A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.     

Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.     

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.     

Genome-wide and molecular evolution analyses of the phospholipase D gene family in Poplar and Grape

Background  

The Phospholipase D (PLD) family plays an important role in the regulation of cellular processes in plants, including abscisic acid signaling, programmed cell death, root hair patterning, root growth, freezing tolerance and other stress responses. PLD genes constitute an important gene family in higher plants. However, until now our knowledge concerning the PLD gene family members and their evolutionary relationship in woody plants such as Poplar and Grape has been limited.     

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.     

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

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.     

The mechanism of docosahexaenoic acid-induced phospholipase D activation in human lymphocytes involves exclusion of the enzyme from lipid rafts

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid that inhibits T lymphocyte activation, has been shown to stimulate phospholipase D (PLD) activity in stimulated human peripheral blood mononuclear cells (PBMC). To elucidate the mechanisms underlying the DHA-induced PLD activation, we first characterized the PLD expression pattern of PBMC. We show that these cells express PLD1 and PLD2 at the protein and mRNA level and are devoid of oleate-dependent PLD activity. DHA enrichment of PBMC increased the DHA content of cell phospholipids, which was directly correlated with the extent of PLD activation. The DHA-induced PLD activation was independent of conventional protein kinase C but inhibited by brefeldin A, which suggests ADP-ribosylation factor (ARF)-dependent mechanism. Furthermore, DHA enrichment dose-dependently stimulated ARF translocation to cell membranes. Whereas 50% of the guanosine 5'-3-O-(thio)triphosphate plus ARF-dependent PLD activity and a substantial part of PLD1 protein were located to the detergent-insoluble membranes, so-called rafts, of non-enriched PBMC, DHA treatment strongly displaced them toward detergent-soluble membranes where ARF is present. Collectively, these results suggest that the exclusion of PLD1 from lipid rafts, due to their partial disorganization by DHA, and its relocalization in the vicinity of ARF, is responsible for its activation. This PLD activation might be responsible for the immunosuppressive effect of DHA because it is known to transmit antiproliferative signals in lymphoid cells.     

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

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.     

Molecular diversity of phospholipase D in angiosperms

Background  

The phospholipase D (PLD) family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology.     

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Background

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.

Methodology/Principal Findings

Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for ¹⁴C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in ¹⁴C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of ¹⁴C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate ¹⁴C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate ¹⁴C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.

Conclusions/Significance

These results suggest that AMPK-mediated PLD1 activation is required for ¹⁴C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake.     

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α<Subscript>1A</Subscript> adrenergic receptor

Background  

Phenylephrine (PHE), an α₁ adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α_1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.     

Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.     

Localization and regulation of phospholipase D2 by ARF6

Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.     

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.     

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.     

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

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.     

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

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

Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells.

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.