Overexpression of protein kinase C-epsilon and its regulatory domains in fibroblasts inhibits phorbol ester-induced phospholipase D activity

In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates phospholipase D (PLD)-mediated hydrolysis of both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PKC-alpha-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo PKC-epsilon and its regulatory, catalytic, and zinc finger domain fragments to determine if this isozyme also regulates PLD activity. Overexpression of holo PKC-epsilon inhibited the stimulatory effects of PMA (5-100 nM) on both PtdCho and PtdEtn hydrolysis. Overexpression of PKC-epsilon also was found to inhibit platelet-derived growth factor-induced PLD activity. Expression of the catalytic unit of PKC-epsilon had no effect on PMA-induced PLD activity. In contrast, expression of both the regulatory domain fragment and the zinc finger domain of PKC-epsilon resulted in significant inhibition of PMA-stimulated PtdCho and PtdEtn hydrolysis. Interestingly, although PKC-alpha also mediates the stimulatory effect of PMA on the synthesis of PtdCho by a phosphorylation mechanism, overexpression of holo PKC-epsilon or its regulatory domain fragments did not affect PMA-induced PtdCho synthesis. These results indicate that the PKC-epsilon system can act as a negative regulator of PLD activity and that this inhibition is mediated by its regulatory domain.     

Continual production of phosphatidic acid by phospholipase D is essential for antigen-stimulated membrane ruffling in cultured mast cells

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.     

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.     

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.     

Difference between the calcium- and the phorbol ester-induced association of protein kinase C with phospholipid membrane

1. Membrane association of protein kinase C is thought to be a prerequisite for the activation of the enzyme. 2. We studied the association of the enzyme with liposome. 3. We show here that the mechanisms for the calcium- and the phorbol ester-induced association of protein kinase C with liposome are different from each other. 4. Diacylglycerol is not crucial for the association of the enzyme with liposome.     

Functions and pathophysiological roles of phospholipase D in the brain

Ten years after the isoforms of mammalian phospholipase D (PLD), PLD1 and 2, were cloned, their roles in the brain remain speculative but several lines of evidence now implicate these enzymes in basic cell functions such as vesicular trafficking as well as in brain development. Many mitogenic factors, including neurotransmitters and growth factors, activate PLD in neurons and astrocytes. Activation of PLD downstream of protein kinase C seems to be a required step for astroglial proliferation. The characteristic disruption of the PLD signaling pathway by ethanol probably contributes to the delay of brain growth in fetal alcohol syndrome. The post-natal increase of PLD activities concurs with synapto- and myelinogenesis in the brain and PLD is apparently involved in neurite formation. In the adult and aging brain, PLD activity has antiapoptotic properties suppressing ceramide formation. Increased PLD activities in acute and chronic neurodegeneration as well as in inflammatory processes are evidently due to astrogliosis and may be associated with protective responses of tissue repair and remodeling. ARF-regulated PLD participates in receptor endocytosis as well as in exocytosis of neurotransmitters where PLD seems to favor vesicle fusion by modifications of the shape and charge of lipid membranes. Finally, PLD activities contribute free choline for the synthesis of acetylcholine in the brain. Novel tools such as RNA interference should help to further elucidate the roles of PLD isoforms in brain physiology and pathology.     

Redundant roles of BIG2 and BIG1, guanine-nucleotide exchange factors for ADP-ribosylation factors in membrane traffic between the trans-Golgi network and endosomes

BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.     

The roles of phospholipase D in EGFR signaling

Epidermal growth factor receptor (EGFR) is a representative model of receptor tyrosine kinases (RTKs), and offers a means of understanding their common principles and fundamental mechanisms. Furthermore, EGFR plays an essential role in cell proliferation and migration, and the disruption of EGFR signaling has been implicated in the development and growth of cancer. Phospholipase D (PLD) is a key mediator of EGFR function, and can be directly regulated by upstream binding partners in an EGF-dependent manner. PLD regulates downstream molecules by generating phosphatidic acid (PA), but it also dynamically interacts with a variety of intracellular molecules and these interactions spatiotemporally regulate EGFR function and serve as a hub that orchestrates signaling flow. This review summarizes the interrelationship between PLD and its binding molecules in the context of EGFR signaling, and addresses the roles of PLD in the mediation and coordination of this signaling.     

Inhibition of phorbol ester-induced cell activation in microgravity

T lymphocytes and monocytes were exposed to microgravity and activated to produce interleukin 2 and interleukin 1, respectively. When Jurkat T cells were triggered with monoclonal antibodies directed against the CD3/T cell receptor complex in the presence of THP-1 monocytes used as accessory cells, cell-to-cell contacts took place in microgravity leading to normal production of interleukin 2 and interleukin 1, as compared to ground controls. In contrast, when cells were individually stimulated by soluble substances including a protein kinase C activating phorbol ester, the production of interleukin 1 and interleukin 2 was dramatically inhibited during microgravity exposure. This result indicates that microgravity may affect the cellular target of phorbol ester.     

Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.     

