©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ADP-ribosylation Factor Translocation Correlates with Potentiation of GTPS-stimulated Phospholipase D Activity in Membrane Fractions of HL-60 Cells (*)

(Received for publication, July 18, 1995)

Martin G. Houle Richard A. Kahn (1) Paul H. Naccache Sylvain Bourgoin (§)

From the Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, and Departments of Medicine and Physiology, Faculty of Medicine, Université Laval, Ste-Foy, Québec, GIV 4G2, Canada Laboratory of Medicinal Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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 GTPS, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTPS-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 GTPS-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 GTPS-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 GTPS-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.


INTRODUCTION

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 (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 cellular effects(4) . Thus PLD may play an important role in neutrophil cell physiology.

Many agonists are known to stimulate PLD activity in neutrophils including fMLP, interleukin-8, C5a, leukotriene B4, and platelet activating factor(5, 6, 7, 8) . The receptors for these agonists are composed of seven transmembrane segments and are coupled to heterotrimeric G-proteins (for a review see (9) ), which in turn can initiate a cascade of signaling events that lead to PLD activation. Accordingly, direct activation of G proteins by nonhydrolyzable analogues of GTP stimulates the activation of PLD in cell-free systems. PLD is known to be activated by at least two distinct pathways: a tyrosine kinase-mediated pathway and a protein kinase C-mediated pathway (10, 11, 12, 13) . Stimulation of PLD by agonists such as fMLP is pertussis toxin-sensitive, but activation of protein kinase C by phorbol esters induces a PLD response that is pertussis toxin-insensitive(14, 15) . Increasing tyrosine phosphorylation by use of inhibitors of tyrosine phosphatases also causes a pertussis toxin-insensitive activation of PLD. Therefore PLD must lie downstream of a heterotrimeric G protein and be coupled to protein kinase C as well as to the tyrosine phosphorylation pathways.

Protein kinase C isoenzymes have been reported to stimulate a membrane-associated PLD activity(16) . Synergistic activation of PLD by guanine nucleotides was observed in rabbit platelet membranes when cells were pretreated with protein kinase C activators(17) . The response was not mimicked by exogenously added protein kinase C, suggesting that the synergism might be the result of the translocation of cytosolic components to the membrane, presumably GTP-binding proteins. It was recently discovered that a small GTP-binding protein named ADP-ribosylation factor (ARF) stimulated PLD in in vitro assays using a reconstituted system including partially purified PLD and exogenously added ARF and GTPS(18, 19) . ARF was first identified as a cofactor necessary for the ADP-ribosylation of subunit G(s)alpha by cholera toxin and classified as a G-protein in its own right, belonging to the Ras superfamily of small GTP-binding proteins(20) . ARF is myristoylated on the amino-terminal Gly residue(18) . Myristoylation appears to be essential for ARF functional activity (for a review see (21) ). ARF is required for coatomer assembly on the Golgi, promoting formation and fusion of non-clathrin-coated vesicles(22, 23) . Upon stimulation with various agonists, including phorbol 12-myristate 13-acetate (PMA), ARF is translocated to the Golgi membrane in Hela cells(24) .

In the present study we demonstrate that the treatment of intact HL-60 cells with a phorbol ester, PMA, or the chemotactic peptide, fMLP, induces a potentiation of the GTPS-stimulated PLD in membrane fractions. Using ARF-specific antibodies, we found that ARF translocates to membranes in response to physiological or nonphysiological agonists, paralleling that of PLD activity. These results document a strong correlation between ARF translocation and PLD activation. Such translocation may play major roles in the mechanisms governing the activation of PLD.


