Phosphatidylcholine-specific Phospholipase D Research Articles

Abstract 1255: Role of choline in adaptive mechanisms in choline phospholipid metabolism of human breast cancer cells

Abstract Proton magnetic resonance spectroscopy studies of cancer have consistently detected increased cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis [1]. Enzymes in choline metabolism present attractive targets that can be exploited for treatment [2]. Two of these enzymes, choline kinase-α (Chk) and phosphatidylcholine specific phospholipase D (PC-PLD) have been associated with increased malignancy [1, 3]. Chk is a cytosolic enzyme that is responsible for phosphorylation and converting free choline to phosphocholine. Two mammalian isoforms of PC-PLD, PLD1 and PLD2 have currently been identified. While PLD2 is restricted to cytoplasm, PLD1 shuttles between cytoplasm and membrane upon activation by G proteins such as ARF, Rho and Rac [4]. Here we used small interfering RNA (siRNA) against Chk and PLD1 alone or in combination in triple negative MDA-MB-231 human breast cancer cells to silence these two genes. Effective downregulation was confirmed at both protein and mRNA levels. However, downregulation of Chk with siRNA resulted in increased PLD1 expression, and downregulation of PLD1 resulted in increased Chk expression, typifying the ability of cancer cells to adapt. We sought to further understand mechanisms underlying this interdependence by changing the concentrations of free choline in the media. We observed a reduction in the increase of PLD1 protein expression and mRNA levels following Chk downregulation with higher concentration of free choline (107.1µM), suggesting that free choline plays a role in mediating the increase of PLD1 observed following Chk downregulation. Understanding the complex networks of pathways that participate following downregulation of a specific target is critical in a complex disease like cancer. These participating pathways provide novel opportunities to target in concert with the primary target to achieve improved control and minimize compensatory responses that allow cancer cells to survive or adapt. This is especially important if the downregulation of one ‘oncogenic target’, e.g. Chk, results in the upregulation of another, e.g. PLD1. Acknowledgements: This work was supported by NIH P50 CA103175.

Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase Cα and Extracellular Signal-regulated Kinase Signaling Pathways

Recent studies highlight the existence of a nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PC as a source of second messengers and, particularly, nuclear localization of PC-specific phospholipase D (PLD). In the present study we have identified the nuclear localization sequence (NLS) of PLD1 whose mutation abolished its nuclear import. Recently, we reported that caspase-mediated cleavage of PLD1 generates the N-terminal fragment (NF-PLD1) and C-terminal fragment (CF-PLD1). Here we show that CF-PLD1 but not NF-PLD1, is exclusively imported into the nucleus via its functional NLS, whereas only some portions of intact PLD1 were localized into the nucleus. The NLS of intact PLD1 or CF-PLD1 is required for interaction with importin-β, which is known to mediate nuclear import. The amount of intact PLD1 or CF-PLD1 translocated into nucleus is correlated with its binding affinity with importin-β. Ultimately, nuclear localization of intact PLD1 but not CF-PLD1 mediates the activation of nuclear protein kinase Cα and extracellular signal-regulated kinase signaling pathways. Taken together, we propose that nuclear localization of PLD1 via the NLS and its interaction with importin-β may provide new insights on the functional role of nuclear PLD1 signaling.

Abstract 46: Interdependence of choline kinase and phospholipase D in human breast cancer cells

Abstract One hallmark of cancer is an increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis. Enzymes in choline metabolism present attractive targets that can be exploited for treatment. Choline kinase (Chk) is a cytosolic enzyme that catalyzes the phosphorylation of choline (Cho) to phosphocholine (PC) by using ATP in the presence of magnesium. Over-expression of Chk-α has been observed in breast, prostate and lung cancers, making it an obvious choice for targeting. Down-regulation of Chk-α results in a significant reduction of cell proliferation and increased differentiation in highly invasive MDA-MB-231 human breast cancer cells after siRNA-Chk transfection. Another potential target is phosphatidylcholine-specific phospholipase D (PC-PLD). Two mammalian isoforms of PLD, PLD1 and PLD2 have currently been identified. PLD1 is activated by G proteins such as ARF, Rho and Rac. Elevated PLD1 has been reported in gastric, renal, colon and breast carcinoma. We characterized the expression of PLD1 in metastatic MDA-MB-231 cells, poorly metastatic MCF-7 cells, and nonmalignant MCF-12A cells and used siRNA to transiently down-regulate PLD1 and Chk-α in MDA-MB-231 and MCF-7 cells. Forty-eight hours post-transfection cells were harvested for protein and RNA. To determine the effective knock-down of Chk-α or PLD1, about 30µg of protein was resolved on 7.5% acrylamide gel, transferred to nitrocellulose membrane and probed with antibody specific to either Chk-α or PLD1. The effects of Chk-α downregulation on PLD1 expression and the effects of PLD1 downregulation on Chk-α expression were determined by western blots, and the message was quantified with q-RT-PCR. Downregulation of Chk resulted in an increase of PLD1 in MDA-MB-231 cells stably expressing shRNA against Chk or transiently transfected with Chk siRNA. Similar data were obtained for MCF-7 cells. PLD1 levels were highest in the metastatic MDA-MB-231 cells. Effective downregulation of PLD1 was achieved with transient PLD1 siRNA transfection, which resulted in an increase of Chk-α in these cells. These data demonstrate the adaptability of cancer cells, the interdependence of Chk-α and PLD1, and the importance of multiple targeting to eliminate any adaptive compensatory effects that would allow cancer cells to survive. This work was supported by NIH P50 CA103175. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 46.

