ADP-Ribosylation Factor Translocation Correlates With Potentiation of GTPγS-Stimulated Phospholipase D Activity In Membrane Fractions of HL-60 Cells (original) (raw)
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ADP-ribosylation Factor Proteins Mediate Agonist-induced Activation of Phospholipase D
Journal of Biological Chemistry, 1998
The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes. Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D. Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells. In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity. However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA. These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.
The subcellular localization of phospholipase D activities in rat Leydig cells
Molecular and Cellular Endocrinology, 1999
Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg 2 + -activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP 2 -PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPgS. The peak of oleate Mg 2 + -PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP 2 -PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester-stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [ 14 C]-palmitate and then stimulated for 15 min with 100 nM 4-b-phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [ 14 C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [ 14 C]-phosphatidylbutanol could be inhibited dose-dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF. : S 0 3 0 3 -7 2 0 7 ( 9 9 ) 0 0 0 5 7 -X
European Journal of Biochemistry, 1995
Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa.
Biochemical Journal, 2000
Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium J. Pharmacol. Exp. Ther. 292,[629][630][631][632][633][634][635][636][637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5h-[γ-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-Abbreviations used : PLD, phospholipase D ; PBut, phosphatidylbutanol ; ARF, ADP-ribosylation factor ; PKC, protein kinase C ; PTK, protein tyrosine kinase ; PKA, cAMP-dependent protein kinase ; PIP 2 , phosphatidylinositol 4,5-bisphosphate ; PA, phosphatidic acid ; GST-βARK1, glutathione Stransferase-β-adrenergic receptor kinase 1 (495-689) fusion protein ; QEHA, peptide encoding residues 956-982 in the C2a region of adenylate cyclase ACII ; GTP[S], guanosine 5h-[γ-thio] triphosphate ; GDP [β-S], guanosine 5h-[β-thio] diphosphate ; ET-1, endothelin-1 ; Rho GDI, Rho-guanine nucleotideexchange inhibitor. ).
Journal of Leukocyte Biology, 1999
We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V 4؉-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase C␣ (PKC␣), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V 4؉-OOH only elicited RhoA translocation and PMA induced ARF and PKC␣ translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTP␥Sstimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKC␣ to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKC␣ in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKC␣ were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKC␣ translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATPindependent manner. Furthermore, increased fMLP-induced PKC␣ translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.
Activation of rat brain phospholipase D by ADP-ribosylation factors 1, 5, and 6: …
Proceedings of the …, 1994
Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-SPW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADPribosylation factor (ARF) 1 and guanine 5' [y-thio]triphosphate, required the presence of phosphatidylinositol 4,5bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [y-thioltriphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleateand ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (dass I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.
The Role of ADP-ribosylation Factor and Phospholipase D in Adaptor Recruitment
The Journal of Cell Biology, 1997
AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTP ␥ S stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment . J. Cell Biol. 123:1093-1105. We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTP ␥ S-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTP ␥ S loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTP ␥ S. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTP ␥ S or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.
Journal of Biological Chemistry, 1999
Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADPribosylation 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.
Journal of Biological Chemistry, 1998
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 (ColleyWe 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 (M r 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 4,5bisphosphate. 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 abbreviations used are: PLD, phospholipase D; hPLD1 and hPLD2, human phospholipase D1 and D2, respectively; rPLD1 and rPLD2, rat phospholipase D1 and D2, respectively; mPLD2, mouse phospholipase D2; kb, kilobase pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; Hi5, high five; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; PE, dioleoyl phosphatidylethanolamine; PC, dipalmitoyl phosphatidylcholine; PIP 2 , phosphatidylinositol 4,5-bisphosphate; ARF, ADP-ribosylation factor.
Journal of Biological Chemistry, 1997
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 Mg 2؉dependent but insensitive to changes in free Ca 2؉ 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-␥S-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-␣ 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-␣ 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.