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)

In rabbit aortic vascular smooth muscle cells (VSMC) platelet-derived growth factor BB (PDGF-BB) stimulated the tyrosine phosphorylation of phospholipase C-gamma, p120 GTPase-activating protein, and the p85 alpha subunit of phosphatidylinositol 3'-kinase only at high concentrations (5-25 ng/ml). In contrast, PDGF-BB induced a rapid and concentration-dependent increase in p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, which was half-maximal and maximum at 1 and 2.5 ng/ml, respectively. Saliently, stimulation of p125FAK tyrosine phosphorylation was sustained at up to 100 ng/ml PDGF-BB and for prolonged times of treatment. With similar concentration dependence, PDGF-BB stimulated the tyrosine phosphorylation of the 68-kDa focal adhesion-associated protein, paxillin. PDGF-BB also induced p125FAK and paxillin tyrosine phosphorylation in human aortic VSMC. PDGF-BB caused no detectable disruption of the actin cytoskeleton in VSMC. PDGF-BB stimulated rabbit VSMC migration with a very similar concentration dependence to that for p125FAK and paxillin tyrosine phosphorylation. PDGF-BB was equally effective in stimulating p125FAK and paxillin tyrosine phosphorylation under conditions similar to those used for cell migration. In Swiss 3T3 fibroblasts, PDGF-BB and -AA stimulated p125FAK tyrosine phosphorylation and cell migration only at low concentrations, and stimulation was abolished at 10-25 ng/ml. PDGF-AA failed to stimulate tyrosine phosphorylation, mitogenesis, and chemotaxis in rabbit VSMC, and immunoblot analysis showed that rabbit VSMC expressed PDGF beta-receptors but no alpha-receptors. These results implicate p125FAK in the chemotactic response to PDGF-BB and suggest that the ability of PDGF-BB to trigger the p125FAK pathway may be dependent both upon cell type and receptor isotype expression.
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PMID:Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts. 753 14

Previously, we characterized the prostaglandin (PG) F2 alpha receptor linked to phospholipase C activation and DNA synthesis in NIH-3T3 cells (Nakao, A., Watanabe, T., Taniguchi, S., Nakamura, M., Honda, Z-I., Shimizu, T., and Kurokawa, K. (1993) J. Cell. Physiol. 155, 257-264). To elucidate intracellular events evoked via this receptor, we examined changes caused by PGF2 alpha stimulation in the phosphotyrosine composition of cellular proteins. The addition of PGF2 alpha to cells in quiescent culture rapidly increased the levels of phosphotyrosine in cellular proteins with Mr values of 70,000 (pp70), 85,000 (pp85), 92,000 (pp92), 100,000 (pp100), and 125,000 (pp125); the latter was immunologically identified as p125 focal adhesion kinase. The PGF2 alpha-induced changes in the level of intracellular Ca2+ ([Ca2+]i) elevation, formation of inositol phosphates, and [3H]thymidine incorporation followed a similar dose dependence as PGF2 alpha-induced tyrosine phosphorylation. This tyrosine phosphorylation was independent of extracellular Ca2+, while a [Ca2+]i chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (50 microM), completely inhibited the PGF2 alpha-induced elevation of [Ca2+]i, tyrosine phosphorylation, and [3H]thymidine incorporation. Ionomycin (0.1 microM), which induced [Ca2+]i elevation without formation of inositol phosphates, mimicked the PGF2 alpha-induced tyrosine phosphorylation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) also induced [3H]thymidine incorporation in a dose-dependent manner but had no significant effect on tyrosine phosphorylation. The PGF2 alpha-induced tyrosine phosphorylation could be observed even in the cells pretreated with TPA (5 microM, 24 h). PGF2 alpha exhibited an additive effect on TPA-induced [3H]thymidine incorporation but had no effect on the 32P-phosphorylation of a known 80-kDa protein kinase (PK) C substrate. Both staurosporine and H-7 inhibited the PGF2 alpha-induced increase in [3H]thymidine incorporation and tyrosine phosphorylation in a similar dose-dependent manner whether or not cells were pretreated with TPA (5 microM, 24 h). However, W-7 and KN-62 had no effect on these cellular responses even at the concentration for the almost complete inhibition of Ca2+/calmodulin-dependent PKs (20 microM). These results, taken together, indicate that PGF2 alpha receptor-mediated tyrosine phosphorylation is evoked by a [Ca2+]i-dependent mechanism that is sensitive to staurosporine and H-7 but which is independent of PKC or Ca2+/calmodulin PKs. Finally, the data suggest that this PGF2 alpha-induced signaling pathway is linked to the proliferation of cells.
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PMID:Prostaglandin F2 alpha enhances tyrosine phosphorylation and DNA synthesis through phospholipase C-coupled receptor via Ca(2+)-dependent intracellular pathway in NIH-3T3 cells. 802 Dec 71

