Secondary literature sources for the PLDc domain

For each of the hand selected primary papers associated with the PLDc domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases.

Zhao Y, Stuckey JA, Lohse DL, Dixon JE
Protein Sci. 1997; 6: 2655-8

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endonuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino-terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single-strand DNA and an artificial substrate, bis(4-nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 A resolution.

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

Zhang Y, Altshuller YM, Hammond SM, Hayes F, Morris AJ, Frohman MA
EMBO J. 1999; 18: 6339-48

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.

The transphosphatidylation activity of phospholipase D.

Yu CH, Liu SY, Panagia V
Mol Cell Biochem. 1996; 157: 101-5

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.

Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in cultured human ciliary muscle cells: activation of phospholipase A2.

Yousufzai SY, Abdel-latif AA
Exp Eye Res. 1997; 65: 73-81

In the present study we have examined the effects and mechanisms of endothelin-1 (ET-1) on arachidonic acid (AA) release and prostaglandin (PG) synthesis in human ciliary muscle (HCM) cells. ET-1 stimulated AA release in a time (t1/2=1.5 min) and concentration-dependent (EC50=5 nM) manner, which is primarily mediated through the ETA receptor subtype. The AA liberated by ET-1 appears to derive mainly from the phosphoinositides and phosphatidylcholine. Our data show that phospholipase A2 (PLA2), but not phospholipase C (PLC), plays an important role in ET-1-induced AA release. This conclusion is supported by the following findings: (1) ET-1-evoked AA release was inhibited by the PLA2 inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE2 was inhibited by the cyclooxygenase inhibitors in the following order: Indomethacin>naproxen >ibuprofen>NS-398>aspirin. (2) The phorbol ester, PDBu, an activator of protein kinase C, potentiated ET-1-induced AA release by 39%, but inhibited that of inositol phosphates formation by 62%. (3) Pretreatment of the labeled cells with isoproterenol lowered ET-1-induced inositol phosphates production, but had no effect on AA release. (4) U71322, a PLC inhibitor, inhibited ET-1-induced inositol phosphates production, but had no effect on that of AA release. (5) Pretreatment of the cells with pertussis toxin (0.1 microg ml-1) attenuated the stimulatory effects of ET-1 on AA release and PGE2 formation. These data demonstrate that ET-1 is a potent agonist for AA release and PG synthesis in HCM cells, and that PLA2, but not PLC, plays an important role in ET-1-induced AA release and PG synthesis. In ciliary muscle, AA and its metabolites play important roles in intracellular signalling, modulation of physiological processes, and regulation of intraocular pressure.

Partially purified RhoA-stimulated phospholipase D activity specifically binds to phosphatidylinositol 4,5-bisphosphate.

Yokozeki T, Kuribara H, Katada T, Touhara K, Kanaho Y
J Neurochem. 1996; 66: 1234-9

Phosphatidylinositol 4,5-bisphosphate (PIP2) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP2, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidyicholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP2 for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP2. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP2. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP2 in the vesicles and correlated well with the extent of the enzyme activation. Further-more, it was found that a recombinant peptide of the pleckstrin homology domain of beta-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP2, inhibited the PIP2-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP2, owing to its specific interaction with PIP2, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP2 effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP2 serves as an anchor to localize PLD on plasma membranes in vivo.

Interaction of the small G protein RhoA with the C terminus of human phospholipase D1.

Yamazaki M, Zhang Y, Watanabe H, Yokozeki T, Ohno S, Kaibuchi K, Shibata H, Mukai H, Ono Y, Frohman MA, Kanaho Y
J Biol Chem. 1999; 274: 6035-8

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.

Requirements and effects of palmitoylation of rat PLD1.

Xie Z, Ho WT, Exton JH
J Biol Chem. 2001; 276: 9383-91

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs (HXKX(4)D), denoted HKD, located in the N- and C-terminal halves, which are required for phospholipase D activity. The two halves of rPLD1 can associate in vivo, and the association is essential for catalytic activity and Ser/Thr phosphorylation of the enzyme. In this study, we found that this association is also required for palmitoylation of rPLD1, which occurs on cysteines 240 and 241. In addition, palmitoylation of rPLD1 requires the N-terminal sequence but not the conserved C-terminal sequence, since rPLD1 that lacks the first 168 amino acids is not palmitoylated in vivo, while the inactive C-terminal deletion mutant is. Palmitoylation of rPLD1 is not necessary for catalytic activity, since N-terminal truncation mutants lacking the first 168 or 319 amino acids exhibit high basal activity although they cannot be stimulated by protein kinase C (PKC). The lack of response to PKC is not due to the lack of palmitoylation, since mutation of both Cys(240) and Cys(241) to alanine in full-length rPLD1 abolishes palmitoylation, but the mutant still retains basal activity and responds to PKC. Palmitoylation-deficient rPLD1 can associate with crude membranes; however, the association is weakened. Wild type rPLD1 remains membrane-associated when extracted with 1 m NaCl or Na(2)CO(3) (pH 11), while rPLD1 mutants that lack palmitoylation are partially released. In addition, we found that palmitoylation-deficient mutants are much less modified by Ser/Thr phosphorylation compared with wild type rPLD1. Characterization of the other cysteine mutations of rPLD1 showed that mutation of cysteine 310 or 612 to alanine increased basal phospholipase D activity 2- and 4-fold, respectively. In summary, palmitoylation of rPLD1 requires interdomain association and the presence of the N-terminal 168 amino acids. Mutations of cysteines 240 and 241 to alanine abolish the extensive Ser/Thr phosphorylation of the enzyme and weaken its association with membranes.

Endothelin-1 stimulated phospholipase D in A10 vascular smooth muscle derived cells is dependent on tyrosine kinase. Evidence for involvement in stimulation of mitogenesis.

Wilkes LC, Patel V, Purkiss JR, Boarder MR
FEBS Lett. 1993; 322: 147-50

The mechanism whereby endothelin stimulates mitogenesis of vascular smooth muscle cells is not understood. Here we show that endothelin-1 stimulates phospholipase D by a protein kinase C and tyrosine kinase dependent mechanism, and present evidence that implicate the phosphatidic acid formed by phospholipase D in the mitogenic response.

Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions.

Wang X
Prog Lipid Res. 2000; 39: 109-49

Multiple Phospholipase D (PLD) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of the genetic and biochemical heterogeneity has provided important clues as to the regulation and function of this family of enzymes. Polyphosphoinositides, Ca(2+), and G-proteins are possible cellular regulators for PLD activation. PLD-mediated hydrolysis of membrane lipids increases in response to various stresses. Recent studies suggest that PLD plays a role in the signaling and production of hormones involved in plant stress responses.

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.

Walter M, Reinecke H, Gerdes U, Nofer JR, Hobbel G, Seedorf U, Assmann G
J Clin Invest. 1996; 98: 2315-23

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.

Activation of phospholipase D1 by Cdc42 requires the Rho insert region.

Walker SJ, Wu WJ, Cerione RA, Brown HA
J Biol Chem. 2000; 275: 15665-8

Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120-139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (DeltaL8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest.

Identification and characterization of a gene encoding phospholipase D activity in yeast.

Waksman M, Eli Y, Liscovitch M, Gerst JE
J Biol Chem. 1996; 271: 2361-4

We have identified an open reading frame on chromosome XI of the yeast, Saccharomyces cerevisiae, as encoding a protein with phospholipase D (PLD) activity. We have named this open reading frame, PLD1, and show that yeast bearing a disruption in this gene are unable to catalyze the hydrolysis of phosphatidylcholine. PLD1 encodes a hypothetical protein of 1683 amino acids and has a predicted molecular mass of 195 kDa. Yeast bearing disruptions at the PLD1 locus are morphologically normal and grow vegetatively like wild-type cells. In contrast, homozygous delta pld1 diploid cells are unable to sporulate and do not produce asci under conditions that induce meiosis and sporulation in wild-type cells. Thus, PLD1 is likely to be essential for the meiotic cycle in yeast cells. This is the first identification of a eukaryotic, nonplant, phosphatidylcholine-hydrolyzing phospholipase D gene. Because the biological role of PLD is not well understood, we expect that delta pld1 yeast will become a useful tool for the characterization of PLD functions as well as for the identification of mammalian PLD homologs.

The measurement of phospholipase D-linked signaling in cells.

Wakelam MJ, Hodgkin M, Martin A
Methods Mol Biol. 1995; 41: 271-8

Regulation of the hydrolysis of phosphatidylcholine in Swiss 3T3 cells.

Wakelam MJ, Cook SJ, Currie S, Palmer S, Plevin R
Biochem Soc Trans. 1991; 19: 321-4

Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta.

Vinggaard AM, Provost JJ, Exton JH, Hansen HS
Cell Signal. 1997; 9: 189-96

In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP2 and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 microM GTP gamma S (6-14-fold) and by the small G proteins 1 microM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human placenta, which is stimulated by PIP2, but not by oleate (Vinggaard, A. M. & Hansen, H. S. (1995) Biochim. Biophys. Acta 1258, 169-176). Here we show that oleic acid and alpha-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important for the inhibitory effect. Furthermore, placental membrane PLD was activated by 30 microM GTP gamma S (4-fold) and by mArf3 (1 microM) and RhoA (0.37 nM) by a factor of 3 and 2, respectively. The solubilized membrane phospholipase D was partially purified to a basal specific activity of 25-37 nmol/min/mg. This preparation was devoid of endogenous RhoA and Arf and could not be stimulated by GTP gamma S. However, mArf3 (1 microM) still activated this partially purified membrane PLD, whereas RhoA (0.37 nM) was not able to activate this PLD fraction. In conclusion, our results suggest that the human placenta contains a PLD that is located both in the cytosol and the membranes, and that is activated by PIP2, mArf3 and RhoA but inhibited by oleate.

Increased phospholipase D activity in human breast cancer.

Uchida N, Okamura S, Nagamachi Y, Yamashita S
J Cancer Res Clin Oncol. 1997; 123: 280-5

Phospholipase D is believed to play an important role in cell proliferation and tumorigenesis. One of its major functions is to cause a sustained activation of protein kinase C through the primary production of phosphatidic acid from phosphatidylcholine by the enzyme, followed by dephosphorylation forming diacylglycerol. Protein kinase C is known to be activated or translocated in some tumors including breast tumors. In order to examine phospholipase D activity in breast tumors, surgical specimens of human breast tumors were obtained by mastectomy or wide excision, and their phospholipase D activities were assayed by determining the formation of phosphatidylethanol from phosphatidylcholine and ethanol. Phospholipase D activity was predominantly localized in the microsomal fraction of the tumor tissue and markedly stimulated by oleic acid. We observed a significant increase in phospholipase D activity in 17 out of 19 spontaneous human breast tumors as compared to adjacent histologically normal breast tissue. The mean specific activity in the tumors was 52.9 +/- 41.8 (SD) pmol min-1 mg protein-1 whereas the value for the normal breast tissue was 34.0 +/- 36.2 (SD) pmol min-1 mg protein-1 (P < 0.01; paired Wilcoxon's rank-sum test). The mean tumor/normal activity ratio was 2.37. Among prognostic factors, the nuclear grade, evaluated according to Schnitt et al., was found to be correlated with the activity ratio. Our results suggest a role for phospholipase D in human breast tumors. An elevation in phospholipase D activity is useful as a potential marker for malignant disease in the breast.

Phospholipase D: regulation and functional significance.

Thompson NT, Garland LG, Bonser RW
Adv Pharmacol. 1993; 24: 199-238

PLD is a major route for hydrolysis of PC in most tissues, consistent with it playing an important role in signal transduction. The enzyme appears to be activated by a variety of different mechanisms in different tissues, suggesting there might be several different isoforms. Little, however, is known at present about its enzymology and molecular biology. There is little direct evidence to indicate the functional significance of PLD activation but an accumulation of indirect evidence links PLD with prolonged changes in cell function. In particular, two areas where there is strong evidence for a role for PLD are mitogenesis and leukocyte hyperresponsiveness. An important area for future work will be the investigation of how products from the PLD pathway exert these effects. Current evidence suggests an important role for Ca(2+)-independent PKC isoforms and probably also for novel cellular targets for the putative second messenger PA.

Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

Thaller MC, Schippa S, Rossolini GM
Protein Sci. 1998; 7: 1647-52

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-?YFWHKR)-X-?YFWHNQ?-X( 102,191)-?KRHNQ?-G-D-?FYWHILVMC?-?QNH?-?FWYGP?-D -?PSNQYW?. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.

Phospholipase activities associated with the tonoplast from Acer pseudoplatanus cells: identification of a phospholipase A1 activity.

Tavernier E, Pugin A
Biochim Biophys Acta. 1995; 1233: 118-22

The study of phospholipase activities associated with the tonoplast of Acer pseudoplatanus was performed in vitro with sn-2-[14C]acylphosphatidylcholine (PC) as a substrate. The hydrolysis of radiolabelled PC into [14C]phosphatidic acid and [14C]lyso-PC demonstrated the presence of phospholipase D and A1 activities, respectively, associated with the tonoplast of Acer pseudoplatanus. The vacuolar sap did not show any significant phospholipase activity. In a second step, the properties of the phospholipase A1 activity was studied using tonoplast endogenous PC labelled in vivo with [14C]choline as a substrate. The phospholipase A1 showed an optimal activity at pH about 6-6.5, did not necessarily require divalent cations, but was stimulated by Mg2+ and particularly by Ca2+. This work presents the first evidence for the presence of phospholipases A1 in plant cells.

A continuous spectrophotometric method for monitoring phospholipase D-catalyzed reactions of physiological substrates.

Takrama JF, Taylor KE
J Biochem Biophys Methods. 1991; 23: 217-26

A continuous spectrophotometric method for monitoring phospholipase D-catalyzed hydrolysis of long acyl chain phosphatidylcholines has been formulated at pH 8.0 in a mixed detergent system using the coupling enzymes choline oxidase and peroxidase. Standard curves for phosphatidylcholine determination in both end-point and rate modes are presented and applied to the estimation of that phospholipid in a solubilized human erythrocyte membrane sample. In rate mode the method is suitable for kinetic study of phospholipase D with physiological substrates in micellar form.

Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis.

Suzuki A, Kozawa O, Shinoda J, Watanabe Y, Saito H, Oiso Y
J Cell Physiol. 1996; 168: 209-16

We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)- inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2+ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca2+ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation.

Structural analysis of human phospholipase D1.

Sung TC, Zhang Y, Morris AJ, Frohman MA
J Biol Chem. 1999; 274: 3659-66

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.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Sung TC, Roper RL, Zhang Y, Rudge SA, Temel R, Hammond SM, Morris AJ, Moss B, Engebrecht J, Frohman MA
EMBO J. 1997; 16: 4519-30

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Phospholipase D regulation by a physical interaction with the actin-binding protein gelsolin.

Steed PM, Nagar S, Wennogle LP
Biochemistry. 1996; 35: 5229-37

Increases in intracellular phosphatidic acid levels caused by receptor- mediated activation of phospholipase D (PLD) have been implicated in many signal transduction pathways leading to cellular activation. PLD is known to be regulated by several means, including tyrosine kinase activity, increases in Ca2+, receptor-coupled G proteins, small GTP binding proteins, ceramide metabolisms, and protein kinase C. We have investigated a additional regulatory effect on PLD activity involving nucleoside triphosphates (NTPs). A NTP binding protein copurifies with LPD activity from rabbit brains using a GTP-agarose affinity column, and this protein stimulates PLD activity only in the absence of NPTs. The NTP effect is reversible and labile, and the binding protein is separable from the PLD activity by heparin-agarose chromatography. We identified this protein as the actin- binding protein gelsolin by amino acid sequencing following peptide mapping. This finding was verified by the co-immunoprecipitation of gelsolin and PLD activity as well as by the reconstitution of gelsolin- dependent nucleotide sensitive PLD activity by the addition of purified gelsolin-free PLD. Our data indicate that actin rearrangements and PLD signaling are coordinately regulated through the physical association between PLD and gelsolin and that this interaction may also serve to amplify both PLD signaling and actin reorganization.

Characterization of human PLD2 and the analysis of PLD isoform splice variants.

Steed PM, Clark KL, Boyar WC, Lasala DJ
FASEB J. 1998; 12: 1309-17

Phospholipase D (PLD) cleaves phosphatidylcholine in response to a variety of cell stimuli to release phosphatidic acid, which is associated with a number of cellular responses including regulated secretion, mitogenesis, and cytoskeletal changes. Recent advances in this field include the reports of cDNA sequences for two mammalian PLD isoforms: human PLD1 and rodent PLD2. We report the characterization of cDNA encoding human PLD2. In these experiments, we uncovered alternate splice variants of both human isoforms and evaluated the relative abundance of these messages by reverse transcriptase polymerase chain reaction, thereby indicating the physiologically relevant forms. Further, Northern hybridization experiments defined the tissue distribution of the human PLD messages. Human PLD1 does not appear to be an abundant message in any tissue tested whereas levels of human PLD2 mRNA apparently were higher and more variable. The specific activity and regulation of recombinant human PLD2 are indistinguishable from that of recombinant mouse PLD2. Analysis of the amino acid sequences of both human isoforms revealed important putative Pleckstrin homology domains and identified additional members of the PLD gene family that help to delimit the catalytic domain. The presence of Pleckstrin homology domains in the PLDs resolves several contradictory observations regarding PLD regulation and the domain structure of the proteins.