The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association

Membrane ruffling is the formation of actin rich membrane protrusions, essential for cell motility. The exact mechanism of ruffling is not fully known. Using YFP and CFP fluorescent chimeras, we show for the first time a co-localization of Phospholipase D2 (PLD2) and Growth factor Receptor Bound protein-2 (Grb2) with actin-rich membrane protrusions of macrophages. Grb2 cooperates with PLD2 in enhancing membrane ruffling, whether in resting cells or in cells stimulated with the growth factor M-CSF, although in the latter an increase in dorsal ruffles was observed, consistent with receptor-ligand internalization. Cells transfected with PLD2 mutated in the PH domain (Y169F) or with Grb2 mutated in the SH2 site (R86K) negate this effect, indicating an association PLD2(Y169)-SH2-Grb2 that was confirmed by immunoprecipitation and Western blotting. The association results in enhanced PLD activity, but the lipase activity can only partially explain the formation of membrane ruffles in vivo. A third component involves the Rho-GTPase Rac2 and it is only when Rac2 is overexpressed along with PLD2 and Rac2 that a full biological effect, including actin polymerization in vivo, is obtained. The mechanism involved is, then, as follows: PLD enzymatic action, after having been increased due to the binding to Grb2-SH2 via Y169, cooperates with Rac2, and the three molecules stimulate actin polymerization and consequently, membrane ruffle formation. Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes.     

The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADP-ribosylation factors 1 and 6

Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.     

Production of leukotrienes in phorbol ester-induced acute lung injury

Leukotrienes, when administered into the pulmonary circulation of intact animals or isolated perfused lungs, have been associated with the formation of pulmonary edema. In addition, leukotrienes were identified in edema fluid and in bronchoalveolar lavage fluid (BALF) both from patients with the adult respiratory distress syndrome (ARDS) and from dogs with ethchlorvynol-induced acute lung injury (ALI). To determine whether the identification of leukotrienes in BALF was a finding common to ALI, etiology notwithstanding, we produced acute lung injury in dogs with phorbol myristate acetate (PMA). PMA produces a model of ALI thought to differ mechanistically from ethchlorvynol-induced ALI. Leukotriene C4 (LTC4), D4 (LTD4) and B4 (LTB4) were measured in BALF before and after PMA administration in intact pentobarbital-anesthetized dogs. The intravenous administration of 20 or 30 micrograms/kg of PMA produced increases in pulmonary vascular resistance (PVR) and extravascular lung water (EVLW), whereas, 10 or 15 micrograms/kg caused only a modest increase in PVR with no increase in EVLW. LTD4 and LTB4 were increased in BALF solely in those animals that developed increases in EVLW. These results, when viewed together with those reported in humans with ARDS and in dogs with ethchlorvynol-induced ALI, support the hypothesis that leukotriene detection in BALF is a feature common to ALI, etiology notwithstanding.     

Glycosaminoglycans enhance phorbol ester-induced proteolytic activity and angiogenesis in vitro

Summary It has been reported that endothelial cells suspended in three-dimensional type I collagen gels can be induced to undergo tube formation by 12-o-tetradecanoyl phorbol 13-acetate (TPA). In this report, we show that TPA-induced endothelial cell tube formation can be further enhanced by the addition of other matrix components in the collagen gels. In the presence of TPA, both high molecular weight hyaluronate and chondroitin sulfate elicit a dose-dependent stimulation of tube formation. The enhanced tube formation appears to be due to an increase in the number of cells undergoing morphogenesis as the average length per tube is not obviously increased. Concomitant with the increased cell morphogenesis, there is an increase in proteolytic activity secreted by the cells. Treatment of cells with cycloheximide suppresses hyaluronate- and chondroitin sulfate-enhanced cell morphogenesis and proteolytic activity suggesting that new protein synthesis, perhaps proteases, is necessary for endothelial cell morphogenesis. The possible role of the production of proteolytic activity in endothelial cell tube formation is discussed.     

Effects of activated ADP-ribosylation factors on Golgi morphology require neither activation of phospholipase D1 nor recruitment of coatomer

Nine mutations in the switch I and switch II regions of human ADP-ribosylation factor 3 (ARF3) were isolated from loss-of-interaction screens, using two-hybrid assays with three different effectors. We then analyzed the ability of the recombinant proteins to (i) bind guanine nucleotides, (ii) activate phospholipase D1 (PLD1), (iii) recruit coatomer (COP-I) to Golgi-enriched membranes, and (iv) expand and vesiculate Golgi in intact cells. Correlations of activities in these assays were used as a means of testing specific hypotheses of ARF action, including the role of PLD1 activation in COP-I recruitment, the role of COP-I in Golgi vesiculation caused by expression of the dominant activating mutant [Q71L]ARF3, and the need for PLD1 activation in Golgi vesiculation. Because we were able to find at least one example of a protein that has lost each of these activities with retention of the others, we conclude that activation of PLD1, recruitment of COP-I to Golgi, and vesiculation of Golgi in cells are functionally separable processes. The ability of certain mutants of ARF3 to alter Golgi morphology without changes in PLD1 activity or COP-I binding is interpreted as evidence for at least one additional, currently unidentified, effector for ARF action at the Golgi.     

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.     

Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA

Phospholipase D1 (PLD1) is known to be activated by ADP-ribosylation factor 1 (ARF1). We report here that ARF1 co-immunoprecipitates with PLD1 and that the ARF1-dependent PLD activation is induced by the direct interaction between ARF1 and PLD1. We found that RalA, another member of the small GTP-binding proteins, synergistically enhances the ARF1-dependent PLD activity with an EC₅₀ of about 30 nM. Using in vitro binding assay, we show that ARF1 and RalA directly interact with different sites of PLD1. The results suggest that the independent interactions of RalA and ARF1 with PLD1 are responsible for the synergistic activation.