©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ADP-ribosylation Factor Functions Synergistically with a 50-kDa Cytosolic Factor in Cell-free Activation of Human Neutrophil Phospholipase D (*)

(Received for publication, November 22, 1994; and in revised form, December 13, 1994)

J. David Lambeth (§) Jong-Young Kwak Edward P. Bowman David Perry David J. Uhlinger Isabel Lopez

From the Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.


INTRODUCTION

Phospholipase D has been implicated as an important signal transducing enzyme in a variety of cells and tissues (1) and is activated by a variety of receptor-linked agonists and by phorbol esters. The enzyme catalyzes the hydrolysis primarily of phosphatidylcholine to generate phosphatidic acid. The latter has been implicated as a biologically active molecule (2, 3, 4, 5) and can also be metabolized via phosphatidic acid phosphohydrolase to form diradylglycerol, a protein kinase C activator. In human neutrophils and HL-60 cells, the major source of diradylglycerol is the phospholipase D/phosphatidic acid phosphohydrolase pathway(6, 7) . In human neutrophils, the diradylglycerol generated through this pathway is essential for cell activation(7) .

Phospholipase D can be reconstituted in cell-free systems in both membrane and soluble preparations. Some of these are activated by detergents and fatty acids(8, 9, 10, 11) , but their relationship to receptor-linked PLD (^1)is unclear. A PLD activity in liver plasma membranes was activated by GTPS (12) and by phorbol esters by a protein kinase C-dependent, phosphorylation-independent mechanism(13) . Similar activities have been noted in homogenates from phagocytic cells(14) , but activity was absent in isolated plasma membranes. PLD activity in granulocytes was restored upon recombining cytosol and plasma membrane, and protein factors in both fractions were needed for both the GTPS (15) and phorbol ester responses(16) . The cytosolic factor had an apparent size of 50 kDa by gel exclusion chromatography(17) .

Bowman et al.(17) showed that the GTPS-binding protein in the granulocyte cell-free system is in the plasma membrane, and identified the protein as a Rho family small GTPase protein, based on inhibition by Rho GTPase Dissociation Inhibitor. The GTP-binding species in liver plasma membranes was recently shown to be extracted by Rho GTPase Dissociation Inhibitor, and activity was restored with recombinant Rho A(18) . Thus, in both phagocytic cells and in liver, a protein factor in the plasma membranes is a Rho GTPase.

ADP-ribosylation factor (ARF), another small molecular weight GTP-binding protein in the Ras superfamily, was also implicated as a PLD-activating factor. Using permeabilized, cytosol-depleted HL-60 cells that had lost their PLD activity, brain cytosol reconstituted activity(19) ; the activating factor was purified and identified as ARF (ARF-1 or ARF-3). Brain cytosol also stimulated PLD in isolated HL-60 plasma membranes and membrane extracts, and ARF was likewise isolated as the activating factor(20) .

These studies utilized heterologous systems in which ARF was identified from brain cytosol using HL-60 membranes or permeabilized cells, but it was not shown whether ARF was the primary activating factor in cytosol from granulocytes themselves. We undertook the present studies to investigate whether ARF was present in our human neutrophil fractions, and whether ARF was the same as the cytosolic factor that we identified previously(16) . We find that ARF activity and immunoreactivity, which is present almost exclusively in the cytosol, is distinct from the majority of the cytosolic factor. Unexpectedly, while ARF alone supported PLD activity weakly, it synergized with a low concentration of the cytosolic factor to cause a marked stimulation of PLD activity.


EXPERIMENTAL PROCEDURES

Cell-free Assay of Phospholipase D

Human peripheral blood neutrophils were isolated as described previously(21) . Cells were labeled with [^3H]alkyl-lysophosphatidylcholine (1.5 µCi/ml; 2 times 10^7 cells/ml) for 75 min at 37 °C. Cells were then incubated on ice with diisopropyl fluorophosphate, and subcellular fractions were isolated as described(16) . Incubations were carried out in the presence of 1.6% ethanol, along with agonists as indicated, and the reaction was terminated by transfer to chloroform:methanol (1:2). Lipid was extracted (16) and spotted onto TLC plates (Silica Gel 60), which were developed with chloroform:methanol:acetic acid (90:10:10 by volume). Radioactivity was quantified using a Bioscanner equipped with two-dimensional software. Phosphatidylethanol formed by transphosphatidylation is expressed as the percentage of total counts in a given lane.