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.
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PMID:Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. 866 84

The relationship between receptor number and agonist-induced intracellular responses has been well studied in receptors coupled to adenylate cyclase; however, for receptors coupled to phospholipase C (PLC), very little is known about the effect of receptor number on receptor-mediated processes. To explore this issue, we investigated the effect of the number of receptors for gastrin-releasing peptide (GRP) on ligand affinity and on the ability to activate intracellular messengers [PLC, tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK)] and cause receptor modulation (internalization, desensitization, down-regulation) and ligand degradation. Three BALB 3T3 cell lines were made that stably expressed the gastrin-releasing peptide receptor (GRP-R) with receptor numbers varying by 280-fold (GRP-R-Low, GRP-R-Med, and GRP-R-Hi). Each cell line had the same affinity for agonist. The efficacy for bombesin to increase [3H]inositol phosphates but not tyrosine phosphorylation of p125FAK correlated well with receptor number. In contrast, the EC50 value for [3H]inositol phosphate generation for bombesin was the same in each cell line. Receptor number did not alter internalization. In the absence of protease inhibitors, there was an inverse correlation between receptor number and receptor down-regulation and desensitization. However, with protease inhibitors present, GRP-R-Med and GRP-R-Hi down-regulated significantly less than the GRP-R-Low. Similarly, GRP-R-Low desensitized significantly more than GRP-R-Med or GRP-R-Hi. GRP-R-Hi caused significantly greater ligand degradation than GRP-R-Low, and protease inhibitors completely inhibited degradation by GRP-R-Low and inhibited degradation by 70% for GRP-R-Hi. In conclusion, we show that for the PLC-coupled GRP-R, receptor number had little or no effect on binding affinity, potency for activating PLC, tyrosine phosphorylation of p125FAK, or extent of receptor internalization. In contrast, receptor number had an effect on ligand degradation, down-regulation, desensitization, and efficacy of PLC activation without altering the efficacy of tyrosine phosphorylation of p125FAK. These results demonstrate that the effect of receptor number differs for the different functions mediated by the GRP receptor and differs from that reported for adenylate cyclase-coupled receptors such as receptors mediating the action of adrenergic agents, secretin, and opioids.
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PMID:Effect of gastrin-releasing peptide receptor number on receptor affinity, coupling, degradation, and modulation. 914 10

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.
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PMID:Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. 918 76

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125(FAK)) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21(rho) activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125(FAK) and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125(FAK) and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125(FAK) and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21(rho), caused significant inhibition of CCK-8-stimulated p125(FAK) and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125(FAK) and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21(rho).
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PMID:Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho. 935 17

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4-9-fold increase in [3H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP was concentration-dependent, with an EC50 of 20-35 nM in both cell lines. [D-Phe6,beta-Ala11,Phe13,Nle14]Bn-(6-14) stimulated a 2-3-fold increase in [Ca2+]i, a 3-fold increase in tyrosine phosphorylation of p125(FAK) with an EC50 of 0.2-0.7 nM, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [D-Arg1,D-Trp7,9, Leu11]substance P and [D-Pro4,D-Trp7,9,10]substance P-(4-11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a competitive fashion (Ki = 0.5 microM). Stimulation of p125(FAK) tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+]i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125(FAK), and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [D-Phe6,beta-Ala11, Phe13,Nle14]Bn-(6-14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 microM, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.
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PMID:Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. 959 99

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.
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PMID:Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells. 1053 23

We investigated the relationship between protein tyrosine phosphorylation and bradykinin (BK) receptor activation in rat mesangial cells (MC). Stimulation of the B2 receptor resulted in a dual effect consisting of an independent activation and inhibition of tyrosine kinase activity (TKA). The activation was rapid and transient, reaching a peak value at 30 s whereas the inhibition was observed at 5 min and persisted up to 10 min. Treatments with pertussis-toxin and U73122 showed that only the BK-induced stimulation of TKA is dependent on phospholipase C activation via a pertussis-toxin sensitive G-protein. In addition, BK induced an increase in tyrosine phosphatase activity. Western-blot analysis demonstrated that the dual effect of BK on TKA was associated with both an increase and a decrease in tyrosine phosphorylation of the p125-focal adhesion kinase (FAK). Moreover, BK was able to reduce the maximal stimulated MC cell proliferation induced by fetal calf serum. These data show that in rat MC, B2 receptor stimulation activates and inhibits two independent tyrosine kinase signaling pathways associated with tyrosine phosphorylation of p125-FAK that might be implicated in MC proliferation.
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PMID:Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells. 1061 90

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.
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PMID:Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway. 1065 10


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