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity.

Stadelmann B, Butikofer P, Konig A, Brodbeck U
Biochim Biophys Acta. 1997; 1355: 107-13

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.

Sphingoid bases and phospholipase D activation.

Spiegel S, Milstien S
Chem Phys Lipids. 1996; 80: 27-36

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.

Epidermal growth factor induces the production of biologically distinguishable diglyceride species from phosphatidylinositol and phosphatidylcholine via the independent activation of type C and type D phospholipases.

Song J, Jiang YW, Foster DA
Cell Growth Differ. 1994; 5: 79-85

An early response to epidermal growth factor in A431 cells is the generation of diglyceride, a physiological activator of protein kinase C. By differentially prelabeling cellular phospholipids with [3H]arachidonate and [3H]myristate, which are incorporated primarily into phosphatidylinositol and phosphatidylcholine, respectively, we have found that epidermal growth factor induces an increase in diglyceride levels from both phosphatidylinositol and phosphatidylcholine via distinct mechanisms and kinetics. The epidermal growth factor-induced increase in phosphatidylinositol-derived diglyceride was transient and peaked at 5 min. As diglyceride levels dropped, there was a corresponding increase in phosphatidic acid, suggesting that the diglyceride is efficiently converted to phosphatidic acid by a diglyceride kinase. In contrast, epidermal growth factor-induced increases in phosphatidylcholine-derived diglyceride peaked at 30 min and remained elevated for greater than 2 h. The epidermal growth factor-induced increases in phosphatidic acid detected in [3H]myristate-prelabeled cells paralleled the increase in diglyceride, suggesting that the phosphatidylcholine-derived diglyceride is produced from phosphatidic acid via a phosphatidic acid phosphatase. Consistent with this hypothesis, epidermal growth factor also induced a protein kinase C-independent phospholipase D activity that was specific for phosphatidylcholine. These data suggest that epidermal growth factor induces diglyceride production from phosphatidylinositol and phosphatidylcholine via two distinct mechanisms: a rapid and transient induction of diglyceride that likely involves phospholipase c-gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate and a slower, more sustained induction of diglyceride via a phospholipase D-mediated hydrolysis of phosphatidylcholine to produce phosphatidic acid, which is then converted to diglyceride by a phosphatidic acid phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of LA-N-2 cell phospholipase D by amyloid beta protein (25-35).

Singh IN, Sorrentino G, Kanfer JN
Neurochem Res. 1998; 23: 1225-32

Amyloid beta protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25-35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and APP (25-35) was calcium-independent. The AbetaP (25-35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AbetaP (25-35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AbetaP (25-35). Only NBQX was effective with an IC50 of 75 microM for AbetaP (25-35) and quisqualate. Activation of phospholipase D by AbetaP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AbetaP (25-35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AbetaP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AbetaP (25-35).

Activation of phospholipases D and A by amphiphilic cations of cultured LA-N-2 cells is G protein- and protein kinase C-independent.

Singh IN, Massarelli R, Kanfer JN
J Lipid Mediat. 1993; 7: 85-96

Several amphiphilic cations such as mepacrine, desipramine, didodecyldimethylamine, chlorpromazine, oleylamine and W-7 activated the phospholipase D (PLD) activity of cultured LA-N-2 cells. These compounds, except for oleylamine, provoked the release of fatty acids, suggesting phospholipase A activation. Melittin, a PLA2 stimulator, caused the robust release of the free fatty acids but was a poor PLD activator. Although PLD could be activated by GTP gamma S, the stimulation by these amphiphilic cations was not abolished by GDP beta S, an inhibitor of G protein function. There was no change in the PLD activation by these amphiphilic cations by DiC8, a PKC activator, or by H-7, a PKC inhibitor or in PKC down-regulated cells.

Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity.

Singer WD, Brown HA, Jiang X, Sternweis PC
J Biol Chem. 1996; 271: 4504-10

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.

Phospholipase D in cell signalling and its relationship to phospholipase C.

Shukla SD, Halenda SP
Life Sci. 1991; 48: 851-66

Phospholipases C and D are phosphodiesterases which act on phospholipid head groups. Although the presence of these enzymes in living organisms has long been known, it is only recently that their role in cell signal transduction has been appreciated. The new developments on phospholipases D (PLD) are especially noteworthy, since these enzymes catalyze a novel pathway for second messenger generation. In a variety of mammalian cell systems, several biological or chemical agents have recently been shown to stimulate PLD activity. Depending on the system, activation of PLD has been suggested to be either dependent on, or independent of, Ca2+ and protein kinase C. PLD primarily hydrolyses phosphatidylcholine (PC) but phosphatidylinositol and phosphatidylethanolamine have also been reported as substrates. Different forms of endogenous PLD may also exist in cells. Exogenous addition of PLD causes alterations in cellular functions. In many instances, Ca2+ mobilizing agonists may stimulate both PLC and PLD pathways. Interestingly, several metabolites of these two enzymes are second messengers and are common to both pathways (e.g. phosphatidic acid, diglyceride). This has raised the issue of the interrelationship between these pathways. The regulation of either PLC or PLD by cellular components, e.g. guanine nucleotide binding proteins or protein kinases, is under intense investigation. These recent advances are providing novel information on the significance of phospholipase C and D mediated phospholipid turnover in cellular signalling. This review highlights some of these new discoveries and emerging issues, as well as challenges for future research on phospholipases.

Mechanism of GnRH receptor signaling: from the membrane to the nucleus.

Shacham S, Cheifetz MN, Lewy H, Ashkenazi IE, Becker OM, Seger R, Naor Z
Ann Endocrinol (Paris). 1999; 60: 79-88

The purpose of this review is to update the information concerning the intracellular effect of GnRH. Binding of GnRH to a G-protein coupled receptor leads to stimulation of Gq and/or G11 protein and to activation of phospholipase C beta. Inositol 1-4-5-triphosphate and early diacylylycerol are the second messengers required for conventional protein kinase C activation. Activation of phospholipase A2 and phospholipase D are also involved, as demonstrated by the liberation of Arachidonic Acid and Phosphatidic Acid. Pituitary cells also express atypical protein kinase C isoforms which mode of activation is not known. Hypothesis concerning transcriptional regulation are presented.

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

Seidler L, Kaszkin M, Kinzel V
Pharmacol Toxicol. 1996; 78: 249-53

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.

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

Sciorra VA, Rudge SA, Prestwich GD, Frohman MA, Engebrecht J, Morris AJ
EMBO J. 1999; 18: 5911-21

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.

Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors.

Schmidt M, Rumenapp U, Keller J, Lohmann B, Jakobs KH
Life Sci. 1997; 60: 1093-100

The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C. botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], a substrate and cofactor for PLC and PLD, respectively, were not altered. These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme. In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.

Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors. Evidence for involvement of tyrosine phosphorylation.

Schmidt M, Huwe SM, Fasselt B, Homann D, Rumenapp U, Sandmann J, Jakobs KH
Eur J Biochem. 1994; 225: 667-75

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phopsholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins.

Phospholipase D and phospholipase C in human cholinergic neuroblastoma (LA-N-2) cells: modulation by muscarinic agonists and protein kinase C.

Sandmann J, Wurtman RJ
Adv Second Messenger Phosphoprotein Res. 1990; 24: 176-81

Phosphatidic acid and phospholipase D both stimulate phosphoinositide turnover in cultured human keratinocytes.

Ryder NS, Talwar HS, Reynolds NJ, Voorhees JJ, Fisher GJ
Cell Signal. 1993; 5: 787-94

Phosphatidic acid (PA) induced a rapid dose-dependent increase in production of inositol phosphates in cultured adult human keratinocytes, peaking at 30 s. Natural and dioleoyl PA were equally effective, while other phospholipid classes had no effect. Lipid A was also active. Lyso-PA also induced inositol phosphate production, but contamination of the PA preparation by lyso-PA could not account for the effect of PA. The effect of PA could not be reproduced by treatment of cells with calcium ionophore. PA-induced inositol phosphate production could be inhibited (> 50%) by pre-treatment of cells with either pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate, suggesting the involvement of a GTP-binding protein and a protein kinase C-mediated negative feedback mechanism. PA also stimulated release of arachidonic acid from keratinocytes. Treatment of cells with exogenous phospholipase D similarly induced inositol phosphate production in the keratinocytes. Since PA may be formed by receptor-mediated activation of phospholipase D, or by phosphorylation of diacylglycerol, the results suggest that PA may play a significant role in signalling mechanisms of human keratinocytes.

Characteristics of protein-kinase-C- and ADP-ribosylation-factor-stimulated phospholipase D activities in human embryonic kidney cells.

Rumenapp U, Schmidt M, Wahn F, Tapp E, Grannass A, Jakobs KH
Eur J Biochem. 1997; 248: 407-14

Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbol-ester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently acting via the GTP-binding proteins, ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [3H]oleic acid, GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [3H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD cofactor, phosphatidylinositol 4,5-bisphosphate, decreased basal and GTP[S]- or PMA-stimulated PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[beta-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the nonselective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cytosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1. potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 and bisindolylmaleimide I. Immunoblot analysis demonstrated that both conventional PKC (alpha, beta, gamma) and atypical PKC isozymes (zeta, tau) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S]' action.

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.

Rudolph AE, Stuckey JA, Zhao Y, Matthews HR, Patton WA, Moss J, Dixon JE
J Biol Chem. 1999; 274: 11824-31

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.

SPO14 Separation-of-Function Mutations Define Unique Roles for Phospholipase D in Secretion and Cellular Differentiation in Saccharomyces cerevisiae.

Rudge SA, Pettitt TR, Zhou C, Wakelam MJ, Engebrecht J
Genetics. 2001; 158: 1431-44

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

Phospholipase D signaling is essential for meiosis.

Rose K, Rudge SA, Frohman MA, Morris AJ, Engebrecht J
Proc Natl Acad Sci U S A. 1995; 92: 12151-5

Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation.

Activation of phospholipase D in rat type II pneumocytes by ATP and other surfactant secretagogues.

Rooney SA, Gobran LI
Am J Physiol. 1993; 264: 13340-13340

Surfactant phospholipid secretion can be stimulated by a variety of agonists acting via a number of signal-transduction mechanisms. To determine whether phospholipase D has a role in surfactant secretion, we examined phosphatidylethanol formation in response to surfactant secretagogues in primary cultures of rat type II cells. Phosphatidylethanol formation was stimulated by ATP, 12-O-tetradecanoylphorbol-13 acetate (TPA), and dioctanoylglycerol, surfactant secretagogues that also activate protein kinase C. Surfactant secretagogues that act via other signaling mechanisms had no effect on phosphatidylethanol formation. The effect of ATP on phosphatidylethanol formation was dependent on time, with the maximum stimulation being achieved in approximately 10 min. It was also dependent on ATP concentration. The ATP concentration eliciting 50% of the maximum effect (EC50) was 2.45 x 10(-6) M. This was similar to the EC50 reported for ATP stimulation of surfactant secretion. ATP analogues also stimulated phosphatidylethanol formation with a potency order generally similar to that reported for surfactant secretion. The effects of ATP, TPA, and dioctanoylglycerol were antagonized by protein kinase C inhibitors. We speculate that activation of protein kinase C either directly by TPA and dioctanoylglycerol or indirectly subsequent to phosphoinositide-specific phospholipase C activation by ATP leads to initial stimulation of surfactant secretion as well as activation of phospholipase D. The action of phospholipase D on cellular phospholipids then leads to further generation of diacylglycerols, continued activation of protein kinase C, and sustained surfactant secretion.

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.

Ribbes G, Cane A, Planat V, Breton M, Chap H, Bereziat G, Record M, Colard O
J Cell Biochem. 1996; 62: 56-68

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.

Are cells rescued from 'low density death' by co-operation between phospholipases C AND D?

Rasmussen M, Rasmussen L
Cell Biol Int. 2000; 24: 121-3

Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid-free nutritionally complete medium. This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained. We propose that this takes place in two stages. Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris -phosphate. Diacylglycerol activates protein kinase C, and inositol tris -phosphate via Ca(2+)phospholipase D (PLD). Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates. Should this switching from PI-specific phospholipase C (PLC) to phospholipase D fail, then the cell will die in the course of milliseconds during the minutes following inoculation.

Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium.

Qin W, Pappan K, Wang X
J Biol Chem. 1997; 272: 28267-73

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9. PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha. A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta. Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLDgamma and -beta are most active at &mgr;M Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [3H]PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha. These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha. The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.

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

Qian Z, Drewes LR
FASEB J. 1991; 5: 315-9

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.

Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain.

Qian Z, Drewes LR
J Biol Chem. 1989; 264: 21720-4

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.

Phospholipase D regulation involves extracellular calcium as a conditional requirement for subsequent stimulation by protein kinase C.

Pyne S, Pyne NJ
Biochem Soc Trans. 1995; 23: 199-199

Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle.

Pyne S, Pyne NJ
Biochem Pharmacol. 1993; 45: 593-603

Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.

Interaction between vanilloid receptors and purinergic metabotropic receptors: pain perception and beyond.

Premkumar LS
Proc Natl Acad Sci U S A. 2001; 98: 6537-9

Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions. Phospholipase D-derived diacylglycerol does not activate protein kinase C in porcine aortic endothelial cells.

Pettitt TR, Martin A, Horton T, Liossis C, Lord JM, Wakelam MJ
J Biol Chem. 1997; 272: 17354-9

Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.

Expression of a novel murine phospholipase D homolog coincides with late neuronal development in the forebrain.

Pedersen KM, Finsen B, Celis JE, Jensen NA
J Biol Chem. 1998; 273: 31494-504

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.

Evidence against a role for phospholipase D in mitogenesis.

Paul A, Plevin R
Trends Pharmacol Sci. 1994; 15: 174-5

Cloning and characterization of phospholipase D from rat brain.

Park SK, Provost JJ, Bae CD, Ho WT, Exton JH
J Biol Chem. 1997; 272: 29263-71

The regulation of phospholipase D cloned from rat brain (rPLD) was examined in vivo and in vitro. The enzyme was a shorter splice variant of human phospholipase D 1 (Hammond, S. M., Altshuller, Y. M. , Sung, T.-C., Rudge, S. M., Rose, K., Engebrecht, J. A., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). Its expression in COS-7 cells led to increased phospholipase D (PLD) activity that was further stimulated by constitutively active V14RhoA. V14RhoA had no effect on the endogenous PLD of the COS-7 cells, but constitutively active L71ARF3 increased its activity. In contrast, L71ARF3 did not activate rPLD expressed in the cells. Addition of phorbol ester markedly increased the endogenous PLD activity of COS-7 cells, and there was a further increase in the cells expressing rPLD. In membranes from COS-7 cells expressing rPLD, addition of myristoylated ADP-ribosylation factor (ARF) and RhoA in vitro stimulated PLD activity. The effect of ARF was greater than that of RhoA, although the concentrations for half-maximal stimulation (0.08-0.2 microM) were similar. Membranes isolated from cells expressing rPLD plus L71ARF3 and/or V14RhoA also showed higher PLD activity but no synergism between the two G proteins. Addition of phorbol ester and protein kinase C alpha (PKCalpha) also stimulated PLD activity in membranes from COS-7 cells expressing rPLD, but it had no effect on the activity in control (vector) membranes and did not enhance the effects of constitutively active ARF or Rho. The stimulation by PKCalpha did not require ATP and was not increased by addition of this nucleotide. No synergism between ARF and Rho and between these and PKCalpha on PLD activity was observed when these were added to membranes from cells expressing rPLD. Oleate inhibited the PLD activity of membranes from both control and rPLD-expressing cells. In summary, these results indicate that in vitro, rPLD is stimulated by ARF, RhoA, and PKCalpha and inhibited by oleate. However, in intact COS-7 cells, ARF activates endogenous PLD but not rPLD, whereas the reverse is true for RhoA. In addition, the effects of phorbol ester are much greater in the intact cells. It is concluded that the regulation of rPLD in intact COS-7 cells differs significantly from that seen in vitro; possible reasons for this are discussed.

Definition of the protein kinase C interaction site of phospholipase D.

Park SK, Min DS, Exton JH
Biochem Biophys Res Commun. 1998; 244: 364-7

Serial deletions of the N-terminal 319 amino acids of rPLD1 expressed in COS-7 cells resulted in increased basal PLD activity. Incubation of the cells with phorbol myristate acetate increased the activity of endogenous and wild-type rPLD1. The mutant rPLD1 with deletion of the first 50 amino acids responded to the phorbol ester, however, rPLD1 with deletions of 115 amino acids or more did not. In cells in which constitutively active V14RhoA was co-expressed with the mutant PLDs, stimulation of PLD activity was observed with all deletion mutants. In membranes from COS-7 cells in which the mutant PLDs were expressed, only the mutant with deletion of 50 N-terminal amino acids responded to added protein kinase C-alpha and phorbol ester, in agreement with the in vivo studies. When myristoylated ADP-ribosylation factor 3 (mARF3) was added together with guanosine 5'-3-O-(thio)triphosphate, all mutants showed stimulation of PLD activity. It is concluded that the site of interaction of protein kinase C with rPLD1 is located in the N-terminal region and that Rho and ARF interact at other sites.

