ADP-ribosylation Factor Translocation Correlates with Potentiation of GTPγS-stimulated Phospholipase D Activity in Membrane Fractions of HL-60 Cells

Abstract

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP␥S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP␥S-stimulated PLD activity in the membrane fractions of these cells. Inactive phorbol esters induced no such potentiation. Both fMLP and active phorbol esters induced a 2-3-fold increase in GTP␥S-stimulated PLD in HL-60 membranes. Membranes derived from stimulated HL-60 cells contained 60 -70% more ARF as compared with membranes derived from control cells. Membrane contents of ARF were assessed by Western blotting with the anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation. Therefore, ARF translocation correlates with the potentiation of the GTP␥S-stimulated PLD activity. The effect on PLD activity and ARF membrane content achieved through fMLP stimulation was greatly enhanced by prior treatment of the cells with cytochalasin B. Membranes derived from control and fMLP-stimulated cells were assayed for PLD activity in the presence of exogenous ARF and a 50-kDa fraction known to contain elements implicated in PLD activation. The ability of ARF and the 50-kDa fraction to enhance GTP␥S-sensitive PLD activity was significantly reduced when the membranes were derived from fMLP-stimulated cells. The data indicate that, in addition to ARF, elements of the 50-kDa PLD-inducing factors were likely already translocated to the membranes upon stimulation. We propose that ARF, upon stimulation with agonists such as fMLP or phorbol esters, is translocated to the membrane and in concert with other protein components of the 50-kDa fraction enhances PLD activity.Neutrophils exercise their functions in primary host defense against pathogens through different effector systems including superoxide generation and granular secretion. It is now generally accepted that phospholipases are implicated in the generation of second messengers essential to these effector systems (1). Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5-biphosphate into inositol 1,4,5-triphosphate and diradylglycerol; two important second messengers. PLD 1 hydrolyzes phosphatidylcholine into choline and phosphatidic acid. Phosphatidic acid is possibly involved in superoxide anion production by inducing the dissociation of the Rac-GDP/GTP dissociation inhibitor complex (2), thereby releasing Rac, which can then assemble the NADPH oxidase complex (e.g. for review see Ref.3). Phosphatidic acid can also be converted to diradylglycerol by the enzyme phosphatidic acid phosphatase. Diradylglycerol derived from the PLD and phospholipase C pathways has been shown to cause different patterns of protein kinase C isoform translocation and to have distinct cell...

Characterization of a Rat Brain Phospholipase D Isozyme

“…Although ARF-responsive PLD activity has been found in other subcellular fractions besides Golgi (plasma membrane, nucleus, and cytosol) (1), its role at these sites is unknown. Some studies have shown agonist-induced membrane translocation of ARF (45,46), but the nature of the membrane target is unclear. Rho regulates the formation of actin stress fibers and focal adhesions (23,24) and has also been implicated in the regulation of phosphatidylinositol 3-kinase (25) and phosphatidylinositol 4-phosphate 5Ј-kinase (26), which synthesize PIP 3 and PIP 2 , respectively.…”

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTPγS-stimulated polymorphonuclear leukocytes

“…PLD1 activation is dependent on the small guanosine triphosphateases (GTPases) adenosine diphosphate (ADP)-ribosylation factor (ARF) and RhoA, one or more PKC isoforms, and phosphatidylinositol-bisphosphate (PIP 2 ), whereas PLD2 activity appears not to involve the GTPases but is primarily stimulated by PIP 2 . 10 Activation of PLD in PMN and HL-60 cells is dependent on ARF and RhoA, 11,12 suggesting that PLD1 is the primary isozyme being measured in previous PMN studies. Both ARF1 and ARF6 have been implicated in PLD activation in phagocytic cells.…”

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

“…We have previously demonstrated that ARF translocation correlated with fMLP-induced PLD activation in HL60 granulocytes [15]. More recently, a concomitant PLD activation and rapid enrichment of RhoA in the particulate fraction was documented in LPAstimulated Rat1 fibroblasts [29] and fMLP-stimulated neutrophils [16].…”

“…After extensive washing with buffer B containing 0.025 M NaCl, 0.19 M Tris-HCl, 0.0065 N NaOH, and 0.15% Tween-20, the membranes were incubated with horseradish peroxidase-linked anti-mouse antibody (dilution 1/15,000) or anti-rabbit antibody (dilution 1/15,000), washed again, and then soaked in a chemiluminescence reagent (ECL) for 1 min and then exposed to a film. stimulation of intact cells by PMA or fMLP increased the PLD response to GTP␥S as well as the ARF content in isolated membranes [15]. However, the mechanisms orchestrating the translocation of small GTPases in human granulocytes have yet to be defined.…”

“…We have previously shown that ARF translocated to the membrane fraction of HL60 cells in response to fMLP and that this translocation correlated with an increase in the GTP␥S-stimulated membranebound PLD activity [15]. Phorbol 12-myristate 13-acetate (PMA), a non-physiological PKC agonist, had a similar effect on membrane-bound PLD activity and ARF translocation [15].fMLP in neutrophils [16] and that lysophosphatidic acid (LPA) stimulation induces the enrichment of RhoA in the particulate fraction of Rat1 fibroblasts [17].…”