Phospholipase D Enzymes Research Articles

The C Terminus of Mammalian Phospholipase D Is Required for Catalytic Activity

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.

Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c.

Calphostin-c inhibits protein kinase C (PKC) isoenzymes by covalent modification of the lipid binding regulatory domain. Exposure of cells to calphostin-c elicits PKC independent effects including disruption of intracellular transport, growth inhibition, and stimulation of apoptosis suggesting actions at additional targets. Phospholipase D (PLD) enzymes are targets for activation by PKC. We have investigated the PKC isoenzyme selectivity for activation of two mammalian PLD enzymes, PLD1 and PLD2, by PKC. We examined the sensitivity of this process to widely used PKC inhibitors and report the surprising finding that calphostin-c is a potent direct inhibitor of PLD1 and PLD2. In vitro, calphostin-c inhibits activity of both PLD1 and PLD2 with an IC(50) of approximately 100 nM. Inhibition is not overcome by protein and lipid activators of these enzymes and does not involve blockade of phosphatidylinositol 4,5-bisphosphate-dependent PLD binding to substrate containing liposomes. Studies using a series of deletion and point mutants of the enzymes suggest that calphostin-c targets the PLD catalytic domain. Inhibition of PLD by calphostin-c in vitro involves stable and apparently irreversible modification of the enzyme. Activity of both PLD1 and PLD2 can be inhibited by calphostin-c treatment of intact cells in a manner that is independent of upstream actions of PKC. Our results suggest that inhibition of PLD1 and PLD2 may explain some of the PKC-independent effects of calphostin-c observed when the compound is applied to intact cells.

Influence of phosphatidylinositol 4,5-bisphosphate on human phospholipase D1 wild-type and deletion mutants: is there evidence for an interaction of phosphatidylinositol 4,5-bisphosphate with the putative Pleckstrin homology domain?

Phosphatidylinositol 4,5-bisphosphate (PIP 2) is an essential cofactor of phospholipase D (PLD) enzymes. In order to further characterize its role in PLD activation, we have constructed N-terminal deletion mutants of the human PLD1 (hPLD1) and a mutant lacking the putative pleckstrin homology domain (ΔPH), which has been proposed to be involved in PIP 2 binding. For the N-terminal deletion mutants (up to 303 amino acids) and the ΔPH mutant we found no significant differences compared to the hPLD1 wild-type, except changes in the specific activities: the K m values were about 20 μM for the substrate phosphatidylcholine, and PIP 2 activated the PLD enzymes maximally between 5 and 10 μM. In contrast, preincubation of the PLD proteins with 5–10 μM PIP 2 or PIP 2-containing lipid vesicles inhibited the PLD activity. This inhibition was neither abolished by n-octyl-β- D-glucopyranoside or neomycin nor by the ADP-ribosylation factor, another activator of PLD enzymes. All tested PLD proteins were active without PIP 2 in the presence of 1 M ammonium sulfate. The 303 N-terminal amino acids of hPLD1 are not involved in substrate binding or the interaction with PIP 2. Our data indicate further that the putative PH domain of hPLD1 is not responsible for the essential effects of PIP 2 on PLD activity.

Protein Kinase C-Stimulated Formation of Ethanolamine from Phosphatidylethanolamine Involves a Protein Phosphorylation Mechanism: Negative Regulation by p21 Ras Protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-α (PKC-α)-activated, presently unknown, mechanism. Now we report that addition of a PKC-α-enriched purified PKC preparation or recombinant PKC-α to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly (∼6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-α, ATP, and PMA on [14C]PtdEtn hydrolysis were inhibited by GF 109203X (10 μM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller (∼3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-α is inhibited by p21 Ras.

385 Honey Dew Fruit Senescence is Regulated by Polyamine Inhibition of Lipid Peroxidation

Polyamines are effective scavengers of activated oxygen free radicals produced by lipoxygenase (LOX) and phospholipase-D (PL-D). Activated oxygen free radicals cause peroxidative damage to membranes and hasten senescence. Exogenous polyamine spermidine (SPD) compared to spermine (SPM) at 1 mM or no polyamine was an effective inhibitor of honey dew (Cucumis melo L. var. inodorus) membrane peroxidation, as determined by malondialdehyde (MDA), following dark incubation for 6 or 48 hours of fully abscised fruit hypodermal mesocarp tissue. MDA levels in SPD-treated tissue was lowest in both 6 and 48 hours compared to SPM or no polyamine. SPD was effective in slowing lipid peroxidation as MDA was highly negatively correlated with the loss in total chlorophyll, plasma membrane H+ pumping ATPase activity, and microsomal phospholipid content (r = -0.89, -0.64 and -0.57, respectively). Both LOX and PL-D enzyme activities were not correlated with the total chlorophyll and microsomal membrane phospholipid losses or MDA levels, demonstrating that these enzymes act indirectly in the degradation of membranes through the production of lipid peroxidating free radicals. The results also demonstrate that the effect of polyamines as anti-senescence compounds is through direct inhibition of lipid peroxidation and not by affecting LOX or PL-D free radicle production.