Assay of ARF Using ADP-ribosylation

ARF was assayed essentially according to (22) and (23) , using transfer of ADP-ribose from [^14C]NAD (nicotinamide [U-^14C]adenine dinucleotide) to agmatine (an arginine analog) followed by chromatography on AG1-X2.

Expression and Purification of ARF

Recombinant human ARF-1 was engineered it into the pTrc vector (containing an ampicillin resistance gene) for expression in Escherichia coli. Because ARF is myristoylated at its N terminus, cells were co-transformed with a vector containing the N-myristoyltransferase gene-1 (NMT-1) in the pBB131 vector, which also contains a kanamycin resistance gene (kindly provided by Jeff Gordon, Washington University School of Medicine, St. Louis, MO). Co-transformed cells were selected on the basis of resistance to both ampicillin and kanamycin. After growing to 0.5-0.6 OD units, bacteria were induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 3.5 h in the presence of 200 µM sodium myristate (24) and harvested by centrifugation, and pellets were frozen at -80 °C. After thawing, cells were resuspended in 50 mM Tris HCl, pH 8.0, 30 mM MgCl(2), 30 mM EDTA, 18% sucrose, and 0.05% Triton X-100 containing leupeptin, pepstatin, aprotinin, and diisopropyl fluorophosphate, and sonicated and centrifuged to remove cell debris. The supernatant containing ARF was loaded onto a 200-ml DEAE-cellulose column and washed with 2 liters of 20 mM Tris/HCl buffer. Fractions containing ARF activity eluted under these conditions and were pooled, concentrated to 2 ml (Amicon YM10 ultrafiltration), and chromatographed on Sephacryl S-200 (1.7 times 80-cm column). The yield from 2 liters of cells was approximately 5 mg of protein, which was greater than 98% pure based on SDS-polyacrylamide gel electrophoresis (Fig. 1, lane A). Using a 40-ml culture containing 400 µCi of [^3H]myristate, myristoylation was confirmed using autoradiography of the SDS-gel separated protein (Fig. 1, laneB). Non-myristoylated ARF was prepared in a similar manner, except that the bacteria did not contain the gene for N-myristoyltransferase. Bovine ARF was purified as in (25) .


Figure 1: Expression of myristoylated human ARF-1 in E. coli. Human ARF-1 was expressed and purified as described under ``Experimental Procedures.'' LaneA shows a Coomassie-stained SDS gel of the purified ARF. Lane B is an autoradiograph of the purified material obtained from cultures prelabeled with [^3H]myristate.



Chromatographic Separation of ARF from the Cytosolic Phospholipase D Factor

Cytosol (20 ml from 3 times 10^9 neutrophils) was dialyzed overnight with one change of buffer against Buffer A (25 mM triethanolamine, pH 7.4, containing 10 mM MgCl(2) and 3 mM NaCl), loaded onto a DEAE-cellulose column (2.5 times 10 cm) preequilibrated with Buffer A, and eluted with a linear gradient consisting of 50 ml of Buffer A and 50 ml of Buffer A containing 100 mM KCl. Fractions (5 ml) were collected and each was concentrated to about 100 µl using a Centricon 10, and aliquots were analyzed for ARF and phospholipase D-supporting activity.

Immunochemical Methods

Western blotting was carried out by standard methods(26) , using alkaline phosphatase conjugated second antibody to visualize bands. Antibodies were raised in rabbits against the purified bovine brain ARF (a mixture of isoforms) and against human recombinant ARF-1 and were used in dilutions of 1:250 and 1:5000, respectively.