Molecular and biochemical properties and physiological roles of plant phospholipase D.

Pappan K, Wang X
Biochim Biophys Acta. 1999; 1439: 151-66

Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology.

Plant phospholipase Dalpha is an acidic phospholipase active at near-physiological Ca(2+) concentrations.

Pappan K, Wang X
Arch Biochem Biophys. 1999; 368: 347-53

The conventional plant phospholipase D (PLD) requires Ca(2+) for activity; however, the most distinct and puzzling feature of this PLD is its in vitro need for 20 to 100 mM Ca(2+). This noncytoplasmic Ca(2+) requirement has raised doubt about the role of Ca(2+) in regulating its function in vivo. Using the cloned conventional castor bean PLD, PLDalpha, expressed in Escherichia coli, this study demonstrates that this PLD is active at micromolar, near-physiological concentrations of Ca(2+), and this activity at low Ca(2+) requires an acidic pH (4.5-5.5). By comparison, the newly cloned PLDbeta and -gamma were active only at neutral pH under the same Ca(2+) concentrations. This study also shows that PLDalpha activity at low Ca(2+) needs substrates presented as a mixture of membrane lipids. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate are equally effective in stimulating the acidic PLDalpha activity, whereas phophatidylinositol is inactive. These results suggest that the conventional plant PLD in vivo is an acidic phospholipase that is active at near-physiological Ca(2+) concentrations. The possible physiological significance of these findings will be discussed.

Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis.

Pappan K, Qin W, Dyer JH, Zheng L, Wang X
J Biol Chem. 1997; 272: 7055-61

A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLDbeta for this PIP2-dependent PLD and PLDalpha for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity. The PLDbeta cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLDbeta cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDbeta, but not PLDalpha, activity. The activity of the expressed PLDbeta is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLDbeta is evolutionarily divergent from PLDalpha and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.

Biochemistry and cell biology of phospholipase D in human neutrophils.

Olson SC, Lambeth JD
Chem Phys Lipids. 1996; 80: 3-19

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.

Phospholipase D development during differentiation of human promyelocytic leukemic HL60 cells.

Ohguchi K, Nakashima S, Nozawa Y
Biochim Biophys Acta. 1999; 1439: 215-27

Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum.

Ogino C, Negi Y, Matsumiya T, Nakaoka K, Kondo A, Kuroda S, Tokuyama S, Kikkawa U, Yamane T, Fukuda H
J Biochem (Tokyo). 1999; 125: 263-9

Phospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Stv. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.

Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity.

Ogino C, Negi Y, Daido H, Kanemasu M, Kondo A, Kuroda S, Tanizawa K, Shimizu N, Fukuda H
Biochim Biophys Acta. 2001; 1530: 23-31

A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum. The enzyme has a monomeric structure with a molecular size of about 37 kDa, being the smallest among the enzymes so far reported. The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol. Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain [C. Ogino et al., J. Biochem. 125 (1999) 263-269] and is therefore regarded as a novel prokaryotic PLD.

Study of phospholipases D and C in maturing and germinating seeds of Brassica napus.

Novotna Z, Valentova O, Martinec J, Feltl T, Nokhrina K
Biochem Soc Trans. 2000; 28: 817-8

Different forms of phospholipase D (dependent on and independent of the presence of phosphatidylinositol 4,5-bisphosphate, PIP(2)) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP(2)-dependent activity increased continuously during seed germination, while PIP(2)-independent activity appeared mostly at the very beginning of seed maturation. PIP(2)-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP(2)-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.

Signalling pathways activated by endothelin stimulation of renal cells.

Nord EP
Clin Exp Pharmacol Physiol. 1996; 23: 331-6

1. Endothelin mediates its effects in a variety of renal cells via a multiplicity of intracellular signalling pathways. 2. Stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC), resulting in the activation of inositol trisphosphate and diacylglycerol, can be detected even at picomolar concentrations of peptide. 3. Endothelin activation of cPLA2 is sensitive to ambient [Ca2+]i, is not contingent upon protein kinase C activation and is independent of PI-PLC stimulation, being coupled to the endothelin receptor in a yet to be determined manner. 4. Activation by endothelin of phosphatidylcholine-specific phospholipase D is under the dual regulation of protein kinase C and [Ca2+]i, with protein kinase C being the major regulator and [Ca2+]i playing a secondary, modulatory role. 5. Phosphatidylcholine-specific phospholipase C (PC-PLC) is stimulated by endothelin and accounts for the prolonged activation of diacylglycerol by this peptide. PC-PLC activity is critically dependent upon [Ca2+]i, whereas protein kinase C plays no role in modulating the activity of this enzyme. 6. Endothelin enhances the phosphorylation of protein tyrosine kinases, with evidence that phosphorylation of pp60 Src may be an important early event.

Regulation of phospholipase D by tyrosine kinases.

Natarajan V, Scribner WM, Vepa S
Chem Phys Lipids. 1996; 80: 103-16

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells. Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well. Activation of PLD in response to an external stimulus may involve PKC, Ca2+, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca2+, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor.

Naro F, Perez M, Migliaccio S, Galson DL, Orcel P, Teti A, Goldring SR
Endocrinology. 1998; 139: 3241-8

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.

Possible role of phospholipase D in cellular differentiation and apoptosis.

Nakashima S, Nozawa Y
Chem Phys Lipids. 1999; 98: 153-64

Phospholipase D (PLD) is widely distributed in mammalian cells and is implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, two related PLD isozymes, PLD1 and PLD2, were cloned. The former activity is regulated in vitro by protein kinase C and small molecular weight GTP-binding proteins (Arf and Rho family). By contrast, the basal activity of the latter is high and it is unresponsive in vitro to these activators. The cellular PLD activity and mRNA levels of these PLD isozymes drastically changed during differentiation and apoptosis in several types of cells. The general trend was that the mRNA level of PLD1 increased during differentiation, as did the observed GTP gamma S-dependent PLD activity which presumably derived from PLD1-specific catalysis. In contrast, the PLD activity and mRNA level of PLD1 were down-regulated during apoptosis. In addition to these PLD isozymes, there exists another PLD isozyme which is activated by unsaturated fatty acids such as oleic acid, although its molecular nature and physiological roles are not well defined. We have observed that this type of PLD activity is drastically increased during apoptosis of Jurkat T cells, which mainly possess this kind of PLD activity. These results suggest the possibility that PLD activity is controlled at the transcriptional level in certain circumstances, and that PLD plays roles in differentiation, survival and apoptosis in mammalian cells.

Differential expression of protein kinase C isozymes and small GTP-binding proteins during HL60 cell differentiation by retinoic acid and cyclic AMP: relation with phospholipase D (PLD) activation.

Nakashima S, Iwasaki Y, Mizutani T, Ohguchi K, Nagata K, Kitajima Y, Nozawa Y
Immunobiology. 1996; 196: 588-98

The differential expression of protein kinase C (PKC) isozymes and small GTP-binding proteins, and their relation to O2 generation and phospholipase D (PLD) activation were analyzed during the differentiation of human promyelocytic HL60 cells to neutrophil-like cells induced by either retinoic acid (RA) or dibutyryl cyclic AMP (dbcAMP). In response to either one of the inducers, nitroblue tetrazolium (NBT) reduction activity time-dependently increased. Although PLD activity was upregulated by dbcAMP-treatment, only a slight increase was observed in RA-treated cells. Small GTP-binding proteins Rac1, Rap1, and RhoA, which are reported to be implicated in O2- generation or PLD activation, were already expressed in undifferentiated HL60 cells and their significant changes were not detected during differentiation. The mRNAs of the cytosolic components of NADPH oxidase system, p47phox and p67phox, were present in trace amounts in undifferentiated cells. However, they rapidly increased in response to RA or dbcAMP. In response to either RA or dbcAMP, the increases were observed in cPKC isozymes (alpha, beta I, beta II) but not in other subtypes (delta, epsilon, theta, zeta) by both RT-PCR and Western blot analyses. In dbcAMP-treated cells PKC alpha increased remarkably, whereas PKC beta I and beta II mainly elevated in RA-treated cells. These results suggest the possibility that cPKCs are closely related to cell differentiation and that PKC alpha is involved in PLD activation.

[Roles of phospholipase D in cell functions and its regulatory mechanisms]

Nakashima S, Banno Y, Nozawa Y
Tanpakushitsu Kakusan Koso. 1995; 40: 877-88

Requirement of GM2 ganglioside activator for phospholipase D activation.

Nakamura S, Akisue T, Jinnai H, Hitomi T, Sarkar S, Miwa N, Okada T, Yoshida K, Kuroda S, Kikkawa U, Nishizuka Y
Proc Natl Acad Sci U S A. 1998; 95: 12249-53

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by beta-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792-815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

[Mechanism of phospholipase D regulation]

Nakamura S
Tanpakushitsu Kakusan Koso. 1999; 44: 1007-12

Inhibition of tyrosine phosphorylation by wortmannin in human neutrophils. Dissociation from its inhibitory effects on phospholipase D.

Naccache PH, Caon AC, Gilbert C, Gaudry M, Roberge CJ, Poubelle PE, Bourgoin S
Lab Invest. 1993; 69: 19-23

BACKGROUND: Recent studies have indicated that the regulation of the activation of human neutrophils depends on tyrosine phosphorylation and on phospholipase D. Furthermore, a tentative causal relationship between these two signalling pathways has been indirectly implied derived through the use of inhibitors of tyrosine kinases. The fungal metabolite, wortmannin is at present the only compound known to inhibit the receptor-mediated activation of phospholipase D in human neutrophils. Its mechanism of action is presently unknown. EXPERIMENTAL DESIGN: The ability of peripheral blood neutrophils to respond to various agonists with an increase in activity of phospholipase D and an enhancement of tyrosine phosphorylation in the absence or presence of wortmannin was monitored. RESULTS: Wortmannin was found to inhibit the stimulation of tyrosine phosphorylation by fMet-Leu-Phe, and by the inflammatory microcrystals monosodium urate and calcium pyrophosphate dihydrate. This effect of wortmannin was not secondary to inhibition of phospholipase D as U73122, a previously described phospholipase C inhibitor, was also found to inhibit phospholipase D without affecting tyrosine phosphorylation. CONCLUSIONS: The results make it likely that one of the earliest sites of action of wortmannin in human neutrophils is at the level of tyrosine phosphorylation which then exerts a modulatory influence on the activation of phospholipase D.

Phospholipid signalling in plants.

Munnik T, Irvine RF, Musgrave A
Biochim Biophys Acta. 1998; 1389: 222-72

Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants.

Munnik T, de Vrije T, Irvine RF, Musgrave A
J Biol Chem. 1996; 271: 15708-15

We provide evidence that phosphatidic acid (PtdOH) formed during signaling in plants is metabolized by a novel pathway. In much of this study, 32Pi-labeled Chlamydomonas cells were used, and signaling was activated by adding the G-protein activator mastoparan. Within seconds of activation, large amounts of [32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the level of [32P]PtdOH subsequently decreased, an unknown phospholipid (PLX) increased in radiolabeling; before activation it was barely detectable. The chromatographic properties of PLX resembled those of lyso-PtdOH and CMP.PtdOH but on close inspection were found to be different. PLX was shown to be diacylglycerol pyrophosphate (DGPP), the product of a newly discovered enzyme, phosphatidate kinase, whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography with a standard and by degradation analysis as follows: [32P]DGPP was deacylated, and the product (glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or pyrophosphatase to produce GroP and Pi as the only radioactive products. Since DGPP is the pyrophosphate derivative of PtdOH and is formed as the concentration of PtdOH decreases, we assumed that PtdOH was converted in vivo to DGPP. This was confirmed by showing that during a short labeling protocol while the specific radioactivity of DGPP was increasing, the specific radioactivity of the 32Pi derived from DGPP as above was higher than that of [32P]GroP. DGPP was also formed in suspension cultures of tomato and potato cells, and its synthesis was activated by mastoparan. Moreover, it was also found in intact tissues of a number of higher plants, for example, carnation flower petals, vetch roots, leaves of fig-leaved goosefoot, and common persicaria and microspores of rape seed. Our results suggest that DGPP is a common but minor plant lipid that increases in concentration when signaling is activated. Possible functions of DGPP in phospholpase C and D signaling cascades are discussed.

Regulation and functions of phospholipase D.

Morris AJ, Hammond SM, Colley C, Sung TC, Jenco JM, Sciorra VA, Rudge SA, Frohman MA
Biochem Soc Trans. 1997; 25: 1151-7

1,25(OH)2-vitamin D3 stimulation of phospholipases C and D in muscle cells involves extracellular calcium and a pertussis-sensitive G protein.

Morelli S, Boland R, de Boland AR
Mol Cell Endocrinol. 1996; 122: 207-11

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] activates in chick myoblasts the breakdown of phosphoinositides by phospholipase C and the hydrolysis of phosphatidylcholine by phospholipase D. Extracellular Ca2+ requirement and GTP-binding protein mediation of 1,25(OH)2D3-dependent activation of phospholipases C and D were investigated in cells prelabelled with [3H]glycerol or [3H]arachidonic acid. Generation of diacylglycerol by phospholipase C and phosphatidylethanol by phospholipase D were shown to be dependent on extracellular calcium, since both responses were suppressed by EGTA and the Ca(2+)-channel blockers nifedipine and verapamil, and were mimicked by the calcium ionophore A23187. The G-protein activators guanosine 5'-O-(3-thiotriphosphate) and AlF4- strongly enhanced diacylglycerol and phosphatidylethanol release in myoblasts while guanosine 5'-O-(2-thiodiphosphate), which inhibits G-protein-mediated signals, abolished 1,25(OH)2D3-dependent diacylglycerol and phosphatidylethanol release. Bordetella pertussis toxin pretreatment suppressed the hormone action. These results suggest that 1,25(OH)2D3-stimulation of phosphoinositide-specific phospholipase C and phospholipase D in chick myoblasts is mediated by a pertussis-sensitive GTP-binding protein(s) and the influx of extracellular calcium.

Characterization of a rat brain phospholipase D isozyme.

Min DS, Park SK, Exton JH
J Biol Chem. 1998; 273: 7044-51

We have recently cloned a cDNA encoding a phospholipase D (PLD) from rat brain and named it rPLD1. It shows 90% amino acid identity with the human PLD isoform hPLD1b. We have expressed rPLD1 as a histidine-tagged fusion protein in insect (Sf9) cells using the expression vector pBlueBacHis and purified the recombinant protein to homogeneity by Ni2+-agarose affinity chromatography. Phosphatidylinositol 4,5-P2 and phosphatidylinositol 3,4,5-P3 activated the PLD equipotently, but other acidic phospholipids were ineffective. The activity of rPLD1 was dependent on both Mg2+ and Ca2+. It was specific for phosphatidylcholine and showed a broad dependence on pH with optimum activity at pH 6.5-7.5. The enzyme was inhibited by oleate and activated by the small G proteins ARF3 and RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Protein kinase C (PKC)-alpha and -betaII, but not PKC-gamma, -delta, -epsilon, or -zeta, activated rPLD1 in a manner that was stimulated by phorbol ester but did not require ATP. Neither synergistic interactions between ARF3 and RhoA nor between these G proteins and PKC-alpha or -betaII were observed. Recombinant PKC-alpha and -betaII phosphorylated purified rPLD1 to high stoichiometry in vitro, and the phosphorylated PLD exhibited a mobility shift upon electrophoresis. Phosphorylation of the PLD by PKC was correlated with inhibition of its catalytic activity. rPLD1 bound to concanavalin A-Sepharose beads, and its electrophoretic mobility was altered by treatment with endoglycosidase F. The amount of PLD bound to the beads was decreased in a concentration-dependent manner when tunicamycin was added to the Sf9 expression system. Tunicamycin also decreased membrane localization of rPLD1. These results suggest that rPLD1 is a glycosylated protein and that it is negatively regulated by phosphorylation by PKC in vitro.

Characterization of the plasma glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD).

Metz CN, Schenkman S, Davitz MA
Cell Biol Int Rep. 1991; 15: 875-82

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

Meacci E, Vasta V, Donati C, Farnararo M, Bruni P
FEBS Lett. 1999; 457: 184-8

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.

Modification of catalytically active phospholipase D1 with fatty acid in vivo.

