Mechanism(s) of activation of secretory phospholipase A(2)s in mouse keratinocytes.

The differential activation of different members of the phospholipase A(2) (PLA(2)) superfamily and their regulation are important as one or more of them regulates the production of eicosanoids and others may contribute to the formation of other lipid mediators. We previously reported the existence of two forms of secretory or sPLA(2) in mouse keratinocytes, namely type I and type II sPLA(2). We show here that mouse keratinocyte sPLA(2)s were potently activated by protease treatment and inhibited by protease inhibitors. We also observed that G protein effectors induced substantial release of oleic acid (OA) from prelabeled mouse keratinocytes. A G(i)/G(0) protein activator significantly enhanced the hydrolysis of OA and this increase was not responsive to either pertussis toxin or cholera toxin treatment. Although there was a significant negative correlation between intracellular cAMP levels and OA hydrolysis, experimentally increasing cAMP with forskolin treatment had no effect on sPLA(2) activity. Arachidonic acid but not its metabolites was also shown to marginally activate keratinocyte sPLA(2) by 1.5-fold. These results lead to the conclusion that mouse keratinocyte sPLA(2)s can be regulated primarily by proteolytic activation and a G protein pathway.     

Phospholipase D activation by norepinephrine is mediated by 12(s)-, 15(s)-, and 20-hydroxyeicosatetraenoic acids generated by stimulation of cytosolic phospholipase a2. tyrosine phosphorylation of phospholipase d2 in response to norepinephrine

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.     

Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.     

Rapid protein kinase C-dependent activation of phospholipase D leads to delayed 1,2-diglyceride accumulation

We have shown previously that the major source of diglyceride (DG) formed following muscarinic receptor (mAChR) stimulation of 1321N1 astrocytoma cells is phosphatidylcholine (PC) rather than the phosphoinositides (Martinson, E. A., Goldstein, D., and Brown, J. H. (1989) J. Biol. Chem. 264, 14748-14754). We have also noted that there is a delay of several minutes before significant DG accumulation is observed. In the present work, we examine the time course and mechanism of PC hydrolysis in response to mAChR stimulation. Treatment of 1321N1 cells with carbachol results in increases in radiolabeled choline, phosphatidic acid (PA) and phosphatidylethanol (PEt), metabolites that are products of phospholipase D (PLD) action on PC. These products are all formed within 15 s of mAChR stimulation and reach a plateau within 30-60 s. The time course of PEt formation suggests that PLD is no longer activated after several minutes of mAChR stimulation. Thus there is a discrepancy between the rapid and transient activation of PLD and the delayed accumulation of DG. It appears that most of the DG is formed through the action of PLD, since propranolol (which inhibits the conversion of PA to DG) and down-regulation of protein kinase C (which prevents activation of PLD by carbachol) both markedly inhibit DG production. Using a protocol in which cells are stimulated with carbachol for only one minute (a period during which PLD and PA formation are maximally activated), we show that DG mass continues to increase following removal of agonist. We suggest that the rapid and transient activation of PLD results in delayed accumulation of DG due to the relatively slow conversion of PA to DG by PA phosphatase.     

Dehydroepiandrosterone (DHEA) stimulates glucose uptake in rat adipocytes: activation of phospholipase D

We examined the effect of dehydroepiandrosterone (DHEA) on glucose uptake and phospholipase D (PLD) activation in rat adipocytes. DHEA (1 microM) provoked a twofold increase in [3H]2-deoxyglucose (DG) uptake for 30 min. Incorporation of [3H]glycerol into diacylglycerol was increased 150% above basal level for 20 min after stimulation with 1 microM DHEA. DHEA increased PLD activity, measured by the incorporation into [3H]phosphatidylethanol in [3H]palmitate labelled rat adipocytes, or by [3H]choline release in [methyl-(3)H]choline labeled rat adipocytes. Our results suggest that DHEA stimulates glucose uptake with activation of PLD in rat adipocytes.     

