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At least 19 recordsLinked to original sources
PubMed2000

Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGF-stimulation of phospholipase C, phospholipase D, phospholipase A(2), phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells.

Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wild-type fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-(&ggr;). Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A(2) and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A(2) as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A(2) and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation.

Animals↗
PubMed2004

Phospholipase D.

Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. Phospholipase D activities have been detected in simple to complex organisms from viruses and bacteria to yeast, plants, and mammals. Although enzymes with broader selectivity are found in some of the lower organisms, the plant, yeast, and mammalian enzymes are selective for phosphatidylcholine. The two mammalian phospholipase D isoforms are regulated by protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families. Mammalian and yeast phospholipases D are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. This review discusses the identification, characterization, structure, and regulation of phospholipase D. Genetic and pharmacological approaches implicate phospholipase D in a diverse range of cellular processes that include receptor signaling, control of intracellular membrane transport, and reorganization of the actin cytoskeleton. Most ideas about phospholipase D function consider that the phosphatidic acid product is an intracellular lipid messenger. Candidate targets for phospholipase-D-generated phosphatidic acid include phosphatidylinositol 4-phosphate 5-kinases and the raf protein kinase. Phosphatidic acid can also be converted to two other lipid mediators, diacylglycerol and lyso phosphatidic acid. Coordinated activation of these phospholipase-D-dependent pathways likely accounts for the pleitropic roles for these enzymes in many aspects of cell regulation.

Animals↗
PubMed1976

Isolation of human eosinophil phospholipase D.

Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B.

Blood Coagulation Factors↗
PubMed2006

Reconstitution system based on cytosol-depleted cells to study the regulation of phospholipase D.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membrane-associated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes--PLD1 and PLD2--have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5'-[gamma-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.

Animals↗
PubMed2000

Phospholipase D.

Phospholipase D is an ubiquitous enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid and choline. Its cellular actions are related to the production of phosphatidic acid and include alterations to cell growth, shape, and secretion. There are two mammalian phospholipase D genes whose products (PLD1 and PLD2) are alternatively spliced. Both forms have two highly conserved HKD motifs that are essential for catalysis and dimerization. PLD1 is regulated in vitro and in vivo by protein kinase C and small GTPases of the Rho and ARF families, whereas PLD2 shows a higher basal activity with little or no response to these proteins. The cellular locations and specific functions of the two PLD isoforms remain to be established.

Animals↗
PubMed1991

Synthesis of 6-phosphatidyl-L-ascorbic acid by phospholipase D.

Phospholipase D (EC 3.1.4.4) of Streptomyces species was found to catalyze transphosphatidylation to L-ascorbic acid from phosphatidylcholine (PC) in a biphasic reaction system. The product was identified as 1,2-diacyl-sn-glycero-3-phospho-6'-L-ascorbic acid (PA-AsA) by mass spectrometry and nuclear magnetic resonance spectroscopy. The optimal pH of transphosphatidylation was 4.5 and the rate of PA-AsA formation increased as concentrations of L-ascorbic acid increased. The conversion of PC to PA-AsA was greater than 80%. PA-AsA was found to be more resistant to hydrolysis by phospholipase D than was PC.

Ascorbic Acid↗
PubMed1996

Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D.

Phospholipase D of rat brain synaptosomal membranes was tested with phosphatidylcholine as the substrate for its specificity in the use of primary alcohols as transphosphatidylation co-substrates. The efficiency of the reaction was related to the hydrophobicity and the membrane penetrating capacity of the alcohol molecule. Phosphatidylalcohol formation could be detected up to 1-octanol but not for alcohols with longer hydrocarbon chains (C(9), C(10)). With increasing alcohol concentration the transphosphatidylation activity of the phospholipase D reached an optimum and then declined abruptly. Alcohol concentrations required for maximal transphosphatidylation reaction generally decreased with increasing hydrophobicities of the alcohols. Nevertheless 1-butanol and 4-chloro-1-butanol were the most efficient cosubstrates, sharing identical optimal conditions. Transphosphatidylation works at the cost of phosphatidic acid formation. Phosphatidic acid itself was transformed to diacylglycerol, probably by a contaminating phosphatidic acid phosphohydrolase.

Alcohols↗
PubMed1996

Conversion of lysophospholipids to cyclic lysophosphatidic acid by phospholipase D.

Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine in aqueous 1% Triton X-100 solution. In situ monitoring of this reaction by 31P NMR revealed the formation of cyclic lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an intermediate which was hydrolyzed further by the enzyme at a functionally distinct active site to lysophosphatidic acid (lyso-PA). Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was found to be stable in aqueous neutral solutions at room temperature. It was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate which was approximately 4-fold slower than the rate of formation of cyclic lyso-PA. The addition of 5-10 mM sodium vanadate could partially inhibit the ring opening reaction and thus increase substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act as a dormant configuration of the physiologically active lyso-PA or may even possess specific activities which await verification.

Hydrolysis↗
PubMed1991

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

Cells, Cultured↗
PubMed1991

Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain.

Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses.

Animals↗
PubMed1998

[Molecular cloning of Candida albicans phospholipase D].