Manifava M, Sugars J, Ktistakis NT
J Biol Chem. 1999; 274: 1072-7

Phospholipase D1 (PLD1) was covalently labeled with 3H when expressed transiently in COS cells and immunoprecipitated following labeling of the cells with [3H]palmitate. Labeling of PLD1 was abolished by treatment with hydroxylamine at neutral pH, indicating that the fatty acid is linked via thioester to the enzyme. In pulse-chase studies the label persisted over a 3-h chase, indicating a slow rate of turnover. A catalytically inactive point mutant of PLD1 that changes serine at position 911 to alanine (S911A) was partially but not entirely redistributed to the cytosol, and it contained no detectable palmitate label. Similarly, N- and C-terminal domain fragments of the protein, encompassing in combination the entire coding region and all expressed to levels comparable with the wild type protein, showed no label with palmitate. Treatment of immunoprecipitated PLD1 with hydroxylamine diminished catalytic activity to background levels in a dose response manner that paralleled the removal of label from [3H]palmitate-labeled protein. We suggest that modification of PLD1 with palmitate is related to its catalytic activity and may be an important requirement for the function of this enzyme.

Enhanced phospholipase D activity and altered morphology in RhoA-overexpressing RAT1 fibroblasts.

Malcolm KC, Sable CL, Elliott CM, Exton JH
Biochem Biophys Res Commun. 1996; 225: 514-9

Small GTP-binding proteins of the Rho family are implicated in the regulation of phospholipase D (PLD). However, few studies have addressed their role in agonist-stimulated PLD activity in vivo. Stable lines of Rat1 fibroblasts overexpressing RhoA were shown to have altered morphology. Moreover, they demonstrated increased PLD activity when stimulated with lysophosphatidic acid, platelet-derived growth factor, and phorbol ester, compared with vector-transfected cells. However, phosphoinositide phospholipase C activity was unaltered by overexpression of RhoA. These data indicate a critical downstream role for RhoA in agonist-stimulated PLD activity in intact cells.

Inhibition of phospholipase D activity by fodrin. An active role for the cytoskeleton.

Lukowski S, Lecomte MC, Mira JP, Marin P, Gautero H, Russo-Marie F, Geny B
J Biol Chem. 1996; 271: 24164-71

Phospholipase D (PLD) is a major enzyme implicated in important cellular processes such as secretion and proliferation. The knowledge of its regulation is essential to understand the control of these phenomena. Several proteins activating PLD have been described in the last years. In this report, we chromatographed bovine brain cytosolic proteins to identify fodrin, the non-erythroid spectrin, as the first described inhibitor of PLD. A cytosolic fraction with an inhibitory effect on PLD activity loses its capacity after immunoprecipitation of fodrin. Moreover, at 1 nM, purified fodrin blocks fully and quickly PLD activity, whatever the stimuli used. In contrast, fodrin has no effect on adenylate cyclase activity. Fodrin-analogous proteins like dimeric or tetrameric erythroid spectrin have the same inhibitory effect on PLD, at higher concentrations. Other cytoskeletal proteins, actin and vimentin, are inefficient on PLD inhibition. The mechanisms implicated in PLD modulation such as post-translational modifications of fodrin and the role of small G-proteins on the cytoskeleton regulation are discussed. In conclusion, this study reveals that fodrin is involved in the control of PLD activity, suggesting that the cytoskeleton could have an active role in control of secretion and proliferation.

Glycosylphosphatidylinositol: an anchor for eukaryotic membrane proteins.

Low MG
Biochem Soc Trans. 1988; 16: 271-2

Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2.

Lopez I, Arnold RS, Lambeth JD
J Biol Chem. 1998; 273: 12846-52

Phospholipase D (PLD) has been implicated in a variety of cellular processes including vesicular transport, the respiratory burst, and mitogenesis. PLD1, first cloned from human, is activated by small GTPases such as ADP-ribosylation factor (ARF) and RhoA. Rodent PLD2, which is approximately 50% identical to PLD1 has recently been cloned from mouse embryo (Colley, W., Sung, T., Roll, R., Jenco, J., Hammond, S., Altshuller, Y., Bar-Sagi, D., Morris, A., and Frohman, M. (1997) Curr. Biol. 7, 191-201) and rat brain (Kodaki, T., and Yamashita, S. (1997) J. Biol. Chem. 272, 11408-11413). We describe herein the cloning from a B cell library and expression of human PLD2 (hPLD2). The open reading frame is predicted to encode a 933-amino acid protein (Mr of 105,995); this corresponds to the size of the protein expressed in insect cells using recombinant baculovirus. The deduced amino acid sequence shows 53 and 90% identity to hPLD1 and rodent PLD2, respectively. The mRNA for PLD2 was widely distributed in various tissues including peripheral blood leukocytes, and the distribution was distinctly different from that of hPLD1. hPLD1 and hPLD2 both showed a requirement for phosphatidylinositol 4,5-bisphosphate. Both isoforms showed optimal activity at 10-20 mol % phosphatidylcholine in a mixed lipid vesicle system and showed comparable basal activities in the presence of phosphatidylinositol 4,5-bisphosphate. Unexpectedly, ARF-1 stimulated the activity of hPLD2 expressed in insect cells about 2-fold, compared with a 20-fold stimulation of hPLD1 activity. Thus, not only PLD1 but also hPLD2 activity can be positively regulated by both phosphatidylinositol 4,5-bisphosphate and ARF.

The C terminus of mammalian phospholipase D is required for catalytic activity.

Liu MY, Gutowski S, Sternweis PC
J Biol Chem. 2001; 276: 5556-62

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.

Localization and possible functions of phospholipase D isozymes.

Liscovitch M, Czarny M, Fiucci G, Lavie Y, Tang X
Biochim Biophys Acta. 1999; 1439: 245-63

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.

Enzymology of mammalian phospholipases D: in vitro studies.

Liscovitch M, Chalifa-Caspi V
Chem Phys Lipids. 1996; 80: 37-44

The existence of multiple forms of phopholipase D was clearly established in a large number of biochemical studies that described and characterized the enzymological properties of the different PLD activities. This review summarizes the in vitro evidence showing differential subcellular localization and chromatographic properties of putative PLD isozymes, their phospholipid and alcohol substrate specificities, their modulation by various divalent cations, small G proteins and protein kinase c isozymes, and the role of phosphatidylinositol 4,5-bisphosphate as a cofactor of phospholipase D.

Phospholipase D-mediated hydrolysis of phosphatidylcholine: role in cell signalling.

Liscovitch M, Ben-Av P, Danin M, Faiman G, Eldar H, Livneh E
J Lipid Mediat. 1993; 8: 177-82

Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD) by a variety of receptor agonists and in many cell types. The signal-dependent formation of phosphatidic acid (PA), by PLD-catalyzed hydrolysis of phosphatidylcholine (PC), may represent a novel and ubiquitous signal transduction pathway in mammalian cells. The mode(s) of coupling between agonist receptors and PLD activation are not well understood. Studies utilizing NIH-3T3 fibroblasts indicated that PLD activation by different mitogens involves distinct mechanisms. Protein kinase C (PKC) seems to play a role both as a mediator and as a modulator of PLD activation. The role of PKC was further examined in Swiss/3T3-derived fibroblasts which stably overexpress PKC-alpha. In these cells, both basal and agonist-stimulated PLD activity are higher than in control cells. In vitro analysis of PLD activity in detergent-solubilized cell membranes, utilizing exogenous C6-NBD-PC as fluorescent substrate, showed nearly 2-fold higher activity in membranes from cells that overexpress PKC-alpha. These results suggest that PKC-alpha may play a role in regulating PLD expression. The PLD product PA was identified as a precursor of 'late phase' diacylglycerol which, at least in some cases, was temporally correlated and causally related to the sustained activation of PKC. However, PA may itself act as an intracellular messenger in its own right, although immediate targets for its action have not yet been identified. Activation of phosphoinositide-phospholipase C, PLD and phospholipase A2 seems to comprise a signaling cascade which is typically utilized by most (if not all) Ca(2+)-mobilizing agonists.

Phospholipase D: role in signal transduction and membrane traffic.

Liscovitch M
J Lipid Mediat Cell Signal. 1996; 14: 215-21

The activation of phospholipase D (PLD) in response to cell stimulation by extracellular signal molecules is a widespread phenomenon. A variety of extracellular signal molecules cause a rapid and dramatic stimulation of PLD activity. G proteins and protein kinases appear to be involved in the receptor-mediated regulation of PLD. There is indirect evidence for the existence of multiple PLD subtypes, both membrane-associated and cytosolic. Recent studies indicate that PLD activities require a lipid cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2). Addition of PIP2 at physiological concentrations stimulates both membrane-associated and partially purified PLD activity. Other acidic phospholipids have little or no effect. Neomycin, a high affinity ligand of PIP2, inhibits membrane PLD activity, presumably by binding to endogenous PIP2. A monoclonal antibody to phosphatidylinositol 4-kinase inhibits PIP2 synthesis in permeabilized U937 cells and blocks PLD activation by GTP gamma S and TPA. These results indicate that PIP2 synthesis is required for G protein- and protein kinase C-mediated activation of PLD in the cells. Recent evidence has implicated PLD and phosphoinositide kinases in vesicular trafficking. The main lipid mediator produced by PLD, phosphatidic acid, could regulate membrane traffic events by direct regulation of target proteins involved in vesicle targeting, docking and fusion. In addition, under certain circumstances, the formation of phosphatidic acid may lead to changes in lipid bilayer properties that would facilitate vesicle budding and fusion events in the course of intracellular membrane traffic.

Phospholipase D in rat myocardium: formation of lipid messengers and synergistic activation by G-protein and protein kinase C.

Lindmar R, Loffelholz K
Biochem Pharmacol. 1998; 56: 799-805

Activation of phospholipase D (PLD) and phosphoinositide-specific phospholipase C (PI-PLC) by fluoride, to stimulate heterotrimeric G-proteins, and by phorbol esters, to stimulate protein kinase C (PKC), was studied in rat atria. Fluoride and 4beta-phorbol-12beta,13alpha-dibutyrate (PDB), in contrast to 4beta-phorbol-13alpha-acetate (PAc), activated PLD, catalyzing the formation of [3H]-phosphatidylethanol ([3H]-PETH), [3H]-phosphatidic acid ([3H]-PA), choline and sn-1,2-diacylglycerol (DAG). Basal PLD activity was resistant to drastic changes in Ca2+ and to Ro 31-8220, a PKC inhibitor, but was decreased by genistein, an inhibitor of tyrosine kinase, and increased by vanadate, a tyrosine phosphatase inhibitor; both effects were, however, very small. Fluoride-evoked PLD activity was resistant to Ro 31-8220 and to genistein, but was Ca2+-dependent. The rate of fluoride-induced PLD activation was maintained for at least 60 min. In contrast, PDB-mediated PLD activity was blocked by Ro 31-8220 and was resistant to extracellular Ca2+-depletion and desensitized within ca. 15 min. PDB markedly potentiated the fluoride-evoked generation of [3H]-phosphatidylethanol and of choline, but inhibited the formation of [3H]-inositol phosphates ([3H]-IP(1-3)). Ethanol (2%) blocked the PDB-evoked generation of both [3H]-phosphatidic acid and of sn-1,2-diacylglycerol, whereas fluoride-evoked responses were reduced only to approximately 50%. In conclusion, the trimeric G-protein-PLD pathway in heart tissue did not enclose PKC activation and was long-lasting and Ca2+-dependent; there was no evidence for an involvement of tyrosine phosphorylation. However, PKC activation modulated G-protein-coupled PLD and PI-PLC activities in opposite directions. PLD activity significantly contributed to the mass production of sn-1,2-diacylglycerol in the heart. The evidence for a pathophysiological role of PLD activation in cardiac hypertrophy and in ischemic preconditioning is discussed.

Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride.

Lindmar R, Loffelholz K
Naunyn Schmiedebergs Arch Pharmacol. 1992; 346: 607-13

Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF-4; 10 mM NaF plus 10 microM AlCl3). Atria of rats and chickens were incubated with [3H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [3H]-phosphatidylethanol (PETH) was rapidly formed at the expense of [3H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca(2+)-free, EGTA-containing medium. The phorbol ester 4 beta-phorbol-12 beta, 13 alpha-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4 beta-phorbol-13 alpha-acetate (PAc) was ineffective. PDB (0.2 microM), in contrast to PAc, also increased the formation of [3H]-PtdOH and, in the presence of ethanol, of [3H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2-0.6 microM, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Ca(2+)-dependency, i.e., both effects were blocked by a Ca(2+)-free, EGTA-containing medium, but not by a Ca(2+)-free medium without EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis.

Lennartz MR
Int J Biochem Cell Biol. 1999; 31: 415-30

Phagocytosis, the process by which leukocytes recognize and destroy invading pathogens, is essential for host defense. The binding of foreign organisms to phagocytic leukocytes initiates a complex signaling cascade which ultimately results in the entrapment and destruction of the pathogen. The signal transduction pathway mediating phagocytosis is the subject of intense investigation and is known to include protein tyrosine kinases, GTP-binding proteins, protein kinase C (PKC), actin polymerization and membrane movement. A rapidly expanding body of evidence suggests that phospholipases play an integral role in phagocytosis by generating essential second messengers. Here we review the data linking activation of phospholipase A2 (PLA2), phospholipase C (PLC) phospholipase D (PLD), and phosphoinositide 3-OH kinase (PI(3)K) to antibody (IgG)-mediated phagocytosis. Evidence is presented that (1) PLA2-derived arachidonic acid (AA) stimulates NADPH oxidase and membrane redistribution during phagocytosis, (2) the inositol-3,4,5-triphosphate (IP3) and diacylglycerol (DAG) products of PLC activate NADPH oxidase and PKC, and (3) sequential activation of PLD and phosphatidic acid phosphohydrolase may provide an alternative pathway for generation of DAG. Additionally, considerable evidence exists that wortmannin, a PI(3)K inhibitor, depresses phagocytosis. This finding is discussed in the context of the extensive effects PI(3)K products have on endocytosis and exocytosis and the potential role of membrane redistribution in phagocytosis. Finally, a model is presented which integrates data obtained from a variety of phagocytic systems and illustrates potential interactions that may exist between phospholipase-derived second messengers and signaling events required for phagocytosis.

Identification and measurement of rat eosinophil phospholipase D. Its activity on schistosomula phospholipids.

Lempereur C, Capron M, Capron A
J Immunol Methods. 1980; 33: 249-60

A sensitive assay, using [14C]lecithin as a substrate, has been developed for the measurement of phospholipase activity in rat peritoneal polymorphonuclear leukocytes. Cell extracts were found to contain a phospholipase D activity and indirect evidence suggested that eosinophils are responsible for the cleavage of lecithin. Intact peritoneal cells were also able to hydrolyze exogenous [14C]lecithin in vitro. When [3H]choline-labeled schistosomula were used as targets in antibody-dependent cytotoxicity experiments, the radioactivity of lecithin decreased more rapidly in a complete cytotoxicity system than in controls, suggesting that hydrolysis of schistosomula phospholipids occurred during the killing process.

The first crystal structure of a phospholipase D.

Leiros I, Secundo F, Zambonelli C, Servi S, Hough E
Structure Fold Des. 2000; 8: 655-67

BACKGROUND: The phospholipase D (PLD) superfamily includes enzymes that are involved in phospholipid metabolism, nucleases, toxins and virus envelope proteins of unknown function. PLD hydrolyzes the terminal phosphodiester bond of phospholipids to phosphatidic acid and a hydrophilic constituent. Phosphatidic acid is a compound that is heavily involved in signal transduction. PLD also catalyses a transphosphatidylation reaction in the presence of phosphatidylcholine and a short-chained primary or secondary alcohol. RESULTS: The first crystal structure of a 54 kDa PLD has been determined to 1.9 A resolution using the multiwavelength anomalous dispersion (MAD) method on a single WO(4) ion and refined to 1.4 A resolution. PLD from the bacterial source Streptomyces sp. strain PMF consists of a single polypeptide chain that is folded into two domains. An active site is located at the interface between these domains. The presented structure supports the proposed superfamily relationship with the published structure of the 16 kDa endonuclease from Salmonella typhimurium. CONCLUSIONS: The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function. The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.

Cloning and direct G-protein regulation of phospholipase D from tobacco.

Lein W, Saalbach G
Biochim Biophys Acta. 2001; 1530: 172-83

Phospholipase D (PLD) and heterotrimeric G-proteins are involved in plant signal transduction pathways at the plasma membrane. There is evidence suggesting that PLD acts downstream from G-proteins, but a direct interaction of specific members has not been shown. In the present paper, a PLD cDNA clone was isolated from tobacco, expressed as a GST fusion in bacteria, and the recombinant protein was purified by glutathione affinity. Its enzymatic properties identified it as an alpha-type PLD. The alpha-subunit of a G-protein from tobacco was isolated in a similar way. Both proteins were functional in biochemical assays. When the G-protein was included in the PLD assay, a strong dosage-dependent inhibition of the PLD activity was observed. Different control proteins did not exhibit this inhibitory effect. When GST-NtGPalpha1 was activated by incubation with GTPgammaS the inhibitory activity was greatly reduced. These results provide a first indication for a direct regulation of PLDalpha by a heterotrimeric G-protein alpha-subunit in plants.