CCR5 signaling through phospholipase D involves p44/42 MAP‐kinases and promotes HIV‐1 LTR‐directed gene expression

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.

A role of p44/42 mitogen‐activated protein kinases in formylpeptide receptor‐mediated phospholipase D activity and oxidant production

Phosphatidylcholine-specific phospholipase D (PLD) is a major cellular source of phosphatidic acid and choline, which regulate various physiopathological processes. PLD activation mediated by chemoattractants involves protein phosphorylation. This study provides pharmacological and biochemical evidence of a major role of p44/42 MAP kinases (ERK1/2) in PLD activation induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). ERK1/2 inhibition by the MEK1/2 antagonist U0126 in neutrophilic HL-60 cells or HEK 293T cells stably expressing fMLP receptors abolished fMLP-mediated PLD activity. Conversely, a constitutively activated MEK1 mutant expressed in HEK 293T cells potentiated fMLP-induced PLD activity. Expression of inactive PLD mutants showed that PLD2, but not PLD1, contributed to fMLP-mediated PLD activity. PLD2 co-immunoprecipitated with ERK1/2 and became phosphorylated on MAP kinase consensus sites in fMLP-stimulated cells. In cell-free systems, ERK2 gave rise to strong ATP-dependent PLD activity and directly phosphorylated PLD2 that generated two phosphopeptides only after tryptic digestion. Finally, pharmacological inhibition of ERK activation and the inhibition of PLD expression by antisense oligonucleotides in HL-60 cells suggest that the ERK/PLD2 pathway contributes to fMLP-mediated oxidant production. In conclusion, the fMLP-mediated PLD activity is regulated by ERK1/2, involving a predominant contribution of PLD2. The ERK/PLD2 coupling may provide potential pharmacological targets to control PLD-associated cellular dysfunctions.

Uronema marinum: Identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca 2+ and 2.5 mM Mg 2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca 2+ and 2.5 mM Mg 2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.

Interactions of phospholipase D and cytochrome P450 protein stability

Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

Activation of phospholipases by N‐acetylchitooligosaccharide elicitor in suspension‐cultured rice cells mediates reactive oxygen generation

The generation of reactive oxygen species (ROS) is a central component of the elicitor‐induced defence reactions in cultured cells as well as the resistance responses of plants to pathogen challenge. We show that N‐acetylchitooligosaccharide elicitor induces rapid and transient activation of phosphatidylinositol‐specific phospholipase C (PI‐PLC) and that phosphatidylcholine‐specific phospholipase D (PC‐PLD) in suspension‐cultured rice cells and their products, phosphatidic acid (PA) and diacylglycerol (DG), especially the former, play an important role in the elicitor‐induced ROS generation based on the following observations: (1) the amount of PA and DG in rice cells was rapidly increased by the elicitor treatment. (2) Elicitor‐induced activation of PI‐PLC and PC‐PLD in the membrane fraction was confirmed by the analysis of enzymatic products from radio‐labelled phospholipids as well as by 1‐butanol (1‐ButOH)‐specific formation of phosphatidylbutanol (PtdBut) (for PC‐PLD). Inhibitors of these phospholipases at least partly inhibited the elicitor‐induced ROS generation. (3) Exogenously applied PA and DG could induce ROS generation in the rice cells in the absence of the elicitor. (4) PA phosphohydrolase (PAPH) and diacylglycerol kinase (DGK) activities, which catalyse the conversion of PA and DG with each other, are present in the rice cells and the inhibitors of these enzymes inhibited/stimulated the elicitor‐induced ROS generation depending on the direction of the PA accumulation. These results indicate the important role of PI‐PLC/PC‐PLD and their products, especially PA, in the signal transduction cascade downstream of the N‐acetylchitooligosaccharide receptor.

Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 μmol/kg wet weight. Within 3–5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [ 14C] or [ 3H]glycerol and [ 14C] or [ 3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1–2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [ 3H]oleic acid/[ 14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.

D609–phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca 2+ influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na + entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na + concentration (approx. 1–2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na + influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na + influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na + concentration. Both thapsigargin- and DOG-induced Na + increases were not affected in the presence of Na +/H + antiport inhibitor, HOE609, or Na +/Ca 2+ antiport inhibitor, dimethylthiourea, as well as in the presence of Co 2+ and Ni 2+, which block store-operated Ca 2+ entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na + influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I CRANC). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na + entry.

Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C.

Activation of phosphatidylcholine-specific phospholipase D (PLD) constitutes an important part of the cellular response to agonist signaling. PLD1 is stimulated in vitro in a direct and synergistic manner by protein kinase C (PKC), ADP-ribosylation factor (ARF) and Rho family members. However, the direct and specific role of each of these effectors in agonist-stimulated PLD activation is poorly understood. We have used transposon mutagenesis to generate a library of PLD1 alleles containing random pentapeptide insertions. Forty-five alleles were characterized to identify functionally important regions. Use of an allele unresponsive to PKC, but otherwise seemingly normal, to examine coupling of PLD1 to a subset of G-protein-coupled receptors demonstrates for the first time direct stimulation of PLD1 in vivo by PKC and reveals that this direct stimulation is unexpectedly critical for PLD1 activation.

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells.

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1-enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.

Structural Analysis of Human Phospholipase D1

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been proposed to play roles in numerous cellular pathways including signal transduction and membrane vesicular trafficking. We previously reported the cloning of two mammalian genes, PLD1 and PLD2, that encode PLD activities. We additionally reported that PLD1 is activated in a synergistic manner by protein kinase c-alpha (PKC-alpha), ADP-ribosylation factor 1 (ARF1), and Rho family members. We describe here molecular analysis of PLD1 using a combination of domain deletion and mutagenesis. We show that the amino-terminal 325 amino acids are required for PKC-alpha activation of PLD1 but not for activation by ARF1 and RhoA. This region does not contain the sole PKC-alpha interaction site and additionally functions to inhibit basal PLD activity in vivo. Second, a region of sequence unique to PLD1 (as compared with other PLDs) known as the "loop" region had been proposed to serve as an effector regulatory region but is shown here only to mediate inhibition of PLD1. Finally, we show that modification of the amino terminus, but not of the carboxyl terminus, is compatible with PLD enzymatic function and propose a simple model for PLD activation.

Molecular Analysis of Mammalian Phospholipase D2

The mammalian phosphatidylcholine-specific phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play roles in signal transduction and membrane vesicular trafficking in distinct subcellular compartments. PLD1 is activated in a synergistic manner in vitro by protein kinase C-alpha, ADP-ribosylation factor 1 (ARF1), and Rho family members. In contrast, PLD2 is constitutively active in vitro. We describe here molecular analysis of PLD2. We show that the NH2-terminal 308 amino acids are required for PLD2's characteristic high basal activity. Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF proteins and displays a modest preference for activation by ARF5. Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive region is in the PLD carboxyl terminus. We also inserted into PLD2 a region of sequence unique to PLD1 known as the "loop" region, which had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low basal activity characteristic of PLD1. The insertion decreased PLD2 activity, consistent with the latter finding. Finally, we show that the critical role undertaken by the conserved carboxyl terminus is unlikely to involve promoting PLD association with membrane surfaces.

Cloning and expression analysis of murine phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells.

Phosphatase activity regulates superoxide anion generation and intracellular signaling in human neutrophils

Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O 2 −) generation. To investigate the role of dephosphorylation by phosphatases in regulating O 2 − production, human neutrophils were treated with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, prior to stimulation. Calyculin A alone did not stimulate O 2 − production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O 2 − production in comparison to cells stimulated with FMLP alone (28.63±7.00 versus 8.69±3.69 nmol O 2 −/1.5×10 6 neutrophils/5 min, respectively, n=18, p<0.001), with an increased duration of O 2 − production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, ≥16 nM). We next examined the effect of calyculin A on products of the phosphatidylcholine-specific phospholipase D (PLD) pathway by assaying the mass levels of phosphatidic acid (PA), choline and diacylglycerol (DAG). Calyculin A increased both PA and choline production to 224±28% and 315±61% of FMLP-stimulated controls, respectively ( p<0.01, n=7) without significantly increasing DAG. Also, membrane protein kinase C activity increased more than 10-fold in FMLP-stimulated cells exposed to calyculin A but decreased in cells stimulated with PMA following calyculin A pre-treatment. These results suggest that phosphatases exert variable and stimulus-dependent effects on pathways leading to O 2 − production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.

