©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Phospholipase D in HL60 Cells
EVIDENCE FOR A CYTOSOLIC PHOSPHOLIPASE D (*)

Abdur R. Siddiqi (1), Jennie L. Smith (1), Annette H. Ross (1), Rong-Guo Qiu (2), Marc Symons (2), John H. Exton (1)(§)

From the (1) Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and (2) Onyx Pharmaceuticals, Richmond, California 94806

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D (PLD) activity that was stimulated by guanosine 5`- O-(3-thiotriphosphate) (GTPS) was detected in cytosol and membranes of HL60 cells. GTPS-stimulated PLD activity was detected in the membranes when exogenous labeled phosphatidylcholine was used in the presence of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate, but not when [H]myristic acid-labeled endogenous substrate was used. Cytosolic PLD co-chromatographed with small GTP-binding proteins on anion-exchange columns, but subsequent chromatography separated these. Reconstitution studies demonstrated ADP ribosylation factor (ARF) as a regulator of cytosolic PLD, whereas the Rho proteins RhoA and CDC42Hs were ineffective. The cytosolic enzyme showed very little activity in the absence of GTPS and was stimulated by 2 m M Ca, whereas the membrane enzyme had significant basal activity and was inhibited by Ca.

Rho-specific GDP dissociation inhibitor inhibited GTPS stimulation of membrane PLD activity in the presence and absence of cytosol. The stimulation in GDP dissociation inhibitor-treated membranes could be partially recovered by the addition of recombinant Rho proteins (RhoA, Rac1, CDC42Hs). RhoA and Rac1 were also stimulatory in untreated membranes. However, Western blot analysis of membranes showed the presence of RhoA, but not Rac1 or CDC42Hs, suggesting that RhoA was the endogenous small GTP-binding protein involved in GTP-dependent PLD activity in membranes in the absence of cytosol. ARF also stimulated the membrane PLD in the presence of GTPS, and the combination of RhoA and ARF showed a synergistic effect. These results show the presence of ARF-dependent PLD activity in both cytosol and membranes. The membranes contain another PLD activity for which the endogenous regulator appears to be RhoA. The data suggest the existence of at least two different PLD isozymes in HL60 cells.


INTRODUCTION

Phospholipase D (PLD)() catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline and has been implicated in signal transduction in a variety of cell types (1, 2, 3, 4, 5) . During the last decade, a large number of agonists that stimulate PLD have been identified (1, 2, 3, 4, 5) , and PA has been proposed to regulate DNA synthesis, cell proliferation, and other cellular functions (5, 6, 7, 8, 9, 10, 11) . PA can also be metabolized to diacyglycerol, which in turn stimulates protein kinase C. A unique property of PLD is its catalysis of the transphosphatidylation reaction in which the phosphatidyl moeity of phospholipids is accepted by a primary alcohol (12, 13, 14) .

Although PLD activity has been shown to be associated with membranes in mammalian cells (15, 16, 17, 18, 19) , there is one report of its existence in the cytosol (20) . In HL60 cells, PLD activity was reported in membranes and shown to be regulated by GTPS in the presence of a cytosolic factor (21, 22) , which was proposed to be a G protein (23, 24) . Two recent reports (25, 26) have revealed that the cytosolic factor is the small G protein (SMG) ADP ribosylation factor (ARF). This 21-kDa protein was first identified and purified on the basis of its activity as a cofactor for cholera toxin-catalyzed ADP ribosylation of the -subunit of G(28, 29) . A recent study has also suggested the involvement of a membrane-associated G protein of the Rho family in PLD regulation (27) . This study demonstrated that GTP-dependent PLD activity in HL60 cell preparations was stimulated by a nonspecific GDP dissociation stimulator and inhibited by a Rho-specific GDP dissociation inhibitor (GDI).

The present investigation of PLD activity in HL60 cells has revealed the presence of PLD in cytosol as well as membranes. In addition, it has been found that the membrane-bound PLD can be stimulated by GTPS in the absence of cytosol, and evidence is presented that the G protein involved is RhoA.