[Action of phospholipase D on the lecithin liposome which contains L-alpha-phosphatidylinositol and cholesterol]

Lee EO, Nakagaki M
Yakugaku Zasshi. 1986; 106: 371-7

Receptor-mediated signalling pathways acting through hydrolysis of membrane phospholipids in cardiomyocytes.

Lamers JM, De Jonge HW, Panagia V, Van Heugten HA
Cardioscience. 1993; 4: 121-31

The aim is to summarize briefly the evidence for the existence and possible functions of receptor-mediated activity of phospholipases C and D in the myocardium. Muscarinic, alpha 1-adrenergic, angiotensin II, endothelin-1, thrombin, adenine nucleotide and opioid peptide receptors are all linked through GTP-binding proteins to phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) in the myocardium. Events that are not linked to receptors, such as mechanical loading (stretching) of cardiomyocytes, can also activate phospholipase C. The high capacity for resynthesis of PIP2 maintains the pool of PIP2, even during maximal activation of phospholipase C. Activation of phospholipase C by endothelin-1, alpha 1-adrenoceptor and angiotensin II, is subject to different rates of homologous desensitization. Protein kinase C is probably not involved in the desensitization of the response to endothelin-1. One of the products of the hydrolysis of PIP2, inositol 1,4,5-trisphosphate (IP3), releases Ca2+ from the sarcoplasmic reticulum. This intracellular response seems to be causally related to positive inotropy. The phosphorylated product of IP3, inositol 1,3,4,5-tetrakisphosphate (IP4), is believed to play a role in the handling of intracellular Ca2+, as well as in the inotropic response; however, its formation is controversial. At present the oscillations in the level of intracellular Ca2+ underlying, for example, the positive inotropy induced by alpha 1-adrenoceptors or endothelin are not clearly identified. The other product of phospholipase C, 1,2-diacylglycerol, activates Ca(2+)-dependent protein kinase C and potentially controls a wide array of cellular functions such as ion transport, myofibrillar Ca2+ sensitivity, "cross-talk" between phospholipases C and D, gene expression, protein synthesis and hypertrophic cell growth. Alterations in the fatty acid composition, particularly the polyunsaturated fatty acids, modify the phosphoinositide response induced by hormones. Cultured cardiomyocytes, incubated in sera containing the fatty acids 18:2n-6 or 20:5n-3, but not 18:0 and 18:1n-9, show a decrease in the phospholipase C responses mediated by alpha 1-adrenoceptors. The fatty acid composition of myocardial phosphatidyl inositol 4-monophosphate (PIP) and PIP2 differs from that of phosphatidylinositol, which indicates that phosphatidylinositol kinases have a certain substrate specificity or have access to localized phosphatidylinositol molecules. The estimation of the level of stimulated 1,2-diacylglycerol is complicated by the contribution of the activity of receptor-mediated phospholipase D. The identification of the molecular species of 1,2-diacylglycerol is crucial in establishing the roles and the sources of 1,2-diacylglycerol. The fatty acids covalently bound in the membrane phospholipids may also influence phospholipases C and D.(ABSTRACT TRUNCATED AT 400 WORDS)

Potent activation of phospholipase D by phenylarsine oxide in rat basophilic leukemia (RBL-2H3) cells.

Kumada T, Nakashima S, Miyata H, Nozawa Y
Biochem Biophys Res Commun. 1994; 199: 792-8

A putative protein tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), potentiated phospholipase D (PLD) activity concentration-dependently in [3H] oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells without significant increase in phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Although PAO induced tyrosine phosphorylation of several proteins, both PAO-induced PLD activation and tyrosine phosphorylation were not affected by a protein tyrosine kinase inhibitor, genistein. Another tyrosine kinase inhibitor, herbimycin A, prevented the PAO-induced PLD stimulation but had no effect on protein tyrosine phosphorylation. However, depletion of protein kinase C (PKC) greatly reduced PAO-stimulated PLD activity. These results indicate that PKC but not tyrosine kinase may be involved in PAO-mediated PLD activation.

Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles.

Ktistakis NT, Brown HA, Waters MG, Sternweis PC, Roth MG
J Cell Biol. 1996; 134: 295-306

Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi-enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evidence that cytosolic ARF is not necessary for initiating coat assembly on Golgi membranes from cell lines with high constitutive PLD activity. Conditions are also described under which ARF is at most a minor component relative to coatomer in coated vesicles from all cell lines tested, including Chinese hamster ovary cells. Formation of coated vesicles was sensitive to ethanol at concentrations that inhibit the production of phosphatidic acid (PA) by PLD. When PA was produced in Golgi membranes by an exogenous bacterial PLD, rather than with ARF and endogenous PLD, coatomer bound to Golgi membranes. Purified coatomer also bound selectively to artificial lipid vesicles that contained PA and phosphatidylinositol (4,5)-bisphosphate (PIP2). We propose that activation of PLD and the subsequent production of PA are key early events for the formation of coatomer-coated vesicles.

Signalling molecules and the regulation of intracellular transport.

Ktistakis NT
Bioessays. 1998; 20: 495-504

A variety of signalling molecules has been implicated over the past 8 years in the regulation of intracellular transport pathways. Those molecules include heterotrimeric GTP binding proteins, members of the protein kinase C family, and members of the Rho subfamily of small GTPases. Until recently, no common theme among the three classes of regulators was apparent. The finding that all three can influence the activity of phospholipase D (PLD), and the fact that members of the Arf subfamily of GTPases (with established roles in intracellular transport) are potent activators of PLD suggests the hypothesis that PLD is a focal point for integration of cellular responses to hormone signalling and for membrane homeostasis. Work during the past 2 years is beginning to uncover some transport pathways where PLD involvement is inferred. It is proposed that, if signalling is required to monitor and adjust transport rates to and from the various membrane organelles, the most economical way to achieve this would be to regulate recycling and allow the concentration of cargo receptors to determine forward transport.

Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes.

Kreger AS, Bernheimer AW, Etkin LA, Daniel LW
Infect Immun. 1987; 55: 3209-12

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.

Effect of retinoic acid on prostaglandin F2 alpha-induced phospholipase D activity in osteoblast-like cells.

Kozawa O, Suzuki A, Shinoda J, Oiso Y
Prostaglandins Leukot Essent Fatty Acids. 1996; 55: 151-4

We previously reported that prostaglandin F2 alpha (PGF2 alpha) activates phosphatidylcholine-hydrolyzing phospholipase D independently from the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells, and reported that pertussis toxin-sensitive GTP-binding protein (G-protein) is involved in the PGF2 alpha-induced phospholipase D activation. In this study, we examined the effect of retinoic acid (RA) on the phospholipase D activity stimulated by PGF2 alpha in these cells. The pretreatment of RA markedly inhibited the formation of choline induced by PGF2 alpha (10 microM) in a dose-dependent manner in the range between 1 nM and 0.1 microM. This inhibitory effect of RA was dependent on the time of pretreatment up to 8 h. However, RA had little effect on the choline formation induced by NaF, a G-protein activator, or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. These results strongly suggest that RA suppresses the phospholipase D activated by PGF2 alpha in osteoblast-like cells and that the effect of RA is exerted at the point between PGF2 alpha receptor and G-protein.

Phospholipase D activity in the Tetrahymena pyriformis GL.

Kovacs P, Csaba G, Nakashima S, Nozawa Y
Cell Biochem Funct. 1997; 15: 53-60

Phospholipase D (PLD) is an enzyme which participates in the signaling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signaling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena.

Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins.

Kotter K, Ji a S, von Eichel-Streiber C, Park JB, Ryu SH, Klein J
Biochim Biophys Acta. 2000; 1485: 153-62

Primary cultures of rat cortical astrocytes express phospholipase D (PLD) isoforms 1 and 2 as determined by RT-PCR and Western blot. Basal PLD activity was strongly (10-fold) increased by 4beta-phorbol-12beta,13alpha-dibutyrate (PDB) (EC(50): 56 nM), an effect which was inhibited by Ro 31-8220 (0.1-1 microM), an inhibitor of protein kinase C (PKC), and by brefeldin A (10-100 microg/ml), an inhibitor of ADP-ribosylating factor (ARF) activation. Pretreatment of the cultures with Clostridium difficile toxin B-10463 (0.1-1 ng/ml), which inactivates small G proteins of the Rho family, led to a breakdown of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50%. In contrast, inactivation of proteins of the Ras family by Clostridium sordellii lethal toxin 1522 did not affect PLD activation. In parallel experiments, serum-induced PLD activation was sensitive to brefeldin A, but not to Ro 31-8220 and not to clostridial toxins. We conclude that, in astrocytes, the PLD isoform which is activated by phorbol ester requires PKC, ARF and Rho proteins for full activity and probably represents PLD1.

Hydrolysis of phospholipid monolayers by phospholipase D at the oil/water interface under the control of the potential drop across the monolayer.

Kondo T, Kakiuchi T, Senda M
Biochim Biophys Acta. 1992; 1124: 1-6

A new method has been proposed for measuring the enzymatic hydrolysis of phosphatidylcholine (PC) monolayers formed at the polarized nitrobenzene(NB)/water(W) interface under the precise control of the potential drop across the interface. As a probe for the hydrolysis, the method utilized the capacitance (Cd1) of the monolayer. Phospholipase D (EC. 3.1.44., PLD) converted L-alpha-dipalmitoylphosphatidylcholine (DPPC) in the monolayer to L-alpha-dipalmitoylphosphatidic acid (DPPA), leading to a drastic decrease in Cdl. This change in Cdl was sensitive enough to monitor the course of enzymatic hydrolysis of the PC monolayer by PLD. The rate of the hydrolysis was markedly dependent on the potential drop across the interface. When the potential of the aqueous phase with respect to that of the NB phase (delta W0 phi) was -140 mV, no hydrolysis was observed, whereas at delta W0 phi = 60 mV the hydrolysis proceeded promptly.

Cloning, expression, and characterization of a novel phospholipase D complementary DNA from rat brain.

Kodaki T, Yamashita S
J Biol Chem. 1997; 272: 11408-13

Phospholipase D (PLD) is implicated in important cellular processes such as signal transduction, membrane trafficking, and mitosis regulation. Recently, cDNA for human PLD1 (hPLD1) was cloned from HeLa cells (Hammond, S. M., Altshuller, Y. M., Sung, T-C., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). hPLD1 is stimulated by phosphatidylinositol 4,5-bisphosphate and the small GTP-binding protein known as ADP-ribosylation factor 1. Here we report the cloning and characterization of cDNA for a different type of PLD (rat PLD2 (rPLD2)) from rat brain. We synthesized highly degenerate amplimers corresponding to the conserved regions of eukaryote PLDs and performed polymerase chain reaction on a rat brain cDNA library. Using the amplified sequence as the probe, we cloned a rat brain cDNA clone that contained an open reading frame of 933 amino acids with an Mr of 105,992. The deduced amino acid sequence showed significant similarity to hPLD1 with a large deletion in the middle of the sequence. When the sequence was expressed in the fission yeast Schizosaccharomyces pombe, PLD activity was greatly increased. The activity was markedly stimulated by phosphatidylinositol 4, 5-bisphosphate, but not by ADP-ribosylation factor 1 and RhoA. Rat brain cytosol known to stimulate small GTP-binding protein-dependent PLD did not stimulate rPLD2 expressed in S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and testis, but at low levels in thymus, liver, and muscle. Only a negligible level was found in spleen and pancreas. Thus rPLD2 is a novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and RhoA.

Dexamethasone-induced reduction of phospholipase D activity in the rat. Possible role of lipocortin.

Kobayashi M, Bansal VS, Singh I, Kanfer JN
FEBS Lett. 1988; 236: 380-2

Subcutaneous injection of dexamethasone resulted in a reduction of demonstrable phospholipase D activity of rat brain and liver microsomes. Partially purified rat lung lipocortin inhibited the activity of both microsomal and partially purified rat brain phospholipase D. These results show that phospholipase D activity is suppressed by dexamethasone and one of the possible mechanisms of inhibition may be a phospholipase inhibitory protein, lipocortin.

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata).

Kim DU, Roh TY, Lee J, Noh JY, Jang YJ, Hoe KL, Yoo HS, Choi MU
Biochim Biophys Acta. 1999; 1437: 409-14

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

Kanoh H, Nakashima S, Zhao Y, Sugiyama Y, Kitajima Y, Nozawa Y
Biochim Biophys Acta. 1998; 1398: 359-64

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

[Molecular cloning of Candida albicans phospholipase D]

Kanoh H, Nakashima S, Kitajima Y, Nozawa Y
Nippon Ishinkin Gakkai Zasshi. 1998; 39: 79-83

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.

Acidic phospholipids inhibit the phospholipase D activity of rat brain neuronal nuclei.

Kanfer JN, McCartney DG, Singh IN, Freysz L
FEBS Lett. 1996; 383: 6-8

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 microM for PIP2 to 200 microM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

The base exchange enzymes and phospholipase D of mammalian tissue.

Kanfer JN
Can J Biochem. 1980; 58: 1370-80

The base exchange enzymes and phospholipase D represent a group of enzymes which alter the polar portion of phospholipids. The base exchange enzymes provide a mechanism of interconverting different species of phospholipids whereas phospholipase D hydrolyzes these phospholipids to phosphatidic acid. Although the occurrence of these activities is widespread, this article is restricted to a description of the mammalian enzymes. Originally it was believed that these catalytic activities resided in a single enzyme. As a result of successes in partial purifications and separations it is clear that separate enzymes exist for the exchange of serine, for the exchange of choline, and for the exchange of ethanolamine resulting in their appearance in the corresponding phospholipid. These enzyme activities are not a reflection of phospholipase D action. Conversely, a partially purified phospholipase D from rat brain was devoid of detectable base exchange activity. However, this enzyme preparation possessed transphosphatidylation activity producing phosphatidylglycerol from glycerol and lecithin. These enzymes are presumed to be functionally significant for cellular homeostasis; however, strong evidence supporting this contention is unavailable. The base exchange enzymes appear to be concentrated in the "microsomal" fraction of tissues. The choline base exchange enzyme is located on the cytoplasmic surface while the serine and ethanolamine base exchange enzymes are located on the luminal surface of the endoplasmic reticulum of rat brain tissue. The lipid environments in which the individual enzymes reside are also distinctly different suggesting that their in situ domains within the microsomal membrane are dissimilar. Therefore it appears that the enzymes are distinctly separate catalytic entities. They also are in topographically different locations on the microsomal membrane and reside in different lipid environments as well. These observations would imply functionally distinct roles for the separate enzymes. Evidence is available which suggests that their existence might be demonstrable in vivo.

Angiotensin II-mediated stimulation of phospholipase D in rabbit kidney proximal tubule cells.

Jung JH, Jung JC, Chung SH
Arch Pharm Res. 1994; 17: 405-10

The present study was undertaken to demonstrate whether or not angiotensin II activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [3H]phosphatidic acid and [3H]phosphatidylethanol, we elucidate the direct stimulation of phospholipase D by angiotensin II. Angiotensin II leads to a rapid increase in [3H]phosphatidic acid and [3H]diacylglycerol, and [3H]phosphatidic acid formation preceded the formation of [3H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from the action of diacylglycerol kinase on the diacylglycerol. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated by extracellular calcium ion. It has also been shown that angiotensin II may activate phospholipase D through protein kinase C-independent pathway.

Glycosyl-phosphatidylinositol-phospholipase type D: a possible candidate for the generation of second messengers.

Jones DR, Avila MA, Sanz C, Varela-Nieto I
Biochem Biophys Res Commun. 1997; 233: 432-7

Membrane associated glycosyl-phosphatidylinositols have been shown to be the precursors of inositol phosphoglycan second messengers. Extraction of human liver membranes and purification by serial thin layer chromatography revealed three glycolipids which co-migrated with glycosyl-phosphatidylinositol from rat liver. These lipidic fractions were partially sensitive to treatment with nitrous acid and to hydrolysis by glycosyl-phosphatidylinositol-specific phospholipase D from bovine serum. In parallel, glycosyl-phosphatidylinositol isolated from rat liver was found to be a substrate for the enzyme generating a biologically active inositol phosphoglycan species (determined by measuring inhibition of protein kinase A activity and stimulation of cell proliferation within the chicken embryo cochleovestibular ganglion). This molecule was recognised by an anti-inositol phosphoglycan antibody. Hence, we propose that glycosyl-phosphatidylinositol-specific phospholipase D could be implicated in cellular signalling.

Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain.

Iwasaki Y, Horiike S, Matsushima K, Yamane T
Eur J Biochem. 1999; 264: 577-81

Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-?6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)-amino]hexanoyl?-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile.

Identification and partial characterization of phospholipase D in the human amniotic membrane.

