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)

The functional significance of phospholipase D (PLD) could most easily be investigated using selective inhibitors. We have isolated a family of fungal metabolites, ketoepoxides, that inhibit chemotactic peptide (formyl-Met-Leu-Phe)-stimulated PLD activation and superoxide generation in granulocytes in the low micromolar range (SCH 49210 having an IC50 of 1.6 microM). Unlike receptor-mediated PLD activation, ketoepoxides were poor inhibitors of phorbol ester-induced PLD activity in granulocytes (IC50 = 43 microM for SCH 49210). Ketoepoxides did not inhibit platelet-derived growth factor-stimulated PLD activity in fibroblasts at up to 50 microM. We also tested the effect of ketoepoxides on in vitro epidermal growth factor receptor and neu tyrosine kinase activities. SCH 49210 (and 49209) did not inhibit the tyrosine kinases at up to 100 microM. These results suggest that ketoepoxides do not inhibit PLD activation due to effects on tyrosine kinase activity. fMLP-induced phospholipase A2 (PLA2) activation is also inhibited by ketoepoxides in the low micromolar range (SCH 49210 having an IC50 of 3.2 microM), but the ketoepoxides were poorer inhibitors of Ca2+ ionophore A23187-induced PLA2 (SCH 49210 having an IC50 of 83 microM). As a measure of phospholipase C (PLC) activity, the generation of inositol-1,4,5 triphosphate in thrombin-stimulated platelets was measured. The ketoepoxides did not inhibit PLC activation indicating that, unlike the aminosteroid U73122, ketoepoxides exhibit some selectivity among receptor-linked phospholipases. The ketoepoxides were also effective inhibitors of tumor cell invasion, as measured by penetration of HT1080 human fibrosarcoma cells into a reconstituted basement membrane matrix. Interestingly, both PLD inhibition and anti-tumor invasion activity correlate closely. These ketoepoxides are, therefore, potential anti-metastatic compounds and may be useful probes to study the role of PLD in cell function.
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PMID:Novel ketoepoxides block phospholipase D activation and tumor cell invasion. 791 2

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.
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PMID:Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster. 796 89

In the mouse keratinocyte line HEL-30 the epidermal mitogen transforming growth factor-alpha (TGF-alpha) stimulated the rapid release of arachidonic acid in a dose- and time-dependent manner. The liberation of arachidonic acid was due to the activation of a Ca(2+)-dependent cytosolic phospholipase A2 (cPLA2). The activation mechanism critically depended on a functionally active epidermal growth factor receptor tyrosine kinase and occurred independently of phospholipase C-mediated increases in cellular diacylglycerol and inositol 1,4,5-trisphosphate concentrations and protein kinase C activation. The activation included an increase in cytosolic PLA2 (cPLA2) activity and an association of the enzyme with the membrane fraction. Both activation steps apparently occurred in the presence of basal cytoplasmic Ca2+ concentrations. Moreover, cPLA2 or a closely associated protein was found to be phosphorylated on tyrosine upon TGF-alpha challenge of the cells. The data suggest that tyrosine phosphorylation is involved in the TGF-alpha-induced activation of cPLA2.
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PMID:Activation of cytosolic phospholipase A2 by transforming growth factor-alpha in HEL-30 keratinocytes. 834 57

Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co-overexpression in human 293 fibroblasts using ET-R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF-R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal-generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase-activating protein (GAP) and the non-catalytic subunit of phosphatidylinositol-3'-kinase, p85, in a ligand-dependent manner. Deletion of 15 C-terminal amino acids, including tyrosine 785 (Y-785) abrogated receptor and substrate phosphorylation activities. Mutation of Y-785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET-YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine-phosphorylated synthetic C-terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C-terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y-785 is a major and selective interaction site for PLC gamma.
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PMID:Tyrosine 785 is a major determinant of Trk--substrate interaction. 838 56

The molecular events that lead from the interaction of insulin with its receptor to the activation of protein serine/threonine kinases are still unknown. In this study, we have examined the role of GTP-binding proteins in this signaling pathway using differentiated 3T3-L1 adipocytes permeabilized with alpha-toxin from Staphylococcus aureus. Addition of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) or insulin to such permeabilized cells markedly increases protein kinase activities in cell lysates using the microtubule-associated protein-2 kinase substrate peptide KRELVE-PLTPSGEAPNQALLR, which contains the threonine 669 phosphorylation site on the epidermal growth factor receptor. Similar stimulations of protein kinase activity by these agents are observed using the peptide KRRRLASLAA, which is selectively phosphorylated by ribosomal protein S6 kinases. The effects of insulin and GTP gamma S are not additive. Importantly, the GTP-binding protein antagonist GDP beta S (guanosine 5'-O-(2-thiodiphosphate)) inhibits the activation of the protein kinase activities by insulin in permeabilized 3T3-L1 adipocytes. These data are consistent with the hypothesis that activation of Ras or other GTP-binding proteins is a key element of the signaling mechanism whereby insulin receptor tyrosine kinase activates the microtubule-associated protein-2 kinase cascade.
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PMID:Activation of protein kinases by insulin and non-hydrolyzable GTP analogs in permeabilized 3T3-L1 adipocytes. 838 15

Recombinant human tumor necrosis factor (TNF)-alpha increased the expression of epidermal growth factor receptor (EGFR) mRNA and protein in all of six human pancreatic carcinoma cell lines tested. In addition, TNF-alpha increased the expression of an EGFR ligand, transforming growth factor (TGF)-alpha, at the mRNA and protein level in all cell lines. Increased expression of EGFR protein was associated with elevated steady-state EGFR mRNA levels. Nuclear run-on analysis showed that increase in EGFR mRNA was due to an increased rate of transcription. Induction of EGFR mRNA expression by TNF-alpha was abrogated by cycloheximide but occurred independently of TNF-alpha-induced production of TGF-alpha protein. Protein kinase A or Gi-type guanine nucleotide-binding proteins were not involved in this process as assessed by using appropriate stimulators and inhibitors of these signal transduction pathways. By contrast, staurosporine, an inhibitor of protein kinase C, partially inhibited, and 4-bromophenacyl bromide, a phospholipase inhibitor, completely inhibited TNF-alpha-dependent EGFR mRNA expression. The phospholipase C-specific inhibitor tricyclodecan-9-yl xanthogenate did not alter TNF-alpha-dependent EGFR mRNA expression, suggesting that phospholipase A2 is involved in the modulation of EGFR expression by TNF-alpha. The simultaneous induction of a ligand/receptor system by TNF-alpha suggests that this cytokine modulates autocrine growth-regulatory pathways in pancreatic cancer cells.
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PMID:Tumor necrosis factor alpha induces the expression of transforming growth factor alpha and the epidermal growth factor receptor in human pancreatic cancer cells. 843 98

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family.
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PMID:Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase. 852 23

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLC gamma affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations.
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PMID:Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation. 859 8

Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2-(dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure-activity relationship studies previously developed for the 4-[(3-bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR.
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PMID:Tyrosine kinase inhibitors. 9. Synthesis and evaluation of fused tricyclic quinazoline analogues as ATP site inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor. 863 15

We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.
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PMID:Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism. 881 94


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