EXPERIMENTAL PROCEDURES

Materials

RPMI 1640, glutamine, penicillin, streptomycin, and fetal bovine serum were obtained from Life Technologies, Inc. Platelet-activating factor (1-O-[^3H] alkyl, mixture of C-16 and C-18 ether alkyls), anti-mouse Ig (sheep) peroxidase-linked antibody and ECL Western blot detection kit were purchased from Amersham Canada Ltd. (Oakland, Canada). The HL-60 cell line was from ATCC (Rockville, MD). Immobilon-P transfer membranes were from Millipore Corp. (Bedford, MA). Phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, and 4alpha-phorbol 12,13-dibutyrate were obtained from Sigma. TLC silica gel 60 plates were purchased from EM Science (Gibbstown, NJ). The synthetic peptide KENLKDCGLF corresponding to the carboxyl terminus of the alpha-subunit of transducin was a gift from Dr. M. F. Crouch (The John Curtin School of Medical Research, Canberra, Australia). ARF4-(2-17) and Rab3A AL-(33-47) peptides were synthesized by the Service de Séquence de Peptide de l'Est du Québec (Centre de Recherche du CHUL, Québec).

Labeling of HL-60 Cells

HL-60 cells were incubated in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin (100 µg/ml), and streptomycin (100 units/ml). Cells were passaged every 3-4 days so that cell density did not exceed 2 times 10^6 cells/ml. To induce differentiation to granulocyte-like cells, cell suspensions were incubated with 1.25% dimethyl sulfoxide for 6-7 days. Differentiated cells were centrifuged and resuspended at 8 times 10^6 cells/ml in RPMI 1640 containing 1-O-[^3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1 mg/ml human serum albumin as described previously by Bourgoin et al.(25) .

Stimulation of HL-60 Cells and Isolation of Membranes

Labeled or washed unlabeled HL-60 cells at 8 times 10^6 cells/ml in RPMI 1640 were treated with 1.1 mM diisofluorophosphate for 30 min at 24 °C. The cell suspension was centrifuged and resuspended in RPMI 1640 at the same cell concentration. The cells were incubated for 5 min at 37 °C and pretreated in the presence or absence of 100 nM PMA, 250 nM PdBu, 250 nM 4alpha-PdBu, or 100 nM fMLP for the indicated periods of time. The incubations were stopped by diluting the cells 1:5 with ice cold RPMI 1640, and membranes were prepared. Briefly, cell suspensions were centrifuged as indicated and resuspended at 1.6 times 10^7 or 2.5 times 10^7 cells/ml in ice-cold KCl-Hepes relaxation buffer (100 mM KCl, 50 mM Hepes, 5 mM NaCl, 1 mM MgCl(2), 0.5 mM EGTA, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 2.5 mM phenylmethylsulfonyl fluoride, pH 7.2) to be used for the PLD assay or Western blotting experiments, respectively. The suspensions were sonicated 2 times 20 s and centrifuged for 7 min at 700 times g. Unbroken cells and nuclei were discarded, and the supernatants were ultracentrifuged at 180,000 times g for 45 min in a Beckman TL-100 ultracentrifuge using a TL-100.4 rotor (65,000 rpm, 4 °C). Membrane pellets were washed once and resuspended in KCl-Hepes buffer (4 times 10^7 cell eq/ml) to be used for the PLD assay.

PLD Activity Assay

The labeled cell membranes were diluted to 1.6 times 10^7 cell equivalent/ml with KCl-Hepes buffer, and sufficient CaCl(2) and MgCl(2) were added to raise their concentrations to 1 µM and 8 mM, respectively. The reaction was initiated by the addition of 1.4% EtOH, and the samples were incubated in the presence or absence of 20 µM GTPS under continuous mixing for 20 min at 37 °C. To terminate the reaction, 1.8 volumes of CHCl(3):MeOH:HCl (50:100:1) and a standard sample of PEt (3 µg). Lipids were extracted essentially according to Bligh and Dyer(26) . After resting overnight at -20 °C, the lower lipid-containing chloroform phase was recuperated and evaporated. The lipid samples were redissolved in 50 µl of CHCl(3):MeOH (2:1) and spotted on TLC plates. Plates were developed using a solvent system of chloroform/methanol/acetic acid (65:15:2, by volume) for separation of PEt. Staining of the plates with Coomassie Blue enabled us to determine the position of PEt on the plate. The plates were then scraped, and lipid-associated radioactivity was counted in a scintillation counter. The results are expressed as a percentage of the total radioactive counts for each sample that is recovered into PEt.