Inamori K, Sagawa N, Hasegawa M, Itoh H, Ueda H, Kobayashi F, Ihara Y, Mori T
Biochem Biophys Res Commun. 1993; 191: 1270-7

The enzymatic activity of phospholipase D and its characteristics have been examined in human amnion tissue. The phospholipase D activity was not Ca(2+)- or Mg(2+)-dependent and was activated by unsaturated fatty acids. The optimal pH of phospholipase D was 5.5. The phospholipase D activity in amnion tissue was highest in the microsomal fraction, and preferentially utilized phosphatidylcholine as a substrate. The phospholipase D activity of the microsomal fraction of amnion tissue obtained at term before labor onset (34.0 +/- 16.3 nmol/hour/mg protein, mean +/- SD, n = 11) was significantly (p < 0.05) higher than the activity in this tissue obtained from women in the mid-trimester (15.0 +/- 7.5 nmol/hour/mg protein, n = 9).

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A.

Huang Y, Zhang XY, Liu F, Chen HL
Mol Cell Biochem. 2000; 207: 3-8

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTPgamma[S], GDP and ATP for maximal activation were 0.1 mM, 5 microM, 1 mM and 1 mM respectively. The activation caused by 1 mM ADP was lower. The enzyme was not activated by 1 mM AMP, but significant activation was observed by the addition of 1 mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A abolished the activation. There were synergic effects between ATP and GTP, ATP and PIP2, but not between ATP and GTPgamma[S], or PIP2 and GTPgamma[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP2 scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP2 is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.

Phospholipase D hydrolyzes ether- and ester-linked glycerophospholipids by different pathways in MDCK cells.

Huang C, Wykle RL, Daniel LW
Biochem Biophys Res Commun. 1995; 213: 950-7

MDCK cells were prelabeled with 1-O-[3H]hexadecyl-2-lyso-GPC and [14C]myristic acid, which selectively labeled the glycerophospholipid subclasses with 93% of tritium in the alkyl-linked subclass and 85% of carbon-14 in the diacyl-linked subclass. By this approach, we have demonstrated that PLD upon activation via PKC pathway selectively catalyzes the degradation of ether-linked glycerophospholipid subclass. In contrast, G-protein regulatory PLD activity seems to preferentially hydrolyze ester-linked subclass. These results suggest that the selective hydrolysis of PLD action may play an important role in cellular signal transduction under physiological and pathological conditions.

Small GTPase-regulated phospholipase D in granulocytes.

Houle MG, Bourgoin S
Biochem Cell Biol. 1996; 74: 459-67

This review examines the functional role of phospholipase D in the neutrophil. Phospholipase D is emerging as an important component in the signal transduction pathways leading to granulocyte activation. Through the second messenger it produces, phosphatidic acid, phospholipase D plays an active role in the regulation of granulocyte NADPH oxidase activation and granular secretion. Many factors from both the cytosol and the membrane are necessary for maximal phospholipase D activation. This paper will focus on the regulation of phospholipase D by low molecular weight GTP-binding proteins, tyrosine kinases, and protein kinase C.

Regulation of phospholipase D by phosphorylation-dependent mechanisms.

Houle MG, Bourgoin S
Biochim Biophys Acta. 1999; 1439: 135-49

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4, 5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Calpha as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.

[Phospholipid metabolism regulated by heterotrimeric G proteins]

Homma Y
Nippon Yakurigaku Zasshi. 1994; 103: 295-304

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 induces the release of Ca2+ from intracellular stores, and diacylglycerol acts as the physiological activator of protein kinase C. Several distinct PI-PLC enzymes have been identified from various cells. Based on the primary sequences, PI-PLC isozymes are divided into three families: PLC-beta, PLC-gamma, and PLC-delta. Substantial evidence has strongly suggested that G proteins regulate PI-PLC in various cell-stimulation systems and that there might be two distinct pathways (pertussis toxin-sensitive and pertussis toxin-insensitive). Recently, it has become apparent that beta-type PLC isoforms are activated by the heterotrimeric G protein subfamily Gq. Careful studies using in vitro and in vivo reconstitution systems have further suggested that the alpha-subunits of Gq/11/16 specifically regulate PLC-beta 1 and PLC-beta 3 and that the beta gamma -subunits of the Gi subfamily interact with PLC-beta 2, which are considered to be responsible for the pertussis toxin-insensitive and the pertussis toxin-sensitive pathways, respectively. In this paper, involvement of G proteins in the regulation of phospholipase A2 and phosphatidylcholine-specific PLC and PLD is also discussed.

Alcohol and membrane-associated signal transduction.

Hoek JB, Rubin E
Alcohol Alcohol. 1990; 25: 143-56

In recent years, ethanol has been shown to interact with membrane-associated signal transduction mechanisms which rely on the reaction of phospholipases with their phospholipid substrates in the membrane. In several cell and membrane preparations, ethanol activates the polyphosphoinositide-specific phospholipase C and triggers the complete battery of intracellular signalling responses that are characteristic for hormones acting through this pathway, including the formation of inositol-1,4,5-trisphosphate, the release of Ca2+ from intracellular storage sites with the consequent activation of cytosolic Ca2(+)-dependent enzymes, and the formation of diacylglycerol leading to the stimulation of protein kinase C. The activation of phospholipase C appears to be due to an interaction of ethanol with the intramembrane complex of receptor-G-protein-phospholipase C, presumably promoting the release of bound GDP and the binding of GTP to activate the G-protein which controls phospholipase C activity. In many intact cells, the phospholipase C is subject to a feedback inhibitory control by protein kinase C. In liver cells, ethanol also triggers this feedback inhibition, leading to a rapid decline in the phospholipase C activation; at the same time, ethanol also causes the desensitization of the response to vasopressin and other phospholipase C-linked agonists. At hormone concentrations in the physiological range, the heterologous desensitization by ethanol of the agonist-mediated phospholipase C activation may be a significant factor at ethanol concentrations that are readily attained in vivo. Further interaction of ethanol with the intracellular second messenger system is mediated through a hormone-sensitive phospholipase D. This enzyme uses phosphatidylcholine to generate phosphatidic acid which can be further converted to diacylglycerol. In the presence of ethanol the enzyme catalyzes the transphosphatidylation to phosphatidylethanol. It is not clear, however, under what conditions this process could affect the normal pattern of formation of second messenger molecules. After chronic ethanol intake, a tolerance can develop at the cellular level to the effects of ethanol on agonist-induced signal transduction processes. However, the mechanism by which this tolerance develops is currently a matter of conjecture. Studies on liver cells indicate that the activity of protein kinase C may play a role in the development of this type of tolerance to ethanol. A better understanding of the interaction of ethanol with these phospholipid-dependent signal transduction processes could point to mechanisms by which ethanol could interfere with physiological control mechanism in a variety of cells and tissues.

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain.

Hodgkin MN, Masson MR, Powner D, Saqib KM, Ponting CP, Wakelam MJ
Curr Biol. 2000; 10: 43-6

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.

Role of phospholipase D in the cAMP signal transduction pathway activated during fibroblast contraction of collagen matrices.

He Y, Grinnell F
J Cell Biol. 1995; 130: 1197-205

Fibroblast contraction of stressed collagen matrices results in activation of a cAMP signal transduction pathway. This pathway involves influx of extracellular Ca2+ ions and increased production of arachidonic acid. We report that within 5 min after initiating contraction, a burst of phosphatidic acid release was detected. Phospholipase D was implicated in production of phosphatidic acid based on observation of a transphosphatidylation reaction in the presence of ethanol that resulted in formation of phosphatidylethanol at the expense of phosphatidic acid. Activation of phospholipase D required extracellular Ca2+ ions and was regulated by protein kinase C. Ethanol treatment of cells also inhibited by 60-70% contraction-dependent release of arachidonic acid and cAMP but had no effect on increased cAMP synthesis after addition of exogenous arachidonic acid or on phospholipase A2 activity measured in cell extracts. Moreover, other treatments that inhibited the burst of phosphatidic acid release after contraction--chelating extracellular Ca2+ or down-regulating protein kinase C--also blocked contraction activated cyclic AMP signaling. These results were consistent with the idea that phosphatidic acid production occurred upstream of arachidonic acid in the contraction-activated cAMP signaling pathway.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Hari T, Butikofer P, Wiesmann UN, Brodbeck U
Biochim Biophys Acta. 1997; 1355: 293-302

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.

Multiple forms of phospholipase D inhibitor from rat brain cytosol. Purification and characterization of heat-labile form.

Han JS, Chung JK, Kang HS, Donaldson J, Bae YS, Rhee SG
J Biol Chem. 1996; 271: 11163-9

Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP2). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70 degrees C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP2, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP2-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.

Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha.

Hammond SM, Jenco JM, Nakashima S, Cadwallader K, Gu Q, Cook S, Nozawa Y, Prestwich GD, Frohman MA, Morris AJ
J Biol Chem. 1997; 272: 3860-8

We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-gammaS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-alpha activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-alpha is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Hammond SM, Altshuller YM, Sung TC, Rudge SA, Rose K, Engebrecht J, Morris AJ, Frohman MA
J Biol Chem. 1995; 270: 29640-3

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

Phospholipase D in platelets and megakaryocytic cells.

Halenda SP, Wu H, Jones AW, Shukla SD
Chem Phys Lipids. 1996; 80: 21-6

Phospholipase D (PLD) is stimulated in platelets by various agents. Phosphatidylcholine is the major substrate for PLD. This enzymatic pathway generates phosphatidic acid selectively. Guanine nucleotides also stimulate PLD in platelet membranes. Furthermore, tyrosine kinase may also be involved in platelet PLD regulation. It appears that multiple signals acting sequentially or in parallel converge on PLD. Among others, PLD has been proposed to play a role in platelet secretion and PLA2 regulation. PLD is also present in platelet percursor megakaryocytric cells and can be activated by platelet agonists. In these cells both PKC and G-proteins (e.g. Rho) may regulate PLD activity. The significance of PLD in megakaryocytes awaits investigation. These recent developments offer new avenues of research to further elucidate the biochemistry of platelet and megakaryocyte function.

Regulation of phospholipase D activity in neuroblastoma cells.

Gustavsson L, Boyano-Adanez MC, Larsson C, Aradottir S, Lundqvist C
J Lipid Mediat Cell Signal. 1996; 14: 229-35

The regulation of phospholipase D was studied in human neuroblastoma cells using phosphatidylethanol as a marker of the enzyme activity. Carbachol induced phospholipase D activity in SH-SY5Y cells. Muscarinic antagonists inhibited the response with potencies suggesting that muscarinic M1 receptors are responsible for the activation. In permeabilized SH-SY5Y cells, both the carbachol- and GTP gamma S-induced Peth formation was inhibited by GDP beta S, indicating that both responses are mediated via a G-protein. The protein kinase C inhibitors, bisindolylmaleimide and staurosporine significantly inhibited the carbachol-induced Peth formation whereas H7 had no effect. Thus, the cholinergic activation of phospholipase D in SH-SY5Y cells is probably mediated via a direct receptor-G-protein coupling but an involvement of protein kinase C cannot be excluded. Calmidazolium, a calmodulin antagonist, induced an increase in phosphatidylethanol formation in both SH-SY5Y and IMR-32 cells. This effect was inhibited by genistein and tyrphostin, indicating a tyrosine kinase dependent pathway for phospholipase D activation in neuroblastoma cells.

ESBRA 1994 Award Lecture. Phosphatidylethanol formation: specific effects of ethanol mediated via phospholipase D.

Gustavsson L
Alcohol Alcohol. 1995; 30: 391-406

Phosphatidylethanol is a unique phospholipid which is formed in cell membranes only in the presence of ethanol. The reaction is catalysed by phospholipase D, an enzyme that normally catalyses the hydrolysis of phospholipids leading to the formation of phosphatidic acid. However, phospholipase D also utilizes short-chain alcohols as substrates resulting in the formation of the corresponding phosphatidylalcohol. This is a specific mechanism through which ethanol may interact with cell function. Phospholipase D is activated by several different receptors and has during recent years been suggested to play a role in cellular signalling. Secretory processes as well as long-term changes of cell function have been associated with the activation of phospholipase D. Since ethanol competes with water as a substrate for this enzyme, phosphatidylethanol is formed at the expense of the normal lipid product, phosphatidic acid, in an ethanol concentration-dependent manner. Therefore, the phospholipase D-mediated signal transduction diverges from production of the normal signalling lipid in the presence of ethanol. However, phosphatidic acid may also be formed by other pathways and their relative contribution to the formation of this lipid depends on the cell and receptor type. Thus, it is important to identify the signalling systems where phospholipase D dominates the lipid messenger production since these may be especially vulnerable to ethanol. In addition to a change in phospholipase D-mediated signal transduction, accumulation of phosphatidylethanol in cell membranes may also induce disturbances in cell function. Significant amounts of this abnormal phospholipid have been detected after ethanol exposure in brain and other organs from rat, in cultured cells as well as in human blood cells. The degradation of phosphatidylethanol is relatively slow and it remains in the cells after ethanol has disappeared. It is possible that an abnormal phospholipid that accumulates in cell membranes affects membrane-associated processes. Phosphatidylethanol is a lipid with a small, anionic head group and its biophysical properties are different compared with other phospholipids. Moreover, this lipid has been demonstrated to influence membrane characteristics, enzyme activities and levels of signalling molecules. Thus, both the inhibition of phospholipase D-mediated signal transduction and the accumulation of phosphatidylethanol represent possible pathways through which ethanol may disturb cell function.

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate.

Gottlin EB, Rudolph AE, Zhao Y, Matthews HR, Dixon JE
Proc Natl Acad Sci U S A. 1998; 95: 9202-7

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

Phospholipase D: a novel major player in signal transduction.

Gomez-Cambronero J, Keire P
Cell Signal. 1998; 10: 387-97

The role of the mammalian phospholipase D (PLD) in the control of key cellular responses has been recognised for a long time, but only recently have there been the reagents to properly study this very important enzyme in the signalling pathways, linking cell agonists with intracellular targets. With the recent cloning of PLD isoenzymes, their association with low-molecular-weight G proteins, protein kinase C and tyrosine kinases, the availability of antibodies and an understanding of the role of PLD product, phosphatidic acid (PA), in cell physiology, the field is gaining momentum. In this review, we will explore the molecular properties of mammalian PLD and its gene(s), the complexity of this enzyme regulation and the myriad physiological roles for PLD and PA and related metabolic products, with particular emphasis on a role in the activation of NADPH oxidase, or respiratory burst, leading to the generation of oxygen radicals.

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

Geng D, Baker DP, Foley SF, Zhou C, Stieglitz K, Roberts MF
Biochim Biophys Acta. 1999; 1430: 234-44

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 (PLD57 approximately 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). PLD42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4-6-fold higher Vmax toward PC substrates than PLD57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD42/20 were cleavage products of PLD57. The larger component included the N-terminal segment of PLD57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD57; this peptide had no PLD activity by itself. Increasing the pH of PLD42/20 to 8.9, followed by chromatography of PLD42/20 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 PLD42/20 indicates that an intact C-terminal region of PLD57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD57.

Unilateral nephrectomy selectively stimulates phospholipase D in the remaining kidney.

Gatalica Z, Moehren G, Hoek JB
Biochim Biophys Acta. 1993; 1177: 87-92

The activation of phospholipase D in the kidney could be detected in vivo in rats treated with ethanol by the accumulation of phosphatidylethanol. Unilateral nephrectomy stimulated the activity of phospholipase D in the remaining kidney as indicated by an increase in the level of phosphatidylethanol. A significant increase in phosphatidylethanol level was observed as early as 5 min after contralateral nephrectomy and peak accumulation (200% of control) was observed after 15 min. The phosphatidylethanol level decreased again to the basal level after 2 h. The accumulation of phosphatidylethanol was specific for kidney and the product was localized primarily in the cortex. Phospholipase D activity in kidney cortical slices from untreated rats was stimulated in vitro by plasma obtained from unilaterally nephrectomized rats, indicating that circulating factors in the plasma are responsible for the activation of phospholipase D. The phospholipase D activation by plasma from uninephrectomized animals was selectively inhibited by the tyrosine kinase inhibitor genistein, but not by the protein kinase C inhibitor H7. It is concluded that phospholipase D activity is stimulated as an early signal transduction event in compensatory kidney growth.

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils.

Garland LG
FEMS Microbiol Immunol. 1992; 5: 229-37

Protein kinase C (PKC) appears to have a central role in the O2- response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP2)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling to PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O2-. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.

Induction of Galphaq-specific antisense RNA in vivo causes increased body mass and hyperadiposity.

Galvin-Parton PA, Chen X, Moxham CM, Malbon CC
J Biol Chem. 1997; 272: 4335-41

Transgenic BDF-1 mice harboring an inducible, tissue-specific transgene for RNA antisense to Galphaq provide a model in which to study a loss-of-function mutant of Galphaq in vivo. Galphaq deficiency induced in liver and white adipose tissue at birth produced increased body mass and hyperadiposity within 5 weeks of birth that persisted throughout adult life. Galphaq-deficient adipocytes display reduced lipolytic responses, shown to reflect a newly discovered, alpha1-adrenergic regulation of lipolysis. This alpha1-adrenergic response via phosphoinositide hydrolysis and activation of protein kinase C is lacking in the Galphaq loss-of-function mutants in vivo and provides a basis for the increased fat accumulation.

Mammalian phospholipase D structure and regulation.