RESULTS AND DISCUSSION

Localization of ARF in Subcellular Fractions

The occurrence of ARF in plasma membrane and cytosol from human neutrophils was investigated. Fig. 2shows that at equivalent protein amounts, most of the ARF immunoreactivity was seen in the cytosol. Cytosol contains approximately 10-fold more protein than does plasma membrane from the same number of cells. Higher concentrations of cytosol could not be examined due to distortion of the electrophoresis pattern, so that it was not technically feasible to visualize protein from an equivalent cell number. However, the data permit the rough extrapolation that well greater than 95% of the ARF in the cell is in the cytosol. Confirming this interpretation, ARF activity using the agmatine assay was detected in cytosol (see below), but no ARF activity was seen in plasma membrane.


Figure 2: Immunochemical visualization of ARF in the cytosol and plasma membrane from human neutrophils. The indicated quantities of plasma membrane (PM), cytosol, recombinant human ARF-1 (rARF), or recombinant human Rac1 (rRac1) were electrophoresed on polyacrylamide gels, transferred to nitrocellulose, and visualized by immunostaining as described under ``Experimental Procedures'' using antibody to the human recombinant ARF-1.



Separation of ARF from the Cytosolic Phospholipase D Factor

Cytosol from human neutrophils was chromatographed on DEAE-cellulose, and ARF was identified in fractions both immunochemically and by activity, using ADP-ribosylation. The distribution of ARF in fractions was compared with the ability of fractions to support GTPS-stimulated PLD. Results (Fig. 3) indicate that ARF activity (opensquares) and immunoreactivity (Western blot shown in inset) eluted in fractions 2, 3, and 4, while greater than 95% of the PLD activity eluted as a broad peak beginning with fraction 6. These latter fractions were used as a source of ARF-depleted factor in subsequent experiments. Interestingly, a small amount of PLD-stimulating activity was seen in early fractions corresponding to ARF. It seems likely that this low activity is that which was previously noted by other groups (19, 20) and does not represent the major cytosolic factor in mature neutrophils. Differences with earlier studies might also be accounted for by differences in assay methods. For example, Brown et al.(20) used phospholipid vesicles to provide phosphatidylcholine substrate, whereas here the substrate is incorporated into the plasma membrane itself. It is possible that the differences in methodology may permit the assay of separate pools of membranes with distinct phospholipase D isoforms.


Figure 3: Chromatographic separation of ARF from the phospholipase D cytosolic factor from human neutrophils. Cytosol from human neutrophils was chromatographed on DEAE-cellulose, as under ``Experimental Procedures.'' Concentrated fractions were assayed for ADP-ribosylation activity (open squares) or stained for ARF immunoreactivity (inset). Fractions were also assayed for GTPS-dependent PLD-supporting activity by assaying in the presence of plasma membrane (filledsquares). The experiment shown is representative of four.



Synergistic Activation of Phospholipase D Activity by ARF and Cytosolic Factor

In Fig. 4, the effect of bovine ARF (primarily ARF-1 and ARF-3, upperpanel) and recombinant myristoylated ARF-1 (lowerpanel) on PLD activity was investigated, either in the absence or presence of ARF-depleted cytosol. The major (second) peak of activity from experiments such as those shown in Fig. 3was utilized as the source of ARF-depleted cytosol. Fractions showing greater than 40% of maximal activity were pooled and concentrated. This material showed a 1.7-fold enrichment in PLD specific activity compared with cytosol. In addition, when the concentrated material was rechromatographed on a Superose 12 column, a single peak was eluted corresponding to a molecular mass of approximately 50 kDa (data not shown).


Figure 4: Synergistic activation of phospholipase D by ARF and cytosolic factor. The concentrations of purified bovine brain ARF (bARF, upperpanel) or purified recombinant human ARF-1 (rARF) were varied as indicated, and phospholipase D-catalyzed transphosphatidylation was monitored by measuring phosphatidylethanol formation (PEth), expressed as a percentage of the total radiolabeled lipids (see ``Experimental Procedures''). Activity was measured in the presence of plasma membranes and GTPS (10 µM), either in the absence of cytosol (filled circles), or the presence of 15 µg (filledsquares) or 50 µg (filledtriangles) of ARF-depleted cytosol. Errorbars represent the range or standard error of two to three determinations for each point.