EXPERIMENTAL PROCEDURES

Materials

Dipalmitoylphosphatidylcholine (dipalmitoyl-PC), phosphatidylethanolamine (PE), PA, and phosphatidylethanol (PEth) were purchased from Avanti Polar Lipids Corp. Phosphatidylinositol 4,5-bisphosphate (PIP) and GTPS were obtained from Boehringer Mannheim. [9,10-H]Myristic acid, dipalmitoyl [2- palmitoyl-9,10-H]phosphatidylcholine, and dipalmitoylphosphatidyl[ methyl-H]choline were from DuPont NEN. HL60 cells were obtained from ATCC. DEAE Sephacel and Superdex 200 were from Pharmacia Biotech Inc. Hydroxylapatite was from CalBiochem.

SMGs and Antisera

The cDNAs of Rac1 and Rho A (gifts of A. Hall, University College, London) and of CDC42 (a gift of S. Munemitsu, Onxy Pharmaceuticals) were provided with a modified Glu-Glu epitope tag at the N terminus, cloned into pAcC13, and transfected into Sf9 cells to produce recombinant baculovirus, and the proteins were purified on a Glu-Glu monoclonal antibody affinity column as described by Malcolm et al. (30) . Since the proteins were prepared from Sf9 cytosol, it is likely that they were partly modified by prenylation.

Soluble ADP ribosylation factor (sARF) (a mixture of ARF1 and ARF3) was purified from bovine or rat brain cytosol (31) . Polyclonal antibodies were raised against sARF according to Tsai et al. (32) . Both were kind gifts of S.-C. Tsai and J. Moss (National Institutes of Health). Antibodies against C-terminal peptides of RhoA, RhoB, Rac1, and Rac2 were from Santa Cruz Biotechnology Corporation. Glutathione S-transferase-fused Rho-specific GDP dissociation inhibitor (GST-Rho-GDI) was purified from E. coli transformed with the relevant plasmid (30) . This plasmid and antiserum against CDC42Hs were gifts from Y. Zheng and R. Cerione (Cornell University).

Culture and Labeling of Cells

HL60 cells were maintained in suspension culture in RPMI 1640 medium (Cell Gro) containing 10% fetal bovine serum (Sigma), at 37 °C in a humidified atmosphere of 5% COand 95% air. Cells were harvested when confluent (1 10cells/ml). Cells were labeled with [H]myristic acid, and membranes were prepared as described (21) . Membranes were aliquoted (0.5-1.0 mg protein/ml) and stored at -80 °C.

Preparation of Membranes and Cytosol

Harvested HL60 cells (4-6 10) were washed twice in buffer. A (25 m M HEPES (pH 7.2), 125 m M NaCl, 2.5 m M KC1, 0.7 m M MgCl, 0.5 m M EGTA, 10 m M glucose). Washed cells were resuspended in buffer B (5 m M HEPES (pH 7.4), 1 m M EGTA, 1 m M dithiothreitol, 250 m M sucrose, 12 µg/ml leupeptin, 10 µg/ml aprotinin, 20 µg/ml 4-amidinophenylmethanesulfonyl fluoride, and 20 µg/ml antipain) and were disrupted in a bomb by rapid decompression after equilibration with Nat 4 °C for 20 min at 500 psi. Unbroken cells and nuclei were removed by centrifugation at 500 g for 10 min. Membranes and cytosol were obtained by centrifugation at 125,000 g for 90 min. Supernatant (cytosol) was used for chromatography, whereas the pellet (membranes) was washed twice in buffer C (50 m M HEPES (pH 7.2), 10 m M KCl, 5 m M NaCl, 0.5 m M EGTA, and 3.5 m M MgCl). Membranes were aliquoted (5-10 mg/ml) and stored at -80 °C.