Frohman MA, Sung TC, Morris AJ
Biochim Biophys Acta. 1999; 1439: 175-86

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.

Conversion of lysophospholipids to cyclic lysophosphatidic acid by phospholipase D.

Friedman P, Haimovitz R, Markman O, Roberts MF, Shinitzky M
J Biol Chem. 1996; 271: 953-7

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.

Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum.

Frank W, Munnik T, Kerkmann K, Salamini F, Bartels D
Plant Cell. 2000; 12: 111-24

Phospholipids play an important role in many signaling pathways in animal cells. Signaling cascades are triggered by the activation of phospholipid cleaving enzymes such as phospholipases C, D (PLD), and A(2). Their activities result in the formation of second messengers and amplification of the initial signal. In this study, we provide experimental evidence that PLD is involved in the early events of dehydration in the resurrection plant Craterostigma plantagineum. The enzymatic activity of the PLD protein was activated within minutes after the onset of dehydration, and although it was not inducible by abscisic acid, PLD activity did increase in response to mastoparan, which suggests a role for heterotrimeric G proteins in PLD regulation. Two cDNA clones encoding PLDs, CpPLD-1 and CpPLD-2, were isolated. The CpPLD-1 transcript was constitutively expressed, whereas CpPLD-2 was induced by dehydration and abscisic acid. Immunological studies revealed changes in the subcellular localization of the PLD protein in response to dehydration. Taken together, the data on enzymatic activity as well as transcript and protein distributions allowed us to propose a role for PLD in the events leading to desiccation tolerance in C. plantagineum.

Ral and Rho-dependent activation of phospholipase D in v-Raf-transformed cells.

Frankel P, Ramos M, Flom J, Bychenok S, Joseph T, Kerkhoff E, Rapp UR, Feig LA, Foster DA
Biochem Biophys Res Commun. 1999; 255: 502-7

Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals. We reported previously that although the transformed phenotype induced by v-Src was dependent upon Raf-1, the PLD activity induced by v-Src was independent of Raf-1. This observation suggested to us that Raf would not likely be an activator of PLD. However, upon examination of PLD activity in v-Raf-transformed cells, surprisingly, we found that PLD activity is elevated to levels that were even higher than that observed in v-Src-transformed cells. To characterize the mechanism of v-Raf-induced PLD activity, we examined the dependence of v-Raf-induced PLD activity upon protein kinase C (PKC) the small GTPases Ral and Rho, which have all been implicated in the activation of PLD. The v-Raf-induced PLD activity was inhibited by dominant negative mutants for both Ral and Rho. The dependence upon Ral was particularly surprising since Ral is a downstream target of Ras, which is an upstream activator of Raf. Depleting cells of PKC by long term phorbol ester treatment actually increased PLD activity in v-Raf-transformed cells, indicating that v-Raf-induced PLD activity is not dependent on PKC. These data describe a novel mechanism for PLD activation by v-Raf that is independent of PKC, but dependent upon both Ral and Rho GTPases.

Regulation of phosphoinositide and phosphatidylcholine phospholipases by G proteins.

Exton JH, Taylor SJ, Blank JS, Bocckino SB
Ciba Found Symp. 1992; 164: 36-42

Two G proteins that regulate phosphoinositide phospholipase C in liver plasma membranes have been purified to homogeneity in both the heterotrimeric and dissociated forms. The heterotrimers contain a 42 kDa or 43 kDa alpha subunit and a 35 kDa beta subunit. The alpha subunits are not ADP-ribosylated by pertussis toxin and are closely related immunologically to members of the recently identified Gq class of G proteins. The specific phosphoinositide phospholipase C isozyme that responds to the G proteins has been determined to the beta 1 isozyme. GTP analogues stimulate phosphatidylcholine hydrolysis in rat liver plasma membranes. The nucleotide specificity and Mg2+ dependency of the response indicate that it is mediated by a G protein. Phosphatidic acid, diacylglycerol, choline and phosphorylcholine are the products, indicating that both phospholipase D and C activities are involved. Activation of phospholipase D is also indicated by the enhanced production of phosphatidyl-ethanol in the presence of ethanol.

Phospholipase D.

Exton JH
Ann N Y Acad Sci. 2000; 905: 61-8

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.

Cell signalling through guanine-nucleotide-binding regulatory proteins (G proteins) and phospholipases.

Exton JH
Eur J Biochem. 1997; 243: 10-20

Phospholipases are important enzymes in cell signal transduction since they hydrolyze membrane phospholipids to generate signalling molecules. Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) play a major role in their regulation by a variety of agonists that activate receptors with seven membrane-spanning domains. Phospholipases of the C type, which hydrolyze inositol phospholipids to yield inositol trisphosphate and diacylglycerol, are regulated by the alpha and betagamma subunits of certain heterotrimeric G proteins as well as by receptor-associated and non-receptor-associated tyrosine kinases. Phospholipases of the D type, which hydrolyze phosphatidylcholine to phosphatidic acid, are regulated by members of the ADP-ribosylation factor and Rho subfamilies of small G proteins, and by protein kinase C and other factors. This review presents recent information concerning the molecular details of G protein regulation of these phospholipases.

Regulation of phospholipase D.

Exton JH
Biochim Biophys Acta. 1999; 1439: 121-33

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.

Regulation and functional significance of phospholipase D in myocardium.

Eskildsen-Helmond YE, Van Heugten HA, Lamers JM
Mol Cell Biochem. 1996; 157: 39-48

There is now clear evidence that receptor-dependent phospholipase D is present in myocardium. This novel signal transduction pathway provides an alternative source of 1,2-diacylglycerol, which activates isoforms of protein kinase C. The members of the protein kinase C family respond differently to various combinations of Ca2+, phosphatidylserine, molecular species of 1,2-diacylglycerol and other membrane phospholipid metabolites including free fatty acids. Protein kinase C isozymes are responsible for phosphorylation of specific cardiac substrate proteins that may be involved in regulation of cardiac contractility, hypertrophic growth, gene expression, ischemic preconditioning and electrophysiological changes. The initial product of phospholipase D, phosphatidic acid, may also have a second messenger role. As in other tissues, the question how the activity of phospholipase D is controlled by agonists in myocardium is controversial. Agonists, such as endothelin-1, atrial natriuretic factor and angiotensin II that are shown to activate phospholipase D, also potently stimulate phospholipase C-beta in myocardium. PMA stimulation of protein kinase C inactivates phospholipase C and strongly activates phospholipase D and this is probably a major mechanism by which agonists that promote phosphatidyl-4,5-bisphosphate hydrolysis secondary activate phosphatidylcholine-hydrolysis. On the other hand, one group has postulated that formation of phosphatidic acid secondary activates phosphatidyl-4,5-bisphosphate hydrolysis in cardiomyocytes. Whether GTP-binding proteins directly control phospholipase D is not clearly established in myocardium. Phospholipase D activation may also be mediated by an increase in cytosolic free Ca2+ or by tyrosine-phosphorylation.

Exploration of the possible roles of phospholipase D and protein kinase C in the mechanism of ischemic preconditioning in the myocardium.

Eskildsen-Helmond YE, Gho BC, Bezstarosti K, Dekkers DH, Soei LK, Van Heugten HA, Verdouw PD, Lamers JM
Ann N Y Acad Sci. 1996; 793: 210-25

Involvement of phosphatidic acid, phosphatidate phosphohydrolase, and inositide-specific phospholipase D in neutrophil stimulus-response pathways.

English D
J Lab Clin Med. 1992; 120: 520-6

Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

El Maarouf H, Carriere F, Riviere M, Abousalham A
Protein Eng. 2000; 13: 811-7

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD alpha were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD alpha yielded a protein (rPLD alpha a, 88 kDa) together with a shorter form (rPLD alpha b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD alpha a and rPLD alpha b showed that rPLD alpha b resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLD alpha a and rPLD alpha b are recognized by a polyclonal antibody previously raised against native soybean PLD alpha. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD alpha is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD alpha b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD alpha b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD alpha a was eluted after chelating calcium ions with EDTA. The purified rPLD alpha yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD alpha constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.

Structural heterogeneity of phospholipase D in 10 dicots.

Dyer JH, Zheng S, Wang X
Biochem Biophys Res Commun. 1996; 221: 31-6

The occurrence of multiple forms of phospholipase D (EC 3.1.4.4) was investigated in different tissues of castor bean (Ricinus communis) and in other plant species. Phospholipase D variants were resolved by nondenaturing and isoelectric focusing polyacrylamide gel electrophoresis and detected by immunoblotting using anti-phospholipase D antibodies and by enzyme activity assay. Three phospholipase D variants were produced differentially in the roots, endosperm, cotyledons, and hypocotyl of 5-day postgermination seedlings of castor bean. Furthermore, different patterns of phospholipase D variants were found in the different regions of hypocotyl (elongated and hook). Multiple phospholipase D forms were found in florets of cauliflower and broccoli, leaves of cabbage, celery, tomato, and potato, and alfalfa sprouts, suggesting that structural heterogeneity of phospholipase D occurs widely in plants.

Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling.

Du G, Altshuller YM, Kim Y, Han JM, Ryu SH, Morris AJ, Frohman MA
Mol Biol Cell. 2000; 11: 4359-68

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.

Alkylphosphate esters as inhibitors of phospholipase D.

Dittrich N, Nossner G, Kutscher B, Ulbrich-Hofmann R
J Enzyme Inhib. 1996; 11: 67-75

Alkylphosphate esters were shown to be potent inhibitors of phospholipase D. Using phosphatidyl choline/sodium dodecylsulfate (2:1) as substrate, IC50 values were determined for alkylphosphocholines of different chain length (C10-C18) and for various octadecylphosphate esters with different polar head groups. The inhibitory potency strongly increased with increasing chain length of the alkyl chain. The substitution of choline for heterocyclic nitrogen compounds or for 2-trimethylarsonio-ethanol also affected the inhibition of phospholipase D. Octadecylphosphocholine proved to be the most efficient inhibitor (IC50 = 6.4 microM).

The presence of phospholipase D in rat central nervous system axolemma.

DeVries GH, Chalifour RJ, Kanfer JN
J Neurochem. 1983; 40: 1189-91

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.

Endothelin stimulates phospholipase D in striatal astrocytes.

Desagher S, Cordier J, Glowinski J, Tence M
J Neurochem. 1997; 68: 78-87

In primary cultures of mouse striatal astrocytes prelabeled with [3H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [3H]phosphatidic acid and [3H]diacylglycerol. In the presence of ethanol, a production of [3H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC50 = 2-5 nM). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a Gi/G(o) protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [3H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally dependent on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.

Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG).

Dawson G, Dawson SA, Post GR
J Neurosci Res. 1993; 34: 324-30

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor-mediated activation of phospholipase C (PL-C) or indirectly via the activation of phospholipase D (PL-D). In a well-characterized human oligodendroglioma (HOG) cell line, PL-C (measured by release of [3H]inositol phosphates) and PL-D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL-C stimulation by carbachol or histamine was completely inhibited by short-term treatment (< 30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL-D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL-D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL-C and the stimulatory effects on PL-D. However, the PKC inhibitors H-7 and galactosylsphingosine did not inhibit the TPA-mediated stimulation of PLD while the less-specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL-C and PL-D activation by carbachol, suggesting homologous desensitization. Time-course studies indicated that PL-D activation (10 sec or less) was at least as fast as PL-C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5-10 microM) was about the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase D hydrolysis of choline phosphoglycerides is selective for the alkyl-linked subclass of Madin-Darby canine kidney cells.

Daniel LW, Huang C, Strum JC, Smitherman PK, Greene D, Wykle RL
J Biol Chem. 1993; 268: 21519-26

Madin-Darby canine kidney (MDCK) cells were used to study the synthesis of diglycerides from choline phospholipids (PC) in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). In this system, diglyceride formation was blocked in the presence of ethanol (0.5%), and a corresponding amount of phosphatidylethanol (PEt) was formed, indicating that phospholipase D is responsible for the diglyceride production. Analysis of the subclasses of phosphatidylethanol revealed 1-O-alkyl-(alkyl), 1-O-alk-1'-enyl-(alkenyl), and 1-acyl species of PEt (38.0, 8.3, and 53.7%, respectively). The molecular species of the alkyl-PEt most closely matched the alkyl-PC. No change in the relative amounts of alkyl- versus acyl-PEt was observed with time after stimulation. Comparison of the alkyl content of PEt (38.0%) and the parent PC (15.2%) indicated a marked selectivity for the alkyl subclass of PC. A cell-free assay (Huang, C., Wykle, R. L., Daniel, L. W., and Cabot, M. C. (1992) J. Biol. Chem. 267, 16859-16865) for phospholipase D was also used to confirm the selectivity of the enzyme for alkyl-PC versus acyl-PC. The predominant molecular species of PEt contained saturated acyl or alkyl chains in position-1 and monounsaturated residues in position-2 accounting for approximately 50% of the total PEt. 1-O-Octadecyl-2-oleoyl-sn-glycerol, a representative alkyl molecular species, was synthesized and tested for its effect upon protein kinase C derived from MDCK cells. This alkyl-diglyceride (DG) neither stimulated protein kinase C nor inhibited its activation by diacylglycerol. In summary, TPA-stimulated phospholipase D is selective for the alkyl-PC subclass in MDCK cells. The alkyl-DG subsequently formed does not appear to function as a second-messenger in activating protein kinase C.

Stimulation of actin stress fibre formation mediated by activation of phospholipase D.

Cross MJ, Roberts S, Ridley AJ, Hodgkin MN, Stewart A, Claesson-Welsh L, Wakelam MJ
Curr Biol. 1996; 6: 588-97

BACKGROUND. Agonist-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, generating the putative messenger phosphatidate (PA). Proposed functions for PA, and hence for PLD, include kinase activation, the regulation of small molecular weight GTP-binding proteins, actin polymerization and secretion. It has not been possible to define a physiological function for PLD activation as it is generally stimulated together with other signalling pathways, such as those involving phospholipases A2 and C, phosphatidylinositide (PI) 3-kinase and the p21(ras)/mitogen-activated protein (MAP) kinase cascade. RESULTS. We report that, in porcine aortic endothelial (PAE) cells, lysophosphatidic acid (LPA) stimulated PLD activity and rapidly generated PA in the absence of other phospholipase, PI 3-kinase or MAP kinase activities. PLD activation was controlled by a tyrosine kinase-regulated pathway. LPA also stimulated actin stress fibre formation, but was inhibited by butan-1-ol; the alcohol also reduced the accumulation of PA. The addition of PA to cells did not stimulate PLD activity, but did cause stress fibre formation in a manner that was insensitive to butan-1-ol. Stimulation of stress fibre formation by LPA and PA was sensitive to genistein, and was inhibited by micro-injection of the Rho-inhibiting C3 exotoxin into PAE cells. CONCLUSIONS. This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.

Phospholipase D: regulation by GTPases and protein kinase C and physiological relevance.

Cockcroft S
Prog Lipid Res. 1996; 35: 345-70

Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular acinar cells.

Chung HC, Fleming N
Pflugers Arch. 1995; 431: 161-8

The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 microM agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P2 by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca2+ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca2+ by inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the phosphoinositide effect. The cell-permeant Ca2+ chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca(2+)-mobilizing arms of the PtdIns(4,5)P2-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P2-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca(2+)-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.

Ablation of Go alpha-subunit results in a transformed phenotype and constitutively active phosphatidylcholine-specific phospholipase C.

Cheng J, Weber JD, Baldassare JJ, Raben DM
J Biol Chem. 1997; 272: 17312-9

Modulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go alpha-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goalpha as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goalpha-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Go in regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme.

Cloning of a cDNA encoding phospholipase D from Pimpinella brachycarpa.

Cha YR, Lee KW, Moon YH, Kim JC, Han TJ, Lee WS, Cho SH
Mol Cells. 1998; 8: 19-26

Phospholipase D (PLD, EC 3.1.4.4) has been known to be related to various cellular processes in plants. To gain an understanding of the property of the enzyme in Pimpinella brachycarpa, the cDNA of the enzyme was isolated by PCR with degenerate primers, cDNA library screening, and 5' RACE. The full-length PLD cDNA is 2859 bp long and contains an open reading frame of 2424 bp coding for a polypeptide of 808 amino acids. The deduced enzyme has a calculated molecular mass of 91.7 kDa and pI of 5.86. The percent identity and similarity values of P. brachycarpa PLD with those of other PLDs in plants are 70 approximately 78 and 84 approximately 95, respectively. It was identified that PLD from P. brachycarpa has HQKIVVVD and HAKMMIVD sequences which were homologous with a duplicated HXKXXXXD motif that has been conserved in PLDs from plants, animals, and yeast. Based on the analysis of amino acid similarity, it is believed that PLD from P. brachycarpa is an alpha form which is distinct from PLD beta reported recently. The N-terminus is homologous to the C2 domain which is present in a number of different proteins involved in signal transduction and membrane trafficking in animals. Southern and northern blot analyses indicated that PLD was expressed from one copy of PLD gene in the genome of P. brachycarpa.