Progesterone triggers the rapid activation of phospholipase D in the amphibian oocyte plasma membrane when initiating the G2/M transition

Previous reports indicate that, in the Rana pipiens oocyte, progesterone triggers a rapid rise in 1,2-diacylglycerol (DAG) derived from phosphatidylcholine (PC) in the plasma membranes. This DAG transient, which appears and is terminated within 60–90 s, is derived both from a phospholipase which we assumed to be phospholipase C and from sphingomyelin (SM) synthase. We now find that progesterone stimulates PC and DAG turnover primarily via the phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) pathways as well as via the SM-ceramide pathway. Rana oocytes were prelabeled with [3H]choline chloride under conditions in which about 70% is incorporated into PC of the plasma membrane of the intact oocyte or with [3H]lysoplatelet activating factor (1-O-octadecyl-sn-glycero-3-phosphocholine, lysoPAF) which is selectively incorporated into plasma membrane PC. Progesterone induced the release of [3H]choline from intact oocytes into the medium within 60–90 s. This choline release was dose-dependent and was not inhibited by a putative PC-specific phospholipase C inhibitor, D609. Progesterone also induced a transient rise in [3H]lysoPAF-derived [3H]DAG within 1–2 min followed by a rise in [3H]PA. In the presence of 20 mM ethanol, progesterone stimulated formation of [3H]lysoPAF-derived phosphatidylethanol, indicating progesterone activation of PC-specific PLD and concomitant formation of PA. A DGK inhibitor (D102) reduced the level of [3H]PA, produced a sustained rise in [3H]DAG and was a weak inducer of meiosis in oocytes not exposed to progesterone. A PA phosphohydrolase inhibitor (propranolol) elevated [3H]PA and completely inhibited the progesterone-induced rise in DAG. Progesterone thus acts at oocyte plasma membrane receptors to release PC-derived DAG via both SM synthase and PC-PLD. The duration of the DAG signal is regulated by the coordinate action of DGK and PAP.

Defective regulation of phosphatidylcholine-specific phospholipases C and D in a kindred with Tangier disease. Evidence for the involvement of phosphatidylcholine breakdown in HDL-mediated cholesterol efflux mechanisms.

The negative correlation between coronary heart disease and plasma levels of HDL has been attributed to the ability of HDL to take up cellular cholesterol. The HDL3-induced removal of cellular cholesterol was reported to be impaired in fibroblasts from patients with familial HDL deficiency (Tangier disease, TD). In addition, we have recently shown that HDL3 stimulates the hydrolysis of phosphatidylcholine (PC) in cholesterol-loaded fibroblasts. To investigate whether this cell signaling pathway is involved in cholesterol efflux mechanisms, we compared the HDL3-induced PC hydrolysis in normal fibroblasts and in fibroblasts from a TD kindred, in whom the HDL3- and apolipoprotein A-I (apo A-I)-induced mobilization of cellular cholesterol was found to be reduced by 50%. The HDL3-induced formation of phosphatidic acid (PA) via PC-specific phospholipase D (PC-PLD) was markedly reduced by 60-80% in these cells, whereas the formation of diacylglycerol (DG) via PC-specific phospholipase C (PC-PLC) was two- to threefold enhanced. Defective regulation of PC-PLC and PC-PLD was similarly observed in response to apo A-I and endothelin, but not in response to the receptor-independent stimulation of PC hydrolysis by PMA. A Tangier-like PA and DG formation pattern could be induced in normal cells after preincubation with pertussis toxin, suggesting the involvement of a G-protein. The impaired mobilization of radiolabeled cellular cholesterol in TD cells could completely be overcome by increasing the PA levels in the presence of the PA phosphohydrolase inhibitor propranolol. Conversely, the inhibition of PA formation in the presence of 0.3% butanol as well as the inhibition of DG formation in the presence of the PC-PLC inhibitor D 609 reduced the mobilization of cellular cholesterol both in normal and in TD cells. Our data indicate that the coordinate formation of PA and DG via PC-PLD and PC-PLC is essential for efficient cholesterol efflux. The molecular defect in this TD kindred appears to affect an upstream effector of protein kinase C responsible for the G-protein-dependent regulation of PC-specific phospholipases.

A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: Identification of duplicated repeats and potential active site residues

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.