Chromatography of HL60 Cytosol

150 ml of cytosol (1 mg of protein/ml) was loaded on a DEAE-Sephacel column (2.5 12 cm). The column was washed with buffer D (25 m M HEPES (pH 7.4), 1 m M EGTA, and 1 m M dithiothreitol), and the bound proteins were eluted with a 200-ml linear gradient of NaCl (0-0.8 M) in buffer D. Fractions (3 ml) were assayed for PLD activity, and active fractions were pooled and used for further purification and characterization of PLD. For gel filtration chromatography, the pool of active fractions was concentrated to 3 ml using a YM10 Amicon membrane and applied to a 150-ml Superdex 200 column in buffer A. This was eluted with the same buffer. Column fractions (2 ml) were assayed for PLD activity in the presence and absence of bovine sARF. Alternatively, a pool of active DEAE fractions (from another cytosol preparation) was diluted with buffer D (without EGTA) to decrease the concentration of NaCl to 50 m M. The diluted pool was then loaded onto a 10-ml hydroxyapatite column. The column was extensively washed with buffer E (25 m M HEPES (pH 7.4), 1 m M dithiothreitol, and 50 m M NaCl), and the adsorbed material was eluted with a linear gradient of potassium phosphate (0-150 m M) in buffer E. Fractions (1 ml) were assayed for PLD activity in presence and absence of sARF.

Assays of PLD

[H]Myristic acid-labeled HL60 membranes were assayed for PLD activity as described (21) . PLD activity was measured in unlabeled membranes, cytosol, and column fractions, using phospholipid (PE/PIP/PC, 16:1.4:1) vesicles prepared according to Brown et al. (25) . Dipalmitoyl[2- palmitoyl-9-10-H]PC (0.5 µCi) (for [H]PEth formation) or dipalmitoyl[ methyl-H]choline PC (0.5 µCi) (for [H]choline release) was included in the phospholipid mixture for each assay. For a 60-µl assay volume, 10 µl of vesicles were added to the assay buffer (50 m M HEPES, (pH 7.2), 3 m M EGTA, 80 m M KCl, 1 m M dithiothreitol, 3 m M MgCl, and 2 m M CaCl) containing membranes, cytosol, or column fractions and were incubated at 37 °C for 30 min (unless otherwise indicated). For the [H]choline release assay, the reactions were stopped by the addition of 200 µl of 10% trichloroacetic acid and 100 µl of 1% bovine serum albumin, and the mixtures were processed as described (25) . For [H]PEth formation, ethanol (1% (v/v) for membranes or 2% (v/v) for cytosol and column fractions) was included in the reaction mixtures, and the reactions were terminated by the addition of 375 µl of chloroform/methanol/HC1 (50:98:2), and the lipids were extracted as described (33) . Separation and Quantitation of [H]PEth and [H]PA-Lipids from the incubation mixtures were resuspended in chloroform/methanol (2:1) and spotted onto thin layer plates (LK6D, Silica gel, Whatman). The plates were developed with ethyl acetate/isooctane/acetic acid/water (50:25:10:50, modified from Ref. 34). This solvent system gives better separation of PA and PEth. Separated phospholipids and standards were visualized on thin-layer chromatography plates by exposure to iodine vapor. Spots corresponding to PA and PEth were scraped, mixed with ready organic scintillant (Beckman), and counted.

SDS-Polyacrylamide Gel Electrophoresis and Western Analysis

SDS-polyacrylamide gel electrophoresis was performed on 14% acrylamide gels or gradient 10-25% acrylamide Novex gels (Novel Experimental Corp.). For Western analysis, proteins were transferred from SDS-polyacrylamide gels onto Immobilon P membranes (Millipore) for 1 h at 15 V using a semi-dry transfer apparatus (Bio-Rad). The immunoblots were blocked overnight with 1% (w/v) bovine serum albumin and 1% (v/v) goat serum and incubated for 2 h with primary antibody in blocking buffer. Blots were developed using the Vector stain alkaline phosphatase ABC kit (Vector Laboratories, Burlingame, CA).

Membrane Extraction

HL60 membranes were incubated with GST-Rho-GDI in buffer F (20 m M HEPES (pH 7.0), 10 m M glutathione) at 4 °C for 60 min. After incubation, membranes were centrifuged at 14,000 g for 10 min. Supernatants were collected, and membranes were washed twice in solution A. For controls, membranes were incubated in buffer F in the absence of GST-Rho-GDI. The supernatants (membrane extracts) and membranes were analyzed by Western blotting using antibodies against small G proteins (RhoA, RhoB, CDC42, and Rac1).


