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)

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.
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PMID:A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. 131 84

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
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PMID:Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. 131 77

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.
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PMID:EBV increases phosphoinositide kinase activities in human B cells. 131 98

Phospholipase D (PLD) has been implicated in signal transduction and membrane traffic. We have previously shown that phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5-P2 as a PLD cofactor. In the present study we extend these observations to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) is greatly potentiated by MgATP. We have utilized this experimental system to test the hypothesis that MgATP potentiates PLD activation by G proteins because it is required for PtdIns-4,5-P2 synthesis by phosphoinositide kinases. As expected, MgATP was absolutely required for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P2 levels in the permeabilized cells. In the presence of MgATP, GTP gamma S further elevated the levels of the phosphoinositides. The importance of PtdIns-4,5-P2 for PLD activation was examined by utilizing a specific inhibitory antibody directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the synthesis of PtdIns-4,5-P2. Anti-PtdIns 4-kinase completely inhibited PtdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P2 levels in the permeabilized cells. In parallel, the anti-PtdIns 4-kinase fully inhibited the activation of PLD by GTP gamma S and caused a 60% inhibition of PLD activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, indicating that elevated PtdIns-4,5-P2 levels are required for PLD activation. This conclusion is supported by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P2, also blocked PLD activation. Furthermore, the activity of PLD in U937 cell lysate was stimulated by PtdIns-4,5-P2 in a dose-dependent manner. The current results indicate that PtdIns-4,5-P2 synthesis is required for PLD activation in permeabilized U937 cells and strongly support the proposed function of PtdIns-4,5-P2 as a cofactor for PLD. In addition, the results further establish PtdIns-4,5-P2 as a key component in the generation of second messengers via multiple pathways including phosphoinositide-phospholipase C, phosphoinositide 3-kinase and PLD.
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PMID:Phosphatidylinositol 4,5-bisphosphate synthesis is required for activation of phospholipase D in U937 cells. 789 Jun 22

Epidermal growth factor (EGF) stimulates phosphatidylinositol PtdIns) hydrolysis in many cell types by effecting the specific interaction between the EGF receptor and phospholipase C gamma. Several studies have suggested that PtdIns 4-kinase activity can also be regulated by EGF, but the mechanism of this stimulation was unclear. We report here that EGF treatment of intact A431 cells increased the association of type II PtdIns kinase with the EGF receptor within 1 min at 37 degrees C. Phosphorylation of immunoprecipitated EGF receptor also increased the association of PtdIns 4-kinase. Furthermore dephosphorylation of phosphoserine residues on the stimulated receptor immune complex led to inactivation of the bound PtdIns 4-kinase, while dephosphorylation of phosphotyrosine residues led to activation. Unlike the stimulated activity measured in total cell and plasma membrane lysates, the changes in activity of the immunoprecipitates were apparent at high substrate concentration. Metabolic labeling was used to show that a 55-kDa phosphoserine and phosphotyrosine-containing protein comigrated with renatured PtdIns 4-kinase activity on SDS-polyacrylamide gel electrophoresis, while in vitro labeling revealed only serine phosphorylation. These data are discussed with reference to the direct regulation of PtdIns 4-kinase by phosphorylation, PtdIns compartmentalization, and the formation of a multienzyme signal transduction complex.
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PMID:Regulation of human type II phosphatidylinositol kinase activity by epidermal growth factor-dependent phosphorylation and receptor association. 798 68

The effects of Ag binding on B cell development and activation are mediated by intracellular signals initiated by the B cell AgR. In this report, we show that the B cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway and the phospholipase C (PLC) pathway. Phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] are produced by PtdIns 3-kinase, an enzyme that appears to be an essential component of tyrosine kinase-mediated signaling. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are likely to function as second messengers in vivo because they can activate the zeta isoform of protein kinase C (PKC) in vitro. We show that cross-linking of the B cell AgR with anti-Ig antibodies caused a five- to sixfold increase in the levels of PtdIns(3,4)P2 in both the mature B cell line BAL 17 and the immature B cell line WEHI-231. PtdIns(3,4)P2 levels increased within 15 s of anti-Ig addition and remained elevated for at least 5 min. AgR cross-linking also caused a slower increase in PtdIns3P levels (approximately 50% over control) and a small, transient increase in PtdIns(3,4,5)P3 levels. Thus, the B cell AgR activates the PtdIns 3-kinase pathway. The other inositol phospholipid signaling pathway involves PLC, which cleaves phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], yielding second messengers that increase intracellular calcium and activate other isoforms of PKC. We analyzed the effects of AgR signaling on PtdIns(4,5)P2 and its precursor, phosphatidylinositol 4-phosphate (PtdIns4P). Consistent with its ability to activate PLC, AgR ligation decreased the levels of PtdIns(4,5)P2. In contrast, AgR cross-linking increased the levels of PtdIns4P. Increased synthesis of PtdIns4P followed by phosphorylation at the D-5 position may prevent depletion of PtdIns(4,5)P2. Thus, signaling by the B cell AgR increases the levels of PtdIns 4-kinase products and PtdIns 3-kinase products. The simplest interpretation of our results is that the B cell AgR activates both PtdIns 3-kinase and PtdIns 4-kinase.
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PMID:Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells. 825 4

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.
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PMID:Differential regulation of muscarinic acetylcholine receptor-sensitive polyphosphoinositide pools and consequences for signaling in human neuroblastoma cells. 947 53

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.
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PMID:Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes. 1040 Jun 87

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) in a signal-terminating reaction. We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P(3) can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P(3), in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P(2). The depletion in cellular PtdIns(4,5)P(2) was confined to a Triton-insoluble cell compartment, enriched in caveolin. In resting cells with elevated Ins(1,4,5)P(3) concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P(2) by 61% in the caveolin-enriched Triton-insoluble compartment. Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolin-enriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P(3) concentrations. Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities. Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P(3) concentrations; however, no reduction in PITP(&agr;) protein expression was detected. These studies indicate that chronic elevation in cellular Ins(1,4,5)P(3) concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolin-enriched agonist-sensitive pool.
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PMID:Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool. 1086 20

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine-xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an *OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of *OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 microM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.
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PMID:Oxidants depress the synthesis of phosphatidylinositol 4,5-bisphosphate in heart sarcolemma. 1105 Oct 96


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