Expression, Characterization, and Mutagenesis of theYersinia pestis Murine Toxin, a Phospholipase D Superfamily Member

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX4DX6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10(4) and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus

Two phospholipase D (PLD) enzymes with both hydrolase and transferase activities were isolated from Streptomyces chromofuscus. There were substantial differences in the kinetic properties of the two PLD enzymes towards monomeric, micellar, and vesicle substrates. The most striking difference was that the higher molecular weight enzyme (PLD 57 ∼57 kDa) could be activated allosterically with a low mole fraction of phosphatidic acid (PA) incorporated into a PC bilayer (Geng et al., J. Biol. Chem. 273 (1998) 12195–12202). PLD 42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4–6-fold higher V max toward PC substrates than PLD 57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD 42/20 were cleavage products of PLD 57. The larger component included the N-terminal segment of PLD 57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD 57; this peptide had no PLD activity by itself. Increasing the pH of PLD 42/20 to 8.9, followed by chromatography of PLD 42/20 on a HiTrap Q column at pH 8.5 separated the 42- and 20-kDa proteins. The 42-kDa complex had about the same specific activity with or without the 20-kDa fragment. The lack of PA activation for the 42-kDa protein and for PLD 42/20 indicates that an intact C-terminal region of PLD 57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD 57.

Expression of a Novel Murine Phospholipase D Homolog Coincides with Late Neuronal Development in the Forebrain

Members of the phospholipase D (PLD) superfamily are defined by the conserved HXKXXXXD motif, which is essential for the catalytic function of mammalian PLD. PLD enzymes are thought to play roles in signal transduction and membrane vesicular trafficking in mammalian cells. Here we describe a 54-kDa novel murine polypeptide (designated SAM-9) that is predicted to be a membrane-associated member of the PLD superfamily. SAM-9 shares 40, 30, and 29% amino acid identity with potential orthologs, in vaccinia virus, Caenorhabditis elegans, and Dictyostelium discoideum, respectively, and belongs to a subclass of PLD homologs in which the second HXKXXXXD motif is imperfect and harbors a conserved Asp to Glu substitution. The sam-9 gene has more than eight exons, and the two HXKXXXXD motifs are encoded by two highly conserved exons. The expression of the sam-9 gene is greater in the brain than in non-nervous tissue and appears to be predominantly of neuronal origin. sam-9 expression is pronounced in mature neurons of the forebrain and appears to be turned on at late stages of neurogenesis as revealed by in situ hybridization analysis of sam-9 expression during postnatal development of the hippocampal formation and the primary somatosensory cortex.

Reconstitution system based on cytosol-depleted cells to study the regulation of phospholipases C and 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.

Human Phospholipase D1 Can Be Tyrosine-phosphorylated in HL-60 Granulocytes

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.

Changes in the activity and mRNA levels of phospholipase D during ceramide-induced apoptosis in rat C6 glial cells.

N-Acetylsphingosine (C2-ceramide), a membrane-permeable analogue, induced apoptosis in C6 glial cells. Phase-contrast micrographs showed that the round cells appeared 3 h after exposure to 25 microM C2-ceramide and the number of floating cells increased time-dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C2-ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPgammaS-dependent PLD activity was down-regulated after ceramide treatment in a time-dependent manner. In the in vitro PLD assay, membrane-associated PLD activation in response to recombinant ADP-ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down-regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription-polymerase chain reaction. Decreases in GTPgammaS-dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.

Enzymatic Spectrophotometric Determination of Glycerol-3-Phosphate and Glycerophosphorylcholine.

ABSTRACT A reliable enzymatic procedure has been devised for the determination of glycerol-3-phosphate and glycero-phosphorylcholine based on spectrophotometry. Glycerol-3-phosphate oxidase and phospholipase-D enzymes are used to produce hydrogen peroxide to oxidize iodide into triiodide at an elevated pH in the presence of Mo(VI), and the absorbance of the triiodide was read at 352 nm.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.