In some experiments partially purified 50-kDa PLD-inducing fractions were prepared as described previously by Bourgoin et al.(25) . Recombinant ARF was produced and purified as described(27) .

ARF Immunoblotting

For immunoblotting experiments, membrane pellets were dissolved in a small volume of buffer A containing 0.25 M Na(2)HPO(4), 0.3 M NaCl, 2.5% sodium dodecyl sulfate, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 2.5 mM phenylmethylsulfonyl fluoride, and samples were assayed for protein content with Pierce Coomassie Brilliant Blue protein assay. The equivalent of 100 µg of protein were loaded and resolved on a 12% SDS-polyacrylamide gel electrophoresis according to Laemmli (28) and were then electrotransferred to Immobilon-P membranes. The membranes were soaked with 2% gelatin for 30 min at 37 °C and then incubated with the monoclonal anti-ARF antibody 1D9 (1:6000) or a polyclonal anti-ARF antibody (1:10,000). After extensive washing with buffer B, containing 0.025 M Tris-HCl, 0.19 M NaCl, 0.0065 N NaOH, and 0.15% Tween-20, the membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit antibody, washed again, and then soaked in a chemiluminescence reagent (ECL) for 1 min and then exposed to a film. Western blot images were captured using an OmniScan hand-held scanner and analyzed with the Macintosh application NIH Image 1.53b42.


RESULTS

Potentiation of PLD Activity in Membrane Fractions of HL-60 Cells in Response to GTPS Induced by Pretreatment with Phorbol Esters and fMLP

The PLD activity measurable in membrane fractions of HL-60 cells pretreated or not pretreated with PMA (100 nM) for various times was examined first. The basal as well as the GTPS-stimulated PLD activity was measured in these samples, and the results are summarized in the data illustrated in Fig. 1. Pretreatment with the phorbol ester caused a slight increase in the basal PLD activity in membranes. Although statistically insignificant, an increase was observed 30 s after the addition of PMA to intact cells, the earliest time sampled. Increasing the duration of the preincubation period up to 10 min did not increase further the basal levels of PEt produced. The addition of GTPS (20 µM) to the membrane fractions enhanced PLD activity. This effect of GTPS was rapidly and significantly potentiated in membranes derived from PMA-treated cells. As shown in Fig. 1A, potentiation of GTPS responses was evident within 30 s, and the amounts of PEt produced more than doubled within 2 min of pretreatment with PMA. The potentiation by PMA of the GTPS-stimulated PLD activity was maintained for at least 5 min and began to decrease by 10 min into the preincubation period. The potentiating effects of PMA were shared by another phorbol ester, PdBu. Pretreatment for 2.5 min with PdBu increased the basal level of PLD activity in HL-60 membranes to a small extent and increased to a larger extent the sensitivity to GTPS, an effect very similar to that observed with PMA. The inactive analogue of PdBu, 4alphaPdBu, was without significant effect on the basal or GTPS-stimulated PLD activity. The PLD activity measured for control, PMA (100 nM), PdBu (250 nM), and 4alphaPdBu (250 nM) stimulated membranes were, respectively, 0.513 ± 0.072, 1.476 ± 0.304, 1.476 ± 0.215, and 0.616 ± 0.075 in the presence and 0.192 ± 0.035, 0.527 ± 0.084, 0.540 ± 0.085, and 0.225 ± 0.024 in the absence of 20 µM GTPS. Furthermore, the addition of PMA to membrane fractions prepared from untreated HL-60 cells had no effect on either basal or GTPS-stimulated PLD activities (data not shown).


Figure 1: Potentiation of GTPS-dependent PLD activity by pretreatment of HL-60 with PMA and fMLP time dependence. Labeled intact HL-60 cells were pretreated for 100 nM PMA (A) or incubated for 5 min with 10 µM CB and stimulated with 0.1 µM fMLP (B) for the indicated times. Membranes were prepared, and PLD activity was assayed in the presence (circle) and absence (bullet) of 20 µM GTPS for 20 min at 37 °C as described under ``Experimental Procedures.'' There were no stimulations at time 0. Values are mean ± S.E. of at least three experiments. *, p < 0.05 for values compared with the control using Student's paired t test.