RESULTS

Distribution of PLD in HL60 Cells

Guanine nucleotide-sensitive PLD activity was detected in both membranes and cytosol of HL60 cells (Fig. 1 A). In contrast to previous reports (25, 26, 27) significant GTPS stimulation of PLD activity was observed in the membranes in the absence of cytosol, but only when exogenous [H]dipalmitoyl-PC was employed as substrate together with PE and PIPin the molar ratio described by Brown et al. (25) . In the absence of PIP, the activity was very low (data not shown, see Ref. 25). The stimulation was enhanced by the addition of cytosol (Fig. 1 A). In contrast, [H]myristic acid prelabeled membranes failed to exhibit GTPS-sensitive [H]PEth formation in the absence of cytosol (Fig. 1 B). Since oleate has been reported to stimulate membrane PLD in some cell types (5) , its effects were tested on HL60 membranes using exogenous [H]dipalmitoyl-PC, but no stimulation was observed (data not shown).


Figure 1: GTPS stimulation of PLD activity in HL60 membranes and cytosol. Panel A, HL60 membranes and cytosol were prepared as described under ``Experimental Procedures.'' HL60 membranes (30 µg of protein) or cytosol (10 µg of protein) or membranes plus cytosol were incubated for 30 min in the presence and absence of 30 µ M GTPS as indicated. [H]PEth formation from [H]palmitoyl-PC was measured in the presence of 1% ethanol under the conditions of Brown et al. (25) as described under ``Experimental Procedures.'' Panel B, HL60 cells were prelabeled with [H]myristic acid, and membranes and cytosol were prepared as described (21). Labeled membranes (20 µg of protein) or labeled membranes plus cytosol (10 µg of protein) were assayed for PLD activity using the conditions described under ``Experimental Procedures.'' Cytosol alone showed no activity. The data shown in panels A and B are representative of two experiments.



Partial Purification of Cytosolic PLD

To study the GTPS-sensitive cytosolic PLD activity, HL60 cytosol was fractionated by DEAE-Sephacel chromatography. A peak of GTPS-dependent PLD activity was eluted in the early part of the NaCl gradient (Fig. 2). In order to investigate the involvement of SMGs, column fractions were also immunoblotted using anti-ARF, anti-RhoA, and anti-CDC42Hs antibodies. As shown in Fig. 2, these SMGs were found in the fractions containing PLD activity. However, ARF immunoreactivity corresponded to the major fractions exhibiting GTPS-dependent PLD activity, although there was a minor component of PLD activity (fractions 30-34) that coeluted with RhoA and CDC42. In addition, we assayed the remaining inactive fractions in the presence of bovine cytosolic ARF (sARF) and detected no ARF-responsive enzyme. The fractions corresponding to the peak of GTPS-stimulated PLD activity were pooled and concentrated using an Amicon YM 10 membrane.


Figure 2: Fractionation of HL60 cytosol on DEAE-Sephacel. Panel A, HL60 cytosol was obtained as described under ``Experimental Procedures.'' It was chromatographed on DEAE-Sephacel, and the fractions were assayed for PLD activity. An aliquot (10 µl) of every second fraction was incubated with 30 µ M GTPS for 30 min, and [H]choline release from phosphatidyl[H]choline was measured as described under ``Experimental Procedures.'' Panel B, an aliquot (10 µl) of every second fraction was diluted with an equal volume of Laemmli SDS sample buffer, and 5 µl of this was subjected to electrophoresis using 14% polyacrylamide gels. After transfer to Immobilon P, proteins were blotted using antisera specific for ARF, RhoA and CDC42Hs.



To determine the molecular size of the ARF-responsive PLD, the concentrated pool was further fractionated by gel filtration using a Superdex 200 column. A lesser peak of GTPS-stimulated PLD activity was observed in the fractions that contained ARF (Fig. 3). Assuming that PLD activity may have separated from endogenous ARF, we assayed the remaining fractions in the presence of sARF. This revealed a peak of GTPS-stimulated PLD activity, which corresponded to a molecular mass of >150 kDa (Fig. 3). The nature of the higher molecular mass material was not determined, but it was assumed to be aggregated enzyme.