Release of a membrane surface glycoprotein from human platelets by phosphatidylinositol specific phospholipase(s) C.

Phosphatidylinositol (PI) specific phospholipase C (PIase C) treatment of human platelets caused release of a surface glycoprotein in the medium. Human blood platelets were isolated by low speed centrifugation and surface glycoproteins were labelled with periodate/[3H]borohydride procedure. Intact surface-labelled platelets were treated with PIase C purified from culture filtrates of Staphylococcus aureus (SA) or Bacillus thuringiensis (BT). After PIase C treatments platelets were spun at low speed, pellet and supernatant were separated. The supernatant was further centrifuged at high speed (140,000 x g) for 30 min. The resulting supernatant and the pellet from low speed were subjected to SDS-PAGE analysis. Protein patterns were obtained by fluorography. Release of a specific glycoprotein of approx. 150 kDa in the medium was observed due to the PIase C treatment. Prolonged incubation of platelets in 0.25 M sucrose and depletion of NaCl concentrations also affected the release of this glycoprotein. BT-PIase C released more approx. 150 kDa protein than SA-PIase C. Western blot experiment with a monoclonal antibody (mAB), epitope SZ2, reactive to human platelet surface glycoprotein Ib (GPIb) complex, confirmed that released 150 kDa glycoprotein reacted with mAB of GPIb. The release of this protein by PIase C was not inhibited by proteinase inhibitors (EDTA, PMSF and leupeptin). Treatment of human platelet membranes with PIase C also caused release of this glycoprotein as evidenced by reactivity to GPIb-mAB. These studies demonstrate that PIase C treatment causes release of 150 kDa glycoprotein from human platelet membrane surface. It is suggested that 150 kDa glycoprotein is anchored to PI in human platelets and that this glycoprotein represents the GPIb complex.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.     

Selective activation of phospholipase D2 by unsaturated fatty acid.

Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate-stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 microM in vitro. Intriguingly, phosphatidylinositol 4,5-bisphosphate (PIP2) synergistically stimulates the oleate-dependent PLD2 activity with an optimal concentration of 2.5 microM. These results provide the first evidence that oleate is a PLD2-specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP2.     

Inhibition of muscarinic receptor-linked phospholipase D activation by association with tubulin

Mammalian phospholipase D (PLD) is considered a key enzyme in the transmission signals from various receptors including muscarinic receptors. PLD activation is a rapid and transient process, but a negative regulator has not been found that inhibits signal-dependent PLD activation. Here, for the first time, we report that tubulin binding to PLD2 is an inhibition mechanism for muscarinic receptor-linked PLD2 activation. Tubulin was identified in an immunoprecipitated PLD2 complex from COS-7 cells by peptide mass fingerprinting. The direct interaction between PLD2 and tubulin was found to be mediated by a specific region of PLD2 (amino acids 476-612). PLD2 was potently inhibited (IC50 <10 nM) by tubulin binding in vitro. In cells, the interaction between PLD2 and tubulin was increased by the microtubule disrupting agent nocodazole and reduced by the microtubule stabilizing agent Taxol. Moreover, PLD2 activity was found to be inversely correlated with the level of monomeric tubulin. In addition, we found that interaction with and the inhibition of PLD2 by monomeric tubulin is important for the muscarinic receptor-linked PLD signaling pathway. Interaction between PLD2 and tubulin was increased only after 1-2 min of carbachol stimulation when carbachol-stimulated PLD2 activity was decreased. The expression of the tubulin binding region of PLD2 blocked the later decrease in carbachol-induced PLD activity by masking tubulin binding. Taken together, these results indicate that an increase in local membrane monomeric tubulin concentration inhibits PLD2 activity, and provides a novel mechanism for the inhibition of muscarinic receptor-induced PLD2 activation by interaction with tubulin.     

Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism

Aluminum fluoride (AlF4-) inhibited guanine nucleotide-activated phospholipase D (PLD) in rat submandibular gland cell-free lysates in a concentration-dependent response. This effect was consistent in permeabilized cells with endogenous phospholipid PLD substrates. Inhibition was not caused by either fluoride or aluminum alone and was reversed by aluminum chelation. Inhibition of PLD by aluminum fluoride was not mediated by cAMP, phosphatases 1, 2A or 2B, or phosphatidate phosphohydrolase. AlF4- had a similar inhibitory effect on rArf-stimulated PLD, but did not block the translocation of Arf from cytosol to membranes, indicating a post-GTP-binding-protein site of action. Oleate-sensitive PLD, which is not guanine nucleotide-dependent, was also inhibited by AlF4-, supporting a G protein-independent mechanism of action. A submandibular Golgi-enriched membrane preparation had high PLD activity which was also potently inhibited by AlF4-, leading to speculation that the known fluoride inhibition of Golgi vesicle transport may be PLD-mediated. It is proposed that aluminum fluoride inhibits different forms of PLD by a mechanism that is independent of GTP-binding proteins and that acts via a membrane-associated target which may be the enzyme itself.     

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.     

Cyclic AMP-elevating agents block chemoattractant activation of diradylglycerol generation by inhibiting phospholipase D activation.

Agents which elevate cellular cAMP (prostaglandin E2, theophylline, and forskolin) or mimic cAMP action (dibutyryl cAMP) are known to inhibit human neutrophil activation (superoxide generation and secretion) by receptor-linked agonists such as formyl-methionyl-leucyl-phenylalanine (fMLP). Herein, we show that these agents also markedly inhibit fMLP-stimulated diradylglycerol generation (assayed by mass methods). The magnitude of inhibition correlated with the ability of a given agent or combination of agents to elevate cAMP. Both 1,2-diacylglycerol and 1-O-alkyl,2-acyl glycerol generation were affected. Effects on the latter species, as well as a lack of effect on fMLP-stimulated inositol phosphate release, implied that cAMP affected diradylglycerol generation from a source other than phospholipase C-dependent phosphoinositide hydrolysis, since phosphatidylinositols do not contain appreciable quantities of the 1-O-alkyl linkage. In cells in which the phosphatidylcholine pool was prelabeled using 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine, prostaglandin E2 plus theophylline inhibited the fMLP-activated rapid generation of [3H]phosphatidic acid and its subsequent conversion to [3H]diradylglycerol, implying an effect at the level of phospholipase D. In the presence of ethanol, the fMLP-activated transphosphatidylation of [3H]phosphatidylcholine to generate [3H]phosphatidylethanol (a phospholipase D-dependent reaction) was also markedly inhibited. In contrast, when phorbol 12-myristate 13-acetate was used to activate cells, cAMP-related agents had no effect on phospholipase D activity, diradylglycerol generation, or superoxide generation. The data indicate an inhibitory effect of cyclic AMP on receptor-mediated phospholipase D activation at a site proximal to phospholipase D (e.g., the receptor or G protein). These studies provide a new example of "cross-talk" among signal transduction systems.     

Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.     

Rapid assay of poly(ADP-ribose) glycohydrolase

We have developed a rapid, highly reproducible assay to determine poly(ADP-ribose) glycohydrolase activity which measures directly the appearance of the reaction product. We also analysed the majority of different techniques which are used to determine poly(ADP-ribose) glycohydrolase activity and found that the apparent activity can vary extensively depending on the method used. Thin-layer chromatography using PEI-F-cellulose was the only method which evaluated directly the specific release of ADP-ribose; by comparison with this method, the other procedures gave an over- or under-estimation of 2- to 10-fold of the enzymatic activity. A rapid method of affinity chromatography has also been developed to synthesize and purify in high yield poly(ADP-ribose) (35% conversion of 1 mM NAD to poly(ADP-ribose)).     

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

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.     

Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.