 739 Oct 2

Angiotensin II AT1 receptor signal transduction has recently been shown to function through the phospholipase C isozyme, PLC-gamma. Since PLC-gamma is known to interact with phosphotyrosine containing proteins through SH2 domains, we examined the phosphorylation state of the AT1 receptor. Immunoprecipitation of the [32P] labeled AT1 receptor from rat aortic smooth muscle cells followed by alkali hydrolysis demonstrated the presence of tyrosine phosphorylation. Phosphoamino acid analysis of the excised bands demonstrated the presence of phosphoserine and phosphotyrosine residues. A fusion protein comprising the intracellular tail of the AT1 receptor was used to screen for candidate kinases, and the src kinase family displayed high activity. In summary, this study shows that the AT1 receptor is serine and tyrosine phosphorylated in vivo and suggests that a soluble kinase related to the src family may be responsible for the tyrosine phosphorylation.
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PMID:The angiotensin II AT1 receptor is tyrosine and serine phosphorylated and can serve as a substrate for the src family of tyrosine kinases. 751 59

We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-beta receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-gamma (PLC-gamma) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-gamma. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-beta receptor in NIH-3T3 cells transformed by c-met protooncogene.
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PMID:PDGF-independent activation of PDGF-beta receptors in NIH-3T3 cells transformed by c-met protooncogene. 751 39

The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase receptor of importance for the normal development of hemopoietic cells, melanoblasts, and germ cells. We recently reported that protein kinase C (PKC) is involved in a negative feedback loop regulating the Kit/SCF-R by direct phosphorylation on serine residues in the receptor. Inhibition of PKC led to increased SCF-induced tyrosine kinase activity and mitogenicity, but PKC was necessary for SCF-induced motility. In this report we have further examined the modulatory role of PKC on SCF-induced signaling. The ligand-activated Kit/SCF-R associated weakly with GRB2 and induced only little tyrosine phosphorylation of phospholipase C-gamma in porcine aortic endothelial cells transfected with Kit/SCF-R. In contrast, the SCF-stimulated Kit/SCF-R associated efficiently with, and induced tyrosine phosphorylation of, the p85 alpha regulatory subunit of phosphatidyl inositide-3'-kinase (PI-3'-kinase). Both receptor association and tyrosine phosphorylation of p85 alpha were increased after inhibition of PKC, while its serine phosphorylation was decreased. Concomitantly, the specific activity of receptor-associated PI-3'-kinase activity was increased. Inhibition of PI-3'-kinase with wortmannin inhibited SCF-induced mitogenicity. SCF-induced phosphorylation of Raf-1 and activation of ERK2 still occurred after PKC inhibition but was not increased. In conclusion, SCF-induced PI-3'-kinase activation paralleled the increased SCF-induced mitogenicity after inhibition of PKC.
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PMID:Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C. 752 Apr 44

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
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PMID:Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. 752 42

The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.
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PMID:Similar rates of phosphatidylinositol hydrolysis following activation of wild-type and truncated rat neurokinin-1 receptors. 753 7

We have recently described an alpha 2-macroglobulin (alpha 2M) signalling receptor which is distinct from the low-density lipoprotein-related protein/alpha 2M receptor (LRP/alpha 2MR). Ligation of the macrophage signalling receptor by alpha 2M-methylamine stimulates production of several second messengers and involves a pertussis toxin-insensitive G-protein. We now report that binding of alpha 2M-methylamine, or the cloned M(r) = 20,000 receptor-binding fragment from rat alpha 1M, to macrophage alpha 2M signalling receptors induces protein phosphorylation. By use of a monoclonal antibody to phospholipase C gamma l (PLC gamma l) we were able to identify it as one target for protein phosphorylation. Phosphorylation was time and concentration dependent, being optimal at about 60 s of incubation and a 100-200 nM ligand concentration. By use of a second monoclonal antibody directed against phosphotyrosine, we were able to demonstrate that at least a portion of the label was incorporated into one or more tyrosine residues. PLC gamma l phosphorylation was then studied in membrane preparations at 4 degrees C in order to minimize serine or threonine modification. Preincubation of macrophage membranes with genistein, a tyrosine kinase inhibitor, drastically reduced phosphorylation of PLC gamma l. Receptor-associated protein, which blocks alpha 2M binding to LRP/alpha 2MR but not to the alpha 2M signalling receptor, had no effect on alpha 2M-methylamine-induced tyrosine phosphorylation of PLC gamma l. Binding of lactoferrin to LRP/alpha 2MR failed to induce phosphorylation of PLC gamma l, further supporting the hypothesis that the alpha 2M signalling receptor and LRP/alpha 2MR are distinct entities. Growth factors which induce tyrosine phosphorylation typically cause a rise in cytosolic pH. Binding of a2M-methylamine to macrophages also gradually increased the intracellular pH in a concentration-dependent manner, being optimal at a 200 nM ligand concentration. The increase in pH was amiloride sensitive. We propose that receptor-recognized forms of a2M may function like growth factors with regard to macrophage regulation.
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PMID:Ligation of the alpha 2-macroglobulin signalling receptor on macrophages induces protein phosphorylation and an increase in cytosolic pH. 754 45

We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating beta-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C-->diacylglycerol-->protein kinase C.
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PMID:Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets. 755 10

Although it is suggested that in the renal proximal tubules, dopamine D1 receptor activation causes inhibition of Na+/K+ATPase via a phospholipase C and protein kinase C coupled pathway, the direct stimulation of protein kinase C by dopamine has not been reported. The present study was designed to examine the effects of dopamine and selective dopamine D1 receptor and dopamine D2 receptor agonists on protein kinase C activity. The renal proximal tubule suspensions were obtained from male Sprague-Dawley rats. The tubules were incubated separately with dopamine and fenoldopam in the presence or absence of dopamine D1 receptor antagonist, SCH 23390 ([(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepine]). The protein kinase C activity was measured by using a kinase target peptide, conjugated to a fluorescent molecule in water. The amino acid sequence of this peptide is, Proline-Leucine-Serine-Arginine-Threonine-Leucine-Serine-Valine-Alanine- Alanine-Lysine(PKSRTLSVAAK). We found that dopamine and fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-di ol] produced concentration-dependent increases in protein kinase C activity, which was blocked by SCH 23390. However, the dopamine D2 receptor agonist, bromocriptine [(5' alpha)-2-bromo-12'-hydroxy-2'-(1-methyl-ethyl)-5'-(2-methylpropyl)erg o- taman-3',6',18-trione] failed to stimulate protein kinase C activity at all the concentrations tested. These results provide direct evidence that dopamine stimulates protein kinase C activity via activation of dopamine D1 receptors.
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PMID:Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D1 receptors. 762 15


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