Figure 3: Gel filtration chromatography of a concentrated PLD activity peak from DEAE-sephacel. Panel A, the concentrated pool of PLD active fractions (see ``Experimental Procedures'') was applied to a Superdex 200 column and eluted as described under ``Experimental Procedures.'' Every second eluted fraction was assayed for [H]PEth formation from [H]palmitoyl-PC with the addition of 30 µ M GTPS in the presence ( closed symbols) and absence ( open symbols) of bovine sARF. Panel B, aliquots (10 µl) of every second fraction were diluted with an equal volume of Laemmli SDS sample buffer, and 5 µl of this was subjected to SDS-polyacrylamide gel electrophoresis and Western blotting as described for Fig. 2 B.



To further explore a role for ARF in the regulation of cytosolic PLD, we also chromatographed the concentrated pool of DEAE fractions on a hydroxylapatite column. Fractions from this column totally failed to show any GTPS-stimulated PLD activity unless reconstituted with bovine sARF (Fig. 4). The ARF-responsive PLD activity eluted in the late phase of the phosphate gradient. Endogenous ARF from the same column also restored the PLD activity, but no activity was detected with fractions containing endogenous Rho A or CDC42.


Figure 4: Hydroxylapatite chromatography of concentrated PLD activity peak from DEAE sephacel. Panel A, the concentrated pool of PLD active fractions, diluted with Buffer E as described under ``Experimental Procedures,'' was applied to a hydroxylapatite column, and eluted fractions (every third) were assayed for [H]PEth formation from [H]palmitoyl-PC in the presence of 30 µ M GTPS and in the presence () or absence () of bovine sARF. Panel B, aliquots (10 µl) of every third fraction were subjected to electrophoresis and Western blotted as described in Fig. 2.



Further Characterization of Cytosolic PLD

The concentrated pool of active PLD fractions from DEAE-Sephacel, hereinafter referred to as the DEAE fraction, was used for further characterization of the cytosolic PLD. The time course of PEth formation in the presence of GTPS was linear for 40 min, but essentially no activity was seen in the absence of the nucleotide (data not shown). The time course was similar to that of the membrane-associated activity (assayed with exogenous [H]PC), except that the latter showed significant activity in the absence of GTPS (data not shown). The GTPS concentration producing half-maximal stimulation of PEth formation by either cytosol or membranes was less than 1 µ M (Fig. 5). This is similar to that observed in cytosol-depleted HL60 cells in the presence of ARF (26) .


Figure 5: GTPS dependence of PLD activation in DEAE fraction and membranes. The DEAE fraction (10 µg of protein) or membranes (30 µg of protein) was incubated with the indicated concentrations of GTPS, and [H]PEth formation from [H]palmitoyl-PC was measured as described under ``Experimental Procedures.'' Data are expressed as the percent of total lipid radioactivity recovered in PEth at the end of the incubations and are representative of two experiments performed in duplicate.



GTPS-dependent PLD activity in the DEAE fraction was linearly proportional to protein concentration up to 10 µg (data not shown). Although millimolar Cahas been reported to stimulate PLD activity in HL60 cell membranes assayed using endogenous substrate (21, 25) , the cation had a negligible stimulatory effect when the membranes were assayed with exogenous PC, and was inhibitory at concentrations of 2 m M and higher (Fig. 6). In contrast, PLD in the DEAE fraction showed a 2-fold stimulation with 2 m M Ca(corresponding to 165 n M free Ca, see the legend to Fig. 6 ), which was lost at higher Caconcentrations.


Figure 6: Effect of Ca on PLD activity in HL60 membranes and DEAE fraction. HL60 membranes (30 µg of protein) or DEAE fraction (10 µg of protein) were incubated with the indicated concentrations of CaClin the presence of 30 µ M GTPS to measure [H]PEth formation for 30 min. The incubation medium contained 3 m M EGTA (see ``Experimental Procedures''), and the concentrations of free Cacorresponding to the added Caconcentrations were calculated to be 165 n M for 2 m M, 8.1 µ M for 4 m M, 25.4 µ M for 6 m M, and 70.6 µ M for 10 m M. Data are representative of two experiments performed in duplicate.



