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 effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC50 values of approximately 45 microM and approximately 1 mM, respectively. Because the IC50 for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP2), these data suggest that negatively charged lipids are required for endosome fusion. A role for negatively charged lipids in membrane traffic has been postulated to involve the activity of a PIP2-dependent phospholipase D (PLD) stimulated by the GTP-binding protein ADP-ribosylation factor (ARF). Although neomycin blocks endosome fusion at a stage of the in vitro reaction that is temporally related to steps inhibited by cytosolic ARFs when they bind guanosine-5'-gamma-thiophosphate (GTPgammaS), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP2-independent PLD does not suppress neomycin inhibition of endosome fusion; moreover, in vitro fusion activity is not affected by the pleckstrin homology domain of phosphoinositide-specific phospholipase C delta1, which binds to acidic phospholipids, particularly PIP2, with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTPgammaS-bound ARF, these effects are unrelated to inhibition of the PIP2-dependent PLD activity stimulated by this GTP-binding protein. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway.
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PMID:Inhibition of in vitro endosomal vesicle fusion activity by aminoglycoside antibiotics. 973 96

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)delta PH domain-green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCdelta PH domain known to form critical contacts with PtdIns(4, 5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol- labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.
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PMID:Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. 978 58

The localization of phospholipase C delta3 (PLC delta3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC delta3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC delta3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC delta3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC delta1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC delta1 is activated by spermine and sphingosine, whereas PLC delta3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta1. A deletion mutant of PLC delta3, lacking the entire pleckstrin homology (PH) domain (residues 1-137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC delta3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC delta3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC delta1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC delta3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC delta3 described in this report suggest that PLC delta3 has a relatively low activity in cellular conditions that fully activate PLC delta1.
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PMID:Localization of phospholipase C delta3 in the cell and regulation of its activity by phospholipids and calcium. 979 16

1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.
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PMID:A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C. 987 7

Angiotensin II (Ang II) receptors of the AT1 subtype are coupled to heterotrimeric G nucleotide-binding proteins, G(q/11), to activate phospholipase C-beta isoforms with production of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. The resultant release of intracellular Ca2+ and increased Ca2+ influx are major determinants of several acute cellular responses initiated by Ang II, including secretion of aldosterone from the adrenal cortex and smooth muscle contraction. However, cellular events related to more prolonged effects of Ang II, such as hypertrophic and hyperplastic responses, are triggered by intracellular signaling cascades that are less dependent on Ca2+ signals. The Ang II-induced activation of Raf-1 kinase, p42 MAP-kinase and c-fos expression in response to Ang II in adrenal glomerulosa cells does not require Ca2+ influx. Moreover, the dose-response relationships for Raf-1 activation, MAP-kinase activation and mitogenesis show significantly higher sensitivity to Ang II than the InsP3, Ca2+-release and aldosterone secretory responses. The sensitivities of both Raf-1 kinase and MAP-kinase stimulation by Ang II to the inhibitors of phosphoinositide kinases, wortmannin and LY 294002, suggest that inositol phospholipids may play a role in these activation events unrelated to their role in Ca2+ signaling. To investigate the changes of various inositides after stimulation at the single cell level, fluorescent probes were developed in which pleckstrin homology domains with distinct binding specificities to inositol phospholipids were fused to the green fluorescent protein and expressed in NIH 3T3 cells. The use of these probes revealed heterogeneity of the inositol lipid pools and their complex relationship to Ca2+ signals. The use of these tools will help to further clarify the complex role of these lipids in initiating Ca2+-dependent and -independent signaling responses.
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PMID:Signaling events activated by angiotensin II receptors: what goes before and after the calcium signals. 988 5

It has been reported that there are two alternatively spliced variants of phospholipase C-delta4 (PLCdelta4), termed ALT I and II, that contain an additional 32 and 14 amino acids in their respective sequences in the linker region between the catalytic X and Y domains (Lee, S. B., and Rhee, S. G. (1996) J. Biol. Chem. 271, 25-31). We report here the isolation and characterization of a novel alternative splicing isoform of PLCdelta4, termed ALT III, as a negative regulator of PLC. In ALT III, alternative splicing occurred in the catalytic X domain, i.e. 63 amino acids (residues 424-486) containing the C-terminal of the X domain and linker region were substituted for 32 amino acids corresponding to the insert sequence of ALT I. Although the expression level of ALT III was found to be much lower in most tissues and cells compared with that of PLCdelta4, it was significantly higher in some neural cells, such as NIE-115 cells and p19 cells differentiated to neural cells by retinoic acid. Interestingly, recombinant ALT III protein did not retain enzymatic activity, and the activity of PLCdelta4 overexpressed in COS7 cells was markedly decreased by the co-expression of ALT III but not by ALT I or II. Moreover, N-terminal pleckstrin homology domain (PH domain) of ALT III alone could inhibit the increase of inositol-1,4, 5-trisphosphate levels in PLCdelta4-overexpressing NIH3T3 cells, whereas a PH domain deletion mutant could not, indicating that the PH domain is necessary and sufficient for its inhibitory effect. The ALT III PH domain specifically bound to phosphatidylinositol (PtdIns)-4,5-P2 and PtdIns-3,4,5-P3 but not PtdIns, PtdIns-4-P, or inositol phosphates, and the mutant R36G, which retained only weak affinity for PtdIns-4,5-P2, could not inhibit the activity of PLCdelta4. These results indicate that PtdIns-4,5-P2 binding to PH domain is essential for the inhibitory effect of ALT III. ALT III also inhibited PLCdelta1 activity and partially suppressed PLCgamma1 activity, but not PLCbeta1 in vitro; it did inhibit all types of isozymes tested in vivo. Taken together, our results indicate that ALT III is a negative regulator of PLC that is most effective against the PLC delta-type isozymes, and its PH domain is essential for its function.
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PMID:A novel phospholipase C delta4 (PLCdelta4) splice variant as a negative regulator of PLC. 991 23

Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.
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PMID:Real-time visualization of PH domain-dependent translocation of phospholipase C-delta1 in renal epithelial cells (MDCK): response to hypo-osmotic stress. 991 30

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.
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PMID:Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth. 1020 27

Activation of phospholipase C (PLC) is a central component of the signal transduction process in numerous cells, including platelets. U73122 has been widely used as a selective PLC inhibitor. In the present study, the effects of U73122 on platelet function have been further examined. Platelets were stimulated with collagen (via PLC-gamma), the stable thromboxane mimetic U46619 (via PLC-beta), or phorbol myristate acetate (PMA) via protein kinase C (PKC). Consistent with inhibition of PLC, U73122 inhibited platelet aggregation and [3H]-serotonin release in response to collagen and U46619 in a concentration-dependent manner. Similarly, U73122 blocked collagen-induced release of thromboxane A2. U73122 also inhibited U46619-induced [32P]phosphatidic acid production and phosphorylation of the major PKC substrate, pleckstrin. U73122 had no effect on PMA-induced pleckstrin phosphorylation, [3H]-serotonin release, or intracellular vacuole formation. However, U73122 did inhibit PMA-induced platelet aggregation and fibrinogen binding. Overall, these results suggest that U73122, in addition to its inhibition of PLC, also affects PKC-independent events that interfere with platelet aggregation.
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PMID:The phospholipase C inhibitor U73122 inhibits phorbol ester-induced platelet activation. 1021 45

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.
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PMID:Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk. 1022 28


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