ARF in the absence of ARF-depleted cytosol caused a small stimulation of PLD, which was 10% or less of that seen with high cytosol. (^2)At a high concentration of ARF-depleted cytosol, PLD activity was maximal and was not further stimulated by either bovine ARF or recombinant ARF-1. However, at a low concentration of cytosol, both bovine and recombinant ARF produced a marked stimulation. The two forms of ARF were approximately equipotent, stimulating maximally at less than 1 µM ARF. The bovine ARF in combination with cytosol stimulated to the same degree as a high concentration of cytosol, while the recombinant ARF-1 was somewhat less effective, perhaps due to incomplete myristoylation or to the presence of more effective isoforms that might be present in the bovine preparation. Non-myristoylated ARF had no effect in this concentration range. The stimulation by ARF was synergistic rather than additive with cytosol, indicating that ARF cooperates in the function of the cytosolic factor.

The present study demonstrates that the major cytosolic factor in human peripheral blood neutrophils is distinct from ARF. The PLD activity is seen in the absence of added ARF, indicating that cytosolic ARF is not essential for the activity. While it is possible that the very small amount of ARF which can sometimes be detected in plasma membrane preparations may account for some activity, this seems unlikely; we estimate that less than 1 nM ARF would be contributed by the presence of plasma membrane, whereas a minimum of 100 nM ARF is required to observe any stimulation with cytosol. Thus, an essential role for ARF in phospholipase D activity is not supported by the present studies.

However, ARF is shown herein to act cooperatively with the cytosolic factor, so that in the presence of ARF, a low concentration of cytosol suffices for near-maximal activation. The mechanism by which ARF synergizes with the cytosolic factor remains unclear. ARF functions not only in stimulation of cholera toxin-dependent ADP-ribosylation of G protein alpha subunits(27) , but also in Golgi membrane trafficking (28, 29, 30, 31) . ARF translocates from cytosol to a putative receptor on Golgi vesicles and triggers coatamer binding. No general themes have yet emerged regarding ARF mechanisms, except that in all its functions, membranes, and/or phospholipids are involved. Given the well known ability of GTP-binding proteins to complex with their effector enzymes, it is reasonable to suggest that ARF functions by forming a complex either with the cytosolic factor or with a PLD-related protein in the plasma membrane. ARF is also known to interact directly with phospholipids, and it is possible that it is functioning by binding to either the substrate (e.g. to make phosphatidylcholine more accessible to phospholipase D) or a regulatory lipid such as phosphatidylinositol 4,5-bisphosphate. However, until the identity and function of the cytosolic factor as well as any other unknown membrane proteins is revealed, any proposed mechanisms remains speculative. Thus, in summary, three or more proteins: a membrane-associated Rho-family GTPase, cytosolic ARF, and a 50-kDa cytosolic factor, participate in GTPS-dependent PLD activity in human neutrophils.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA46508. 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.

(^1)
The abbreviations used are: PLD, phospholipase D; ARF, ADP-ribosylation factor; GTPS, guanosine 5`-3-O-(thio)triphosphate.

(^2)
The basal activity seen in various plasma membranes differed considerably for unknown reasons, as was the case in the upper and lowerpanels of Fig. 4, which utilized two different membrane preparations. Similar experiments with both bovine ARF have been repeated using two different membrane preparations and the experiment with recombinant ARF has been repeated four times. In all cases, the same general results were obtained regardless of the basal activity seen.


ACKNOWLEDGEMENTS

We thank Walter A. Patton, Su-Chen Tsai, and Joel Moss of the Laboratory of Cellular Metabolism, NHLBI, National Institutes of Health for providing the antibody to bovine brain ARF and the cDNA for human ARF-1, and for carrying out preliminary ADP-ribosylation assays.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.