Identification of the SMGs Regulating Membrane PLD

In view of a previous report indicating the involvement of an unidentified Rho family SMG in the regulation of PLD (27) , we utilized HL60 membranes to examine GTPS-stimulated PLD activity as a function of increasing concentrations of GST-Rho-GDI (Fig. 7 A). As observed earlier (27) , GTPS-stimulated membrane PLD activity, assayed using endogenous substrate ([H]myristate-labeled membranes) in the presence of cytosol, was significantly inhibited by micromolar Rho-GDI. However, when assayed in the absence of cytosol using exogenous [H]PC, Rho-GDI was more potent and efficacious in inhibiting the PLD activity (Fig. 7 B). Since treatment of membranes with Rho-GDI has been reported to extract post-translationally modified Rho proteins (35, 36) , Western blotting of the supernatants was performed using antibodies to RhoA, RhoB, CDC42, Rac1, and Rac2. The results showed that RhoA was significantly extracted from the membranes, whereas Rac1, Rac2, or CDC42 were not detected in the extracts (data not shown). The presence of Rac1, Rac2, and CDC42 was also examined in unextracted HL60 membranes, and these were also found to be absent (data not shown). In contrast, HL60 cytosol showed the presence of CDC42 and RhoA, but not Rac1 immunoreactivity ( Fig. 2and data not shown).


Figure 7: Inhibition of GTPS-stimulated PLD activity in membranes by Rho GST-GDI. Panel A, HL60 cells were prelabeled with [H]myristic acid as described previously (21). The labeled membranes (20 µg of protein) and cytosol (10 µg of protein) were preincubated with indicated concentrations of Rho GST-GDI on ice for 15 min. Incubations were continued at 37 °C for 30 min in the presence of 30 µ M GTPS. [H]PEth formation in the presence of 1% ethanol was measured as described under ``Experimental Procedures.'' Panel B, HL60 membranes were preincubated with the indicated concentrations of Rho GST-GDI in the absence of cytosol and assayed for [H]PEth formation from exogenous [H]palmitoyl-PC under the same experimental conditions as in Fig. 1 A. Data for both experiments are representative of two experiments performed in duplicate.



The GDI-extracted membranes were tested for the restoration of GTPS-dependent PLD activity after reconstitution with recombinant RhoA, CDC42, and Rac1. All of these SMGs partially restored the PLD activity in the order of efficacy RhoA > CDC42 > Rac1 (Fig. 8). In the absence of GTPS, none of the SMGs was effective (data not shown). Interestingly, recombinant RhoA and Rac1 were also effective in a dose-dependent manner on unextracted membranes, whereas CDC42 was ineffective (Fig. 9). To further explore the role of RhoA, we also studied the translocation of Rho proteins from cytosol to membranes in the presence of GTPS. Although RhoA and CDC42 were detected in HL60 cytosol, only RhoA was translocated in a time-dependent manner in the presence of GTPS (Fig. 10). PLD activity in unextracted membranes was also tested with bovine sARF, and a stimulation was observed (Fig. 11), as expected (25) . The reconstitution of membranes with a mixture of increasing concentrations of recombinant RhoA and bovine sARF showed a synergistic effect on PLD activity, although this was not marked (Fig. 11).


Figure 9: Effect of recombinant Rho proteins on the PLD activity of unextracted membranes. HL60 membranes (30 µg) were preincubated with indicated concentrations of RhoA, CDC42, and Rac1 in the presence of GTPS (30 µ M) on ice for 15 min. The incubations were continued at 37 °C for 30 min in the presence of 1% ethanol. [H]PEth from [H]palmitoyl-PC was quantitated as described under ``Experimental Procedures.'' A representative experiment of two performed in duplicate is shown.




Figure 10: GTPS-dependent translocation of Rho A from cytosol to membranes. HL60 membranes (0.1 mg) and cytosol (0.05 mg) were incubated in PLD assay buffer, in the presence and absence of GTPS (50 µ M) at 37 °C for the indicated times. After incubation, membranes were centrifuged at 14,000 g for 10 min, and pellets were washed twice in buffer A. Pellets were resuspended in Laemmli SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting as described under ``Experimental Procedures.''