The biological effects of the chemotactic peptide fMLP include the activation of PLD(7, 15) . Therefore, the effects of fMLP on the activity of the membrane-associated PLD were examined next. HL-60 cells were preincubated for 5 min with CB and then stimulated with fMLP for varying times, following which the membrane fractions were prepared. The PLD activity was then assayed in the absence or presence of 20 µM GTPS for 20 min (Fig. 1B). The basal membrane-associated PLD activity (i.e. in the absence of GTPS) remained essentially stable during the length of the experiment and was affected only slightly, if at all, by a preincubation of HL-60 cells with fMLP. On the other hand, the magnitude of the response to GTPS rapidly increased in membranes from cells stimulated with the formylated peptide. The effect of fMLP was observed after 15 s and reached a maximum within the first minute of preincubation. The levels of PEt produced in response to GTPS were maintained for up to a 5-min preincubation with fMLP.

Variations of ARF Content in HL-60 Membranes following Stimulation with Agonists

In granulocytes and HL-60 cells, the activation of a membrane-associated PLD activity required cytosolic cofactors. These findings suggest that a least part of the GTPS-stimulated PLD activity is dependent on the translocation of one or more cytosolic cofactors, which is in accordance with previously reported results(29, 30) . One of the required cytosolic cofactors has been shown to be the small GTP-binding protein, ARF. We therefore considered the possibility that the translocation of ARF to membranes mediated the potentiation of the PLD response to GTPS. This hypothesis was tested first by examining the effects of ARF1-(2-17) and ARF4-(2-17) NH(2)-terminal peptides on the GTPS-sensitive PLD activity in HL-60 membranes. To this end, membranes were prepared from PMA-treated HL-60 cells, and the GTPS-sensitive PLD activity was evaluated in the absence and presence of 50 µg/ml of the NH(2)-terminal peptides of ARF1-(2-17), ARF4-(2-17), Rab3A AL-(33-47) and of the COOH-terminal peptide of the alpha-subunit of transducin. The ARF1 and ARF4 peptides significantly inhibited the GTPS-stimulated PLD activity in membranes from PMA-pretreated HL-60 cells by 50 ± 9.5% and 47 ± 8%, respectively (p < 0.05). On the other hand, neither the Rab3A nor the G(t)alpha peptides had any significant effect. The inhibition of GTPS-stimulated PLD activity by ARF NH(2)-terminal peptides is consistent with a role of ARF in the activation of PLD, suggesting a causal relationship. Caution should nevertheless be exercised when interpreting the data obtained using ARF NH(2)-terminal peptides, which can have nonspecific effects (31) .

In view of the role that has been postulated for ARF in the regulation of PLD activity, we then sought to examine the amounts of ARF in membrane fractions derived from untreated HL-60 cells and from phorbol ester-treated cells. Membranes from untreated HL-60 cells as well as from cells treated for 2.5 min with PMA, PdBu, or 4alphaPdBu were prepared and blotted with anti-ARF antibodies. The blots (Fig. 2B) were analyzed by densitometry, and the results of these experiments are summarized in Fig. 2A. These data demonstrate detectable amounts of ARF in membranes from resting cells. In addition, the levels of membrane-bound ARF were significantly increased in response to the protein kinase C activators PMA and PdBu but not by 4alphaPdBu. When expressed as a percentage of the unstimulated level, the amount of membrane-associated ARF averaged 163 ± 8%, 153 ± 13%, and 97 ± 21% (n = 4) in response to PMA, PdBu, and 4alphaPdBu, respectively.