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine, a major substrate, to phosphatidic acid and choline, and its activity is regulated by a variety of hormones, growth factors, and other extracellular signals in mammalian cells. Thus, it is now recognized as a signal transducing enzyme such as phosphatidylinositol-specific phospholipase C, adenylate cyclase, or protein tyrosine kinases. Furthermore, recent findings that regulation by members of the ADP-ribosylation factor (ARF) and Rho families of monomeric GTP-binding protein suggest roles of PLD in intracellular vesicle traffi-cking, morphological changes, and mitogenic signaling process. In Saccharomyces cerevisiae, PLD gene has been cloned and revealed to be essential for meiosis. In contrast, little is known about PLD in Candida albicans. As a first step to understand possible physiological roles of PLD in C. albicans, we cloned a PLD gene from a C. albicans genomic DNA library. Deduced amino acid sequence analysis showed the structural similarity to mammalian, yeast, and plant PLDs. It was also suggested employing RT-PCR (reverse transcriptase polymerase chain reaction) that an isozyme of C. albicans PLD was present.

Candida albicans↗
PubMed1998

Determination of interaction sites on the small G protein RhoA for phospholipase D.

Phospholipase D (PLD) has been identified as a target of small G proteins of the Rho family. The present study was directed at defining the interaction sites of RhoA with rat brain PLD in vitro using chimeric proteins between RhoA and Ha-Ras or Cdc42Hs and point mutations. The switch I region of RhoA, which is the common effector domain of Ras-like G proteins, was a crucial interaction site for PLD. Mutations in conserved amino acids (Tyr34, Thr37, Phe39) totally abolished PLD activation, while mutations in Val38 or Tyr42 caused partial loss. Two additional sites were responsible for the differential PLD activation ability between RhoA and Cdc42Hs. Changing Asp76 in the switch II region of RhoA to the corresponding amino acid in Cdc42Hs led to partial loss of PLD activation. A chimeric protein with the N-terminal third of Cdc42Hs changed to RhoA showed enhanced PLD activation. Analysis of other Rho/Ha-Ras chimeric proteins and mutations indicated that Gln52 adjacent to the switch II region is responsible for this gain of function. In conclusion, the present study shows that conserved amino acids in the switch I region of RhoA are major PLD interaction sites and that residues in the switch II and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.

Amino Acid Sequence↗
PubMed2004

Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D.

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.

Animals↗
PubMed1998

Identification of lipid inhibitor of mammalian phospholipase D.

Phospholipase D (PLD) is implicated in important cellular processes, such as hormone action, inflammation, secretion, mitogenesis, and neural activity. Recent studies using cell-free systems have shown that the enzyme activity is modulated by both positive and negative regulators. During an attempt to purify PLD from pig colon mucosa, we noted the presence of a PLD inhibitor in the tissue extract. The inhibitor was purified and identified as comprising lysophosphatidylserine, phosphatidylinositol, and lysophosphatidylinositol, of which lysophosphatidylserine was the most potent. These lipids affected all of the PLD isoforms examined, oleate-dependent PLD, ARF-dependent PLD (PLD1a, PLD1b), and phosphatidylinositol 4,5-bisphosphate-dependent PLD (PLD2), in the concentration range of the 1 or 10 microM order. In contrast to lysophosphatidylserine, the diacyl counterpart phosphatidylserine was without effect in the same concentration range. PLD inhibition by lysophosphatidylserine could not be reversed by an increase in the concentration of the substrate phosphatidylcholine or activator phosphatidylinositol 4,5-bisphosphate.

Animals↗
PubMed1999

Regulation of phospholipase D.

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H(2)O(2) treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.

ADP-Ribosylation Factors↗
PubMed1996

Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D.

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report that the use of didecanoyl phosphatidylcholine (C10-PC) in mammalian PLD assays considerably increases the detection limit. C10-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C16-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from placental cytosol. C10-PC was superior to C16-PC by a factor of 2-28 depending on assay conditions and tissue, and it allowed the detection of GTP[S]-and Arf-stimulated PLD activity without addition of phosphatidylinositol 4,5-bisphosphate.

ADP-Ribosylation Factors↗
PubMed2001

[Biochemical and molecular biology aspects of plant phospholipase D].

Phospholipase D (PLD) was first discovered in plants. It plays an important role in the regulation of cell functions not only in plants, but also in animal systems. It is interesting from the aspect of its dominant position in the signalling transduction processes. It controls the utilization of the membrane phospholipids for specific intentions--products of its catalytic activity are involved in intracellular communication processes. The study of the cell-regulation-system shows that PLD is a part of lipid-based signalling via octadecanoid pathway, which leads to the production of jasmonic acid, the basic signalling molecule in plants. PLD-mediated hydrolysis of the membrane phospholipids posed this enzyme into the level of the transmembrane and cell signalling participant. This area is interesting in the aspect of the production of biologically active compounds in plants. Because the signal-regulated synthesis of some secondary metabolites by the lipid signalling pathway has been already shown (induction of the gene expression coding the biosynthetic enzymes of flavonoids and terpenoids), the area of PLD study is perspective. This article is orientated on plant PLD and gives a basic review of its biochemical and molecular-biological properties (enzymology, molecular structure, subcellular localization, isoenzymes) and its physiological functions on the cell level (lipid signalling pathway, phospholipid degradation, phytohormones and cell signalling, growth, development and cell ageing, membrane remodeling).

Animals↗
PubMed2002

Cloning, overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D.

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.

Amino Acid Sequence↗