Figure 11: Effect of RhoA and ARF on membrane PLD stimulation. HL60 membranes (30 µg of protein) were preincubated with the indicated concentrations of recombinant RhoA or bovine sARF or a combination of the RhoA and ARF in the presence of GTPS (30 µ M) on ice for 15 min. The experiment was continued as described in Fig. 10. Data are representative of two experiments.




DISCUSSION

HL60 cells are one of the most extensively studied cell types with respect to the regulation of PLD activity. It is generally believed that the PLD activity in these cells is located in the particulate fraction and that its stimulation by guanine nucleotides is mediated by cytosolic factors. One of the cytosolic factors has recently been identified as ARF. Like previous findings, our studies with [H]myristic acid-labeled cell membranes also indicate the requirement of cytosol for GTPS-dependent PLD activity. However, when we assayed the enzyme with exogenous substrate according to Brown et al. (25) , surprisingly, we found GTPS-dependent activity in the soluble as well as the particulate fraction. Furthermore, we found that the PLD activity in the particulate fraction could be stimulated up to 5-fold with GTPS in the absence of cytosol. These two interesting observations provoked us to further investigate the regulation of PLD activity in both particulate and soluble fractions.

Our observations are consistent with the finding that the stimulation of the hydrolysis of exogenous PC by membrane PLD in the presence of GTPS plus ARF requires the addition of PIP(25, 37) . Also, in agreement with other findings (25, 26, 27) we detected significant stimulation of HL60 membrane PLD by GTPS plus cytosol or plus bovine sARF when [H]myristic acid-labeled membranes were used. However, when exogenous [H]PC was used as substrate in the presence of PIP, PLD stimulation by GTPS was evident in the membranes in the absence of cytosol or ARF. As will be discussed below, these results can be explained by the presence of two PLD isoforms, one responsive to ARF and another to Rho.

The use of exogenous substrate also made it possible to uncover the presence of PLD in the cytosol. Chromatography of this fraction separated the PLD activity from SMGs. Although several SMGs were found, the initial co-elution of PLD activity with ARF immunoreactivity supported the involvement of ARF in the regulation of cytosolic PLD activity. Further evidence was obtained when the PLD activity peak from DEAE was separated from the SMGs by two different chromatographic procedures. In fractions in which PLD was separated from ARF, activity was restored by either endogenous ARF (separated on the column) or exogenous ARF (bovine sARF). However, RhoA and CDC42 were ineffective. It is interesting to note that GTPS-dependent PLD activity could also be detected in bovine brain or rat liver cytosol.() This was barely measurable in crude cytosol but came enriched after DEAE chromatography.

Both membrane and cytosolic PLDs were GTPS-dependent and showed very similar dependence on the nucleotide. However, the basal activity of the membrane PLD was significant, whereas that of the cytosolic PLD was barely detectable. Furthermore, Caions had very different effects on cytosolic and membrane PLD. These data raise the possibility of different PLD isozymes in the two subcellular locations, although this remains speculative until the cytosolic and membrane enzymes are purified and characterized.

The cytosolic PLD was stimulated by ARF but not RhoA or CDC42 (which are all present in the cytosol), whereas the membrane PLD could be stimulated by members of the Rho family (RhoA, Rac1, and CDC42) and also ARF (Figs. 9 and 11). Previously, Bowman et al. (27) showed that GTPS stimulation of the membrane PLD could be inhibited by Rho-specific GDI (27) . We observed similar results and also found that the inhibition by Rho-GDI was more marked in the absence of cytosol (Fig. 7 B). Furthermore, we observed that in membranes extracted with Rho-specific GDI, it was possible to partially restore the GTPS stimulation by reconstitution with recombinant RhoA, CDC42, and Rac1. Bovine sARF also restored PLD activity in the extracted membranes, probably because of the presence of both Rho-specific and ARF-specific PLD isozymes. Surprisingly, the stimulation by Rho family G proteins did not show much specificity, since all tested Rho recombinant proteins (RhoA, CDC42, Rac1) could restore PLD activity in GDI-extracted or untreated membranes, although to varying extents.() However, Western blotting revealed that, of these proteins, only RhoA was detectable in the membranes, implying that this was the Rho family member that exerted endogenous control on the enzyme.