Figure 2: Evaluation of membrane-associated ARF upon stimulation of HL-60 cells with phorbol esters and fMLP. Intact HL-60 cells were treated with 100 nM PMA, 250 nM PdBu, 250 nM 4alphaPdBu, 0.1 µM fMLP, or Me(2)SO for 2.5 min. The membrane content of ARF was determined by resolution of 100 µg of protein samples on 12% SDS-polyacrylamide gel electrophoresis, electrotransfer to polyvinylidene difluoride membrane and immunostaining with the anti-ARF monoclonal antibody 1D9 as described under ``Experimental Procedures.'' A, densitometric evaluation of membrane-associated ARF in phorbol ester-treated cells. B, a blot representative of four independent experiments is shown. C, densitometric evaluation of membrane-associated ARF in fMLP-treated cells. Intact cells were incubated for 5 min at 37 °C with 10 µM CB and stimulated for 1 min with fMLP. D, a blot representative of four similar experiments is presented.



Consistent with the prior observations with phorbol esters, preincubation of HL-60 cells with fMLP 100 nM for 1 min also enhanced the amount of membrane-associated ARF, as can be observed after a SDS-polyacrylamide gel electrophoresis resolution of membrane proteins and Western blotting with the anti-ARF antibody 1D9 (Fig. 2D). Densitometric evaluation of blots statistically confirmed that fMLP induced a translocation or association of cytosolic ARF to the membranes. In six independent experiments, the amount of membrane-bound ARF averaged 165 ± 10% as compared with control membrane fractions, when fMLP was the stimulus (Fig. 2C)

CB Is Required for Maximal Activation of PLD by fMLP and for the Translocation of ARF

CB is a pharmacological agent that is necessary for maximal activation of PLD by fMLP in granulocytes(13) . Experiments were conducted in order to ascertain whether or not CB was essential for activation of membrane-associated PLD by fMLP and, if so, whether or not ARF translocation was affected as well. Fig. 3B shows that although PLD activity in membranes is slightly enhanced by a preincubation with fMLP without CB there was no significant potentiation of the response to GTPS. These results concur with those in Fig. 3A, which demonstrate that there is little translocation of ARF in the absence of CB. By itself, CB has no effect on basal membrane ARF levels. However, CB increases ARF content and potentiates GTPS-stimulated PLD activity in membranes derived from fMLP-treated cells. Next we sought to determine if CB had any effect on PMA-stimulated PLD activity and found that CB had no effect on either PLD activity or ARF translocation in this case. The data in Fig. 3B also show that when PMA and fMLP are used together as agonists they induce a PLD activation and an ARF translocation (Fig. 3, A and B), which is less than or equal to that obtained through stimulation by either fMLP or PMA alone.


Figure 3: Effect of CB on ARF translocation and PLD activity induced by fMLP and PMA. Labeled or unlabeled intact HL-60 cells were either pretreated for 5 min with 10 µM CB or the vehicle (Me(2)SO) and stimulated for 2.5 min with 100 nM fMLP and 100 nM PMA alone or in combination. Membrane fractions were prepared as described under ``Experimental Procedures.'' A, ARF content was determined by resolution of 30-µg protein samples on 12% SDS-polyacrylamide gel electrophoresis, electrotransfer to polyvinylidene difluoride membrane and immunostaining using an anti-ARF polyclonal antibody. A blot representative of two experiments is shown. B, PLD activity was assayed with or without 20 µM GTPS for 20 min at 37 °C. Values are mean ± S.E. of at least three experiments. p values were obtained using Student's paired t test.



Activation of PLD Required Multiple Cytosolic Factors

Recently, a 50-kDa fraction for PLD activation was identified(25) . In an effort to determine the role of this 50-kDa fraction and to ascertain what effect exogenous myristoylated recombinant ARF would have on membranes derived from stimulated and unstimulated cells we added either 50-kDa factors or ARF on membranes derived from control and fMLP-stimulated cells. Labeled cells were pretreated for 5 min with CB and then stimulated with 100 nM fMLP or the vehicle dimethyl sulfoxide for 2 min. The membrane fractions derived from these cells were then assayed for PLD activity in the absence or presence of either 10 µg of the 50-kDa fraction or 1 µM of myristoylated recombinant ARF in the presence or absence of 20 µM GTPS. The addition of the 50-kDa fraction to control membranes greatly enhanced GTPS-stimulated PLD activity (Fig. 4A). The 50-kDa fraction, when added to fMLP-stimulated membranes, also enhances GTPS-stimulated PLD activity. The effects of fMLP pretreatment and of the 50-kDa fraction were less than additive. In fact, the PLD response to GTPS is about the same as that observed when the 50-kDa fraction is added to control membranes (Fig. 4A). Interestingly, the 50-kDa fraction consistently increased basal PLD activity in the absence of GTPS. Moreover, the GTPS-sensitive PLD was further stimulated by the addition of high concentrations of the 50-kDa fraction (50 µg of proteins) to membranes from control and fMLP-stimulated cells (data not shown).