Compound 48/80 activates mast cell phospholipase D via heterotrimeric GTP-binding proteins.

Chahdi A, Fraundorfer PF, Beaven MA
J Pharmacol Exp Ther. 2000; 292: 122-30

Previous studies have indicated the presence of a cholera toxin-sensitive phospholipase D (PLD) in cultured RBL-2H3 mast cells that is synergistically activated via calcium, protein kinase C, and another unidentified signal. Here we identify a third potential signal for activation transduced by a pertussis toxin-sensitive trimeric GTP-binding protein, most likely via G(i2) or G(i3). Quercetin-treated RBL-2H3 cells in which expression of G(alphai2) and G(alphai3) is enhanced more than 7-fold respond to the G(i) stimulant compound 48/80 with the activation of PLD, a transient activation of phospholipase C, and enhanced membrane GTPase activity. The activation of PLD was blocked in pertussis toxin-treated cells and, as with other stimulants of PLD, was enhanced in cholera toxin-treated cells. The PLD response to compound 48/80 was only partially inhibited by calcium deprivation and inhibition of protein kinase C to indicate a component of the response that was independent of calcium, protein kinase C, and, presumably, phospholipase C. Based on these and other data, we hypothesized that betagamma-subunits, released from G(i2) or G(i3) by compound 48/80 or from G(s) by cholera toxin, provide an additional signal for the activation of PLD. Consistent with this hypothesis, recombinant G(beta2gamma2) subunits, but not G(alphai-3) subunits, at concentrations of 50 to 300 nM markedly synergized PLD activation by compound 48/80 in permeabilized RBL-2H3 cells.

Interferon beta mediated intracellular signalling traffic in human lymphocytes.

Cataldi A, Caracino A, Di Baldassarre A, Robuffo I, Miscia S
Cell Signal. 1995; 7: 627-33

The early molecular mechanisms activated by the treatment of human lymphocytes with human interferon beta have been studied. These identify an early increase with respect to control, in diacylglycerol (DG) levels as response to interferon treatment. Such a DG production was derived from the rapid and sequential activation of phosphoinositide specific phospholipase C and phospholipase D pathway. This suggests that a synergistic involvement of phosphatidylinositol-bis-phosphate (PIP2) hydrolysis and phosphatidylcholine (PC) breakdown provide early molecular events upon the interaction between interferon beta and its cell surface receptors. This finally leads to the slowing down of cell growth.

A human homolog of the vaccinia virus HindIII K4L gene is a member of the phospholipase D superfamily.

Cao JX, Koop BF, Upton C
Virus Res. 1997; 48: 11-8

We have identified a human gene encoding a protein with 48% amino acid identity to the vaccinia virus (VV) K4L gene product. Both contain motifs characteristic of the phospholipase D (PLD) protein superfamily. These proteins are also related to vaccinia virus p37, encoded by the F13L gene, which is required for envelopment and spread of the virus. The similarity to phospholipase D provides insight into the mechanisms and evolution of these processes.

Determination of interaction sites of phospholipase D1 for RhoA.

Cai S, Exton JH
Biochem J. 2001; 355: 779-85

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD(1) for RhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD(1) (rPLD(1)) with RhoA. Selection with phage display of different peptides of rPLD(1) confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873-1024 at the C-terminus of rPLD(1). RhoA also associated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD(1). A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD(1), and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD(1) (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD(1) described above failed to bind V14RhoA compared with wild-type rPLD(1), whereas rPLD(1) with mutations outside the region K946-K962 bound V14RhoA normally. It is concluded that basic amino acids in a restricted C-terminal region of rPLD(1) are important for binding of RhoA and its activation of PLD activity.

Generation of phosphatidic acid during calcium-loading of human erythrocytes. Evidence for a phosphatidylcholine-hydrolyzing phospholipase D.

Butikofer P, Yee MC, Schott MA, Lubin BH, Kuypers FA
Eur J Biochem. 1993; 213: 367-75

We have studied the mechanism by which calcium-loading of human erythrocytes stimulates phospholipid turnover and generates diacylglycerol and phosphatidic acid. Using quantitative measurement of individual phospholipid classes, we have demonstrated that the amount of phosphatidic acid generated during calcium-loading of intact red cells exceeds the amount of diacylglycerol formed by phospholipase-C-mediated hydrolysis of the polyphosphoinositol lipids and that addition of the diacylglycerol kinase inhibitor, R59022, only partly inhibited this increase. Thus, in contrast to current explanations, the phosphatidic acid generated following calcium-loading of erythrocytes cannot be solely explained by the action of a polyphosphoinositol-lipid-specific phospholipase C with subsequent phosphorylation of diacylglycerol to phosphatidic acid. Our data demonstrate that calcium-loading of intact erythrocytes, but not of red cell ghost membranes, causes a small but significant decrease in the relative amount of phosphatidylcholine (PtdCho). In order to identify the mechanisms responsible for calcium-mediated hydrolysis of PtdCho, we encapsulated Ptd[Me-14C]Cho-containing rat liver microsomes into erythrocytes and studied the generation of [Me-14C]choline and phospho[Me-14C]choline. We found that choline was the only detectable 14C-labeled product. Furthermore, incubation of erythrocytes with calcium under hypotonic conditions and in the presence of [14C]PtdCho vesicles and ethanol resulted in the formation of [14C]phosphatidylethanol. Together, these results suggest that the loss of PtdCho during calcium-loading of human erythrocytes is caused by a previously unrecognized PtdCho-hydrolyzing phospholipase D, resulting in direct generation of phosphatidic acid. Analysis of the molecular species composition of PtdCho, phosphatidic acid, and diradylglycerol, confirm the simultaneous actions of PtdCho-hydrolyzing and polyphosphoinositol-lipid-hydrolyzing phospholipases in calcium-loaded human erythrocytes.

Inhibition of glycosylphosphatidylinositol (GPI) phospholipase D by suramin-like compounds.

Brunner G, Zalkow L, Burgess E, Rifkin DB, Wilson EL, Gruszecka-Kowalik E, Powis G
Anticancer Res. 1996; 16: 2513-6

A number of proteins are found attached to the plasma membrane of mammalian cells by a glycosylphosphatidylinositol (GPI) anchor that can be cleaved by GPI specific phospholipase D (GPI-PLD). There are no known specific inhibitors of GPI-PLD. We examined some inhibitors of phosphatidylinositol specific phospholipase C (PI-PLC) for their ability to inhibit human serum and human bone marrow cell GPI-PLD. Azo analogues of suramin were found to be potent inhibitors of GPI-PLD. One compound had an IC50 of 3.7 microM that was 10-fold lower than the IC50 required to inhibit PI-PLC. The azo suramin analogues inhibited cancer cell growth at concentrations similar to those required to inhibit GPI-PLD, and below concentrations required to inhibit growth factor binding. It is possible that inhibition of cell growth might be related to the ability of the compounds to inhibit GPI-PLD.

The regulation of agonist-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

Briscoe CP, Wakelam MJ
Biochem Soc Trans. 1993; 21: 492-492

Characterization of phospholipase D activation by muscarinic receptors in human neuroblastoma SH-SY5Y cells.

Boyano-Adanez MC, Lundqvist C, Larsson C, Gustavsson L
Neuropharmacology. 1997; 36: 295-304

The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.

Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation.

Bourgoin S, Grinstein S
J Biol Chem. 1992; 267: 11908-16

To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.

1,25-Dihydroxyvitamin D(3), phospholipase D and protein kinase C in keratinocyte differentiation.

Bollinger Bollag W, Bollag RJ
Mol Cell Endocrinol. 2001; 177: 173-82

1,25-Dihydroxyvitamin D(3), thought to be a physiological regulator of epidermal keratinocyte growth and differentiation, also elicits the complete differentiative program in vitro, with expression of various genes/proteins characteristic of both early and late differentiation. 1,25-Dihydroxyvitamin D(3) functions by interacting with an intracellular receptor that binds to DNA at vitamin D response elements (VDRE) thereby affecting transcription. 1,25-Dihydroxyvitamin D(3) has been demonstrated to alter the expression of several enzymes involved in signal transduction, and presumably this is the mechanism through which the hormone regulates differentiation. It has recently been shown that 1,25-dihydroxyvitamin D(3) specifically increases the expression/activity of phospholipase D-1, an enzyme that hydrolyzes phospholipids to generate lipid messengers, such as diacylglycerol (DAG). DAG, in turn, is known to activate several members of the protein kinase C (PKC) family. It has been proposed that this signaling pathway mediates late differentiation events in epidermal keratinocytes. In this article the data supporting a role for PKC and phospholipase D in keratinocyte differentiation, as well as in the pathogenesis of skin diseases, are reviewed and a model is proposed for the signaling pathways that regulate this process upon exposure to 1,25-dihydroxyvitamin D(3).

Measurement of phospholipase D activity.

Bollag WB
Methods Mol Biol. 1998; 105: 151-60

A role for phospholipase D in control of mitogenesis.

Boarder MR
Trends Pharmacol Sci. 1994; 15: 57-62

How do growth factors that act on G protein-coupled cell-surface receptors communicate with the nucleus? These receptors commonly activate phospholipase C, and it has been assumed that the consequent rise in cytosolic Ca2+ concentrations and activation of protein kinase C mediates the mitogenic response. Recent evidence has demonstrated that phospholipase D (PLD) might be capable of eliciting mitogenesis. This enzyme is stimulated by a variety of growth factors, including those that act on receptors that possess intrinsic tyrosine kinase activity as well as those acting on G protein-coupled receptors. In this review, Michael Boarder considers the evidence that PLD, activated downstream of tyrosine protein kinases by both classes of cell-surface growth factor receptor, is implicated in the mitogenic response. This evidence is related to the possibility of PLD involvement in the regulation of vascular smooth muscle cell proliferation by endothelin-1 and platelet-derived growth factor.

Phospholipase D and cell signaling.

Billah MM
Curr Opin Immunol. 1993; 5: 114-23

Phospholipase D, which hydrolyzes phospholipids (primarily phosphatidylcholine) to generate phosphatidic acid, has emerged as a critical component in cellular signal transduction. Research during the past year has confirmed and extended the view that phosphatidic acid and its dephosphorylated product, sn-1,2-diacylglycerol, are important intracellular second messengers and that the coupling of phospholipase D to specific receptors occurs through multiple mechanisms involving protein kinase C, protein tyrosine kinase, Ca2+ and GTP-binding proteins.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes.

Benistant C, Moehren G, Gustavsson L, Torres-Marquez E, Hoek JB, Rubin R
Biochim Biophys Acta. 1994; 1223: 84-90

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10-1500 microM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C, perhaps at the level of a G protein.

Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis) and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium.

Barksdale L, Linder R, Sulea IT, Pollice M
J Clin Microbiol. 1981; 13: 335-43

A search has been made for corynebacterial phospholipase D, "ovis toxin," a sphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among a wide variety of corynebacteria. Phospholipase D activity has been found in strains exhibiting the biochemical properties characteristic of Corynebacterium pseudotuberculosis or of Corynebacterium ulcerans and in no other species of Corynebacterium. Methods for the assay of phospholipase D as a sphingomyelinase and methods for screening for phospholipase D in the presence of Corynebacterium equi on washed sheep blood agar are discussed.

Involvement of PL-D in the alternate signal tranduction pathway of macrophages induced by an external stimulus.

Bandyopadhyay R, Basu MK
Mol Cell Biochem. 2000; 203: 127-33

The alternate pathway of signal transduction via hydrolysis of phosphatidylcholine, the major cellular phospholipid, has been investigated in murine peritoneal macrophages. A sustained formation of diacylglycerol, is preceded by an enhanced production of phosphatidic acid, when the macrophages were given a stimulus with 12-O-tetradecanoyl phorbol-13-acetate for sixty minutes. Production of choline and choline metabolites are significantly increased too. Propranolol, which inhibits phosphatidate phosphohydrolase, the enzyme responsible for conversion of phosphatidic acid to diacylglycerol, can effectively block the formation of diacylglycerol. Inhibition of protein kinase C either by its inhibitors, staurosporine and H-7 or by depletion, apparently affect the generation of the lipid products. Moreover, based on the results of transphosphatidylation reaction, involvement of a phospholipase D in the phosphatidylcholine-hydrolytic pathway in macrophages is predicted. These observations support the view that probably the phorbol ester acting directly on protein kinase C of the macrophages activate their phosphatidylcholine-specific phospholipase D to allow a steady generation of second messengers, to enable them to participate in the cell signalling process in a more efficient manner than those generated in the phosphoinositide pathway of signal transduction.

RANTES activation of phospholipase D in Jurkat T cells: requirement of GTP-binding proteins ARF and RhoA.

Bacon KB, Schall TJ, Dairaghi DJ
J Immunol. 1998; 160: 1894-900

The chemokine RANTES is a potent agonist of T cell activation. In an investigation of signal-transduction events activated by this chemokine, we have shown that RANTES stimulates dose-dependent phospholipase D (PLD) activity in Jurkat cells. Equilibrium-binding analyses using 125I-labeled RANTES indicated the presence of a receptor for RANTES on these cells, which has a Kd of 0.1 nM, is expressed at approximately 600 sites per cell, and a binding specificity that was not comparable with that of any of the known chemokine receptors, since 125I-labeled RANTES was displaced by macrophage-inflammatory protein-1 beta (but not macrophage-inflammatory protein-1 alpha), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4, and eotaxin. RANTES-induced PLD activation was augmented by GTP gamma S, but not GDP beta S, and inhibited by the protein kinase C inhibitor bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and C3 exoenzyme (Clostridium botulinum), implicating the activation of RhoA. RANTES also induced GTP-GDP exchange of immunoprecipitated RhoA. RANTES-stimulated PLD activity was dependent on an ADP-ribosylation factor(s), as assessed by inhibition studies using a synthetic inhibitory peptide of the N-terminal 16 amino acids of ADP-ribosylation factor 1. These studies indicate the potential existence of a novel receptor-mediated mechanism for activation of T cells by the chemokine RANTES.

Activation of phospholipase D participates in signal transduction pathways responsive to gamma-radiation.

Avila MA, Otero G, Cansado J, Dritschilo A, Velasco JA, Notario V
Cancer Res. 1993; 53: 4474-6

Early responses of mammalian cells to ionizing radiation include the activation of a protein kinase C implicated in the regulation of gene expression, the stimulation of tyrosine kinase activities, and the enhancement of phosphatidylinositol turnover. In the present report we show that clinically relevant doses of gamma-radiation (2 Gy) stimulate phosphatidylcholine hydrolysis in human squamous carcinoma cells. Radiation induced the accumulation of intracellular [3H]choline and the simultaneous increase in [3H]myristoyl-phosphatidic acid, followed by a small increase in the levels of [3H]myristoyl-diacylglycerol. Furthermore, in the presence of ethanol, gamma-radiation stimulated the appearance of [32P]phosphatidylethanol, an indicator of phospholipase D transphosphatidylation activity. These data demonstrate for the first time that phospholipase D activation participates in signaling pathways in response to gamma-radiation.

Endothelin receptors stimulate both phospholipase C and phospholipase D activities in different cell lines.

Ambar I, Sokolovsky M
Eur J Pharmacol. 1993; 245: 31-41

Endothelin (ET) receptor-binding assays using [125I]ET-1 in C6-glioma cells and in Rat-1 and Swiss 3T3 fibroblasts indicated the presence of two binding sites, one of which binds agonists at the pM range and the other at the nM range. All three cell lines exhibited the same pharmacological profile for agonist binding (ET-1 congruent to sarafotoxin-b > ET-3), which suggests that the receptor is of the ETA type. Binding of ET-1 to the receptor resulted in activation of two phospholipases, phospholipase C (PLC) and phospholipase D (PLD). The activation of PLC or PLD by endothelin in the three cell lines was mediated by the high affinity binding site (nM range) and was not significantly affected by either extracellular or intracellular Ca2+. Measurement of PLD activation by ET-1 and/or phorbol 12-myristate 13-acetate (PMA), in the presence and absence of two potent inhibitors of protein kinase C (PKC), strongly suggests that activation of PLD by ET receptor in C6 glioma cells as well as in Rat-1 and Swiss 3T3 fibroblasts involves both PKC-dependent and PKC-independent mechanisms.

Adaptation of signal transduction in brain.

Alling C, Gustavsson L, Larsson C, Lundqvist C, Rodriguez D, Simonsson P
EXS. 1994; 71: 19-28

Cell culture models were used to study the effects of long-term ethanol exposure on neuronal cells. Effects on phospholipase C and phospholipase D mediated signal transduction were investigated by assaying receptor-binding, G protein function, activities of lipases, formation of second messengers and c-fos mRNA. The signal transduction cascades displayed abnormal activities from 2 to 7 days of exposure which differed from the acute effects. Phosphatidylethanol formed by phospholipase D is an abnormal lipid that may harmfully affect nerve cell function.

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line.

Ali H, Choi OH, Fraundorfer PF, Yamada K, Gonzaga HM, Beaven MA
J Pharmacol Exp Ther. 1996; 276: 837-45

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.