To further emphasize the role of RhoA, we also studied the translocation of Rho proteins from cytosol to the membranes in the absence and presence of GTPS (Fig. 10). Among RhoA, RhoB, and CDC42, only RhoA was translocated in the presence of nucleotide in a time-dependent manner. Again, Rac1 could not be detected in either cytosol or membranes. These findings support the involvement of RhoA in the regulation of membrane PLD.

Although, RhoA and ARF synergistically stimulate membrane PLD, stimulation by RhoA does not require the presence of ARF.() Contrary to membrane PLD, cytosolic PLD responds to ARF, but not to Rho, i.e. it must be different from one of the membrane isozymes. The existence of different PLD isozymes makes it possible to explain the different results obtained with membranes using exogenous versus endogenous substrate. For example, although an ARF-responsive isozyme is present in the membranes (25, 26) , it cannot be responsible for the effects of GTPS added alone since ARF is lacking in the membranes. On the other hand, a RhoA-responsive enzyme and RhoA are both present in the membranes ( cf. Fig. 1 A and Fig. 10), and this isozyme could therefore mediate the effects of GTPS. If this isozyme were able to utilize exogenous PC (under the specific assay conditions) efficiently, this could explain the results. The existence of other isozymes in the membranes that utilize PC less efficiently could explain why a GTPS effect is not detectable in the membranes in the absence of cytosol or ARF when the endogenous substrate is labeled.

In summary, our observations support the results of earlier investigations into the control of PLD by ARF and Rho (25, 26, 27) and provide another example of the specific role of RhoA (30) . They strongly suggest the existence of PLD isozymes that differ in cellular location and regulation. The Rho-regulated isozyme appears to be present only in membranes, while the ARF-responsive enzyme is found in both cytosol and membranes. However, since ARF is absent from the isolated membranes, it would have to be translocated there in order to regulate the enzyme, and this may be true for Rho also. Clearly, elucidation of the mechanisms of regulation of PLD by both SMGs will require much further work.


FOOTNOTES

*
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 all correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381.

The abbreviations used are: PLD, phospholipase D; G protein, GTP-binding protein; SMG, small GTP-binding protein; GTPS, guanosine 5`- 0-(3-thiotriphosphate); PC, phosphatidylcholine; PE, phosphatidyl ethanolamine; PIP, phosphatidylinositol 4,5-bisphosphate; PEth, phosphatidylethanol; HAP, hydroxylapatite; ARF, ADP ribosylation factor; sARF, soluble ARF; Rho-GDI, Rho-specific GDP dissociation inhibitor; GST, glutathione S-transferase; PA, phosphatidic acid.

A. R. Siddiqi, J. L. Smith, A. H. Ross, R.-G. Qiu, M. Symons, and J. H. Exton, unpublished results.

Comparison of Figs. 9 and 10 shows that RhoA and Rac1 were unable to completely restore GTPS stimulation of PLD in the GDI-extracted membranes but could significantly enhance the stimulation when added to untreated membranes. This indicates that the GDI treatment procedure produced additional changes that diminished the ability of PLD to respond to these SMGs. Comparison of the figures also shows that the relative potency of RhoA, Rac1, and CDC42 was changed. We do not know the reason for these differences, but they could be due to the extraction of GDP dissociation stimulator(s) and/or other proteins that control the activity of these SMGs.

The weak synergistic interaction between RhoA and ARF could be explained by the presence of two types of PLD in the membranes, one of which responds to Rho or ARF only and another that responds to both ARF and Rho. However, since it has not been shown that PLD is the immediate target of these SMGs rather than another regulatory protein, other explanations are possible.


ACKNOWLEDGEMENTS

We thank Y. Zheng and R. Cerione (Cornell University) for kind gifts of the GST-Rho-GDI plasmid and the antiserum against CDC42Hs, and S.-C. Tsai and J. Moss (National Institutes of Health) for generous gifts of sARF and sARF antibodies. We also thank J. Childs for typing this manuscript.


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