Figure 4: Effects of exogenous recombinant ARF or the 50-kDa factor on PLD activity in membranes derived from control or fMLP-stimulated cells. Labeled intact HL-60 cells were pretreated for 5 min with 10 µM CB and stimulated with either 100 nM fMLP for 2 min or with the vehicle (Me(2)SO). Membrane fractions were prepared, and PLD activity was assayed in the presence and absence of ARF or the 50-kDa fraction. A, where indicated, 10 µg of proteins from the 50-kDa fraction were incubated with or without 20 µM GTPS. B, where indicated, 1 µM myristoylated recombinant ARF was incubated in the presence and absence of GTPS for 20 min at 37 °C. Values are mean ± S.E. of at least three experiments.



The addition of ARF to control membranes in the presence of GTPS increases PLD activity to levels similar to that of membranes derived from fMLP-stimulated cells. By contrast, the addition of ARF to membranes derived from fMLP-treated cells has little or no effect on PLD activity induced by GTPS (Fig. 4B). In the absence of GTPS, ARF was without effect on basal PLD activity.


DISCUSSION

PLD activation requires both cytosolic and membrane factors(29, 30) . Activation of PLD by GTP analogues implies a role for GTP-binding proteins. In granulocytes and HL-60 cells, GTP analogues do not stimulate PLD activity in cytosol-depleted cells. A small GTP-binding protein, ARF, has been identified as an important component in PLD activation. The addition of exogenous recombinant ARF is able to reconstitute GTPS-stimulated PLD activity in cytosol-depleted human granulocytes(19) . Though GTPS-dependent PLD has not been purified to homogeneity, an ARF-dependent PLD activated by ARFs has been characterized(18, 31) . Evidence also suggest that PMA-mediated responses may be dependent on the presence of ARF1(32) . The observation that PMA-pretreatment greatly enhances the ARF-dependent PLD activity suggests the recruitment of additional cytosolic components to the membranes.

The present study was undertaken to determine whether translocation of cytosolic ARF to the membranes is required for maximum PLD activation and if distinct PLD activation pathways, protein kinase C-mediated and receptor-mediated, had a similar downstream effect, which is the association of cytosolic ARF to the membrane fraction. We demonstrated that a pretreatment of intact HL-60 cells with fMLP or PMA enhances the subsequent response to GTPS by 2-3-fold in isolated membrane fractions, suggesting the stable recruitment of cytosolic components at the membranes. The GTP dependence of the effects suggests translocation of GTP-binding proteins. This is in agreement with the observation that the addition of PMA to isolated membrane fractions had no effect on GTPS-stimulated PLD activity (results not shown). Following pretreatment of intact HL-60 cells with two different types of agonists we demonstrated, by Western blotting and densitometric analysis of blots, a 60-70% increase in the levels of membrane-bound ARF. When either PMA or fMLP are used as agonists, similar downstream effects can be observed: the binding of ARF to the membranes and potentiation of the responses to GTPS. Treatment of rat basophils with phorbol esters was similarly reported to promote ARF binding to the Golgi membrane(24) . Maximal stimulation of membrane GTPS-sensitive PLD as well as ARF translocation induced by fMLP required a prior treatment with CB. CB was shown to have no effect on either PLD activity or ARF translocation when used on control cells or PMA-stimulated cells. Our results differ from those of Balsinde et al.(33) in that in the cell-free system no synergy was observed between PMA and fMLP. In fact, when both agonists were used together PLD activity was no higher than when the cells were stimulated by a single agonist.

As described previously by Cockcroft(34) , we found that the incubation of membrane fractions derived from PMA-pretreated cells with NH(2)-terminal peptides of ARF1-(2-17) and ARF4-(2-17) inhibited GTPS-stimulated PLD activity in membrane fractions by approximately 50%, while unrelated peptides such as Rab3A and G(t)alpha peptides had no effect on GTPS responses. Though ARF peptides may have nonspecific effects(34) , the data are consistent with the peptide ARF1-(2-17) being a specific inhibitor in an ARF assay(35) .

Accumulating evidence demonstrates that PLD activation required multiple cytosolic-derived components, including ARF-type small GTPases and other factors contained in a 50-kDa cytosolic fraction(25, 36) . The cytosolic factors implicated in PLD activation have been further investigated by Singer et al.(37) and are now thought to comprise at least two different groups of factors implicated in PLD activation apart from ARF. One group contains RhoA and is GTPS-sensitive, and the other group activates PLD, albeit slightly, in the absence of GTPS. These investigators and others also demonstrated that PLD activity is synergistically activated by ARF and RhoA(36, 37, 38) . Our results show that stimulation of cells by fMLP renders their membrane fractions less sensitive to activation by exogenous ARF or the 50-kDa cytosolic factors, suggesting that these factors have already been translocated to the membrane. However, the magnitude of PLD activation in membrane from control and to a lesser extent from stimulated cells depends largely on exogenously added 50-kDa fraction. The 50-kDa fraction was shown to contain Rho-related proteins and cytosolic factor(s) other than RhoA and Rac2(25) . A faint translocation of RhoA was observed in membranes derived from fMLP-stimulated cells in the presence of CB exclusively and not with PMA alone or in combination with fMLP (data not shown). The data suggest that component(s) of the 50-kDa PLD-inducing fraction are also translocated to the membrane; however, their association may be less stable than that observed with ARF. The amounts of membrane-associated 50-kDa factors is likely the rate-limiting step for maximal PLD activation in membranes derived from stimulated cells.

Maximal activation of the GTP-dependent PLD requires lipid cofactors such as phosphatidylinositol 4,5-biphosphate and possibly phosphatidylinositol 3,4,5-triphosphate(18, 39, 40) . The phosphatidylinositol 4,5-biphosphate dependence is consistent with results showing that acidic phospholipids have complex effects on ARF and ARF-regulatory proteins(41, 42) . Thus, phosphatidylinositol lipids resynthesis may act in concert with ARF in a general mechanism for PLD activation. Accordingly, the activation of PLD by neutrophil agonists has been reported to be inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase(43, 44) . RhoA may have a role in PLD activation through stimulation of phosphatidylinositol 3-kinase (45) and phosphatidylinositol 4-phosphate 5-kinase(46) .

In summary, ARF and possibly other factors such as those contained in the 50-kDa fraction are translocated to the membrane fraction of HL-60 cells when these cells are pretreated with either an active phorbol ester or the chemotactic peptide fMLP. ARF translocation correlates with increased GTPS-stimulated PLD activity in isolated membranes. An essential role for cytosolic ARF in PLD activation is supported by the present study. Cytosolic ARF needs to be associated with the membrane to be active and to interact with its effector, possibly membrane-bound PLD itself. It will be interesting to see if ARF interacts directly with PLD or through the assembly of a multifunctional protein complex upon stimulation of HL-60 cells with appropriate agonists.


FOOTNOTES

*
Supported in part by grants and fellowships from the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, CHUL, Room 9800, 2705 Blvd. Laurier, Ste-Foy, Québec, G1V 4G2, Canada. Tel.: 418-654-2772; Fax: 418-656-2765.

(^1)
The abbreviations used are: PLD, phospholipase D; ARF, ADP-ribosylation factor; fMLP, N-formyl-methionyl-leucyl-phenylalanine; GTPS, guanosine 5`-O-(thiotriphosphate); PEt, phosphatidylethanol; PMA, phorbol 12-myristate 13-acetate; PdBu, phorbol 12,13-dibutyrate; 4alphaPdBu, 4alpha-phorbol 12,13-dibutyrate; CB, cytochalasin B.


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