©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Low Molecular Weight GTP-binding Proteins in HL-60 Granulocytes
ASSESSMENT OF THE ROLE OF ARF AND OF A 50-kDa CYTOSOLIC PROTEIN IN PHOSPHOLIPASE D ACTIVATION (*)

(Received for publication, July 21, 1994; and in revised form, December 19, 1994)

Sylvain Bourgoin (1)(§) Danielle Harbour (1) Yvan Desmarais (1) Yoshimi Takai (2) André Beaulieu (1)

From the  (1)Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Sainte-Foy, Québec G1V 4G2, Canada and the (2)Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTPS (guanosine 5`-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of ^3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTPS-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTPS-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.


INTRODUCTION

Phosphatidylcholine hydrolysis by a type D phospholipase (PLD) (^1)generates two proximal second messengers, phosphatidic acid and diacylglycerol, which in turn control the functional responses of various cell types(1) . Phosphatidic acid may have a messenger role in regulating stimulus-secretion coupling and in activating the respiratory burst in granulocytes(2, 3, 4, 5, 6) . So far, PLD activation has been demonstrated in neutrophils and HL-60 cells upon stimulation with N-formylated peptides, C5a, platelet-activating factor, and leukotriene B(4)(7, 8, 9, 10) . The recent cloning of chemotactic and chemokine receptors revealed that these proteins belong to the family of seven transmembrane G protein-coupled receptors(11) . These receptors appear to be coupled to a pertussis toxin-sensitive G protein in granulocytes. ADP-ribosylation of the G(i) class of G protein by pertussis toxin abrogates a variety of biochemical responses, including PLD activity in fMLP-stimulated neutrophils(12, 13) .

Additional support for the existence of GTP-binding proteins in the regulation of PLD activity comes from the observation that, in permeabilized cells as well as in cell-free systems, PLD can be stimulated by GTPS(14, 15, 16) . In streptolysin O-permeabilized cells, GTPS stimulates PLD independently of phospholipase C(14) . Activation of PLD involves the interactions of several neutrophil components, some located in the plasma membrane and other(s) in the cytosol(17) . A PLD-associated stimulatory GTP-binding protein has been reported to reside in the plasma membrane(18) . More recently, several laboratories reported the activation of PLD by a small GTP-binding protein isolated from brain cytosols(19, 20, 21) . Evidence for the involvement of a small GTP-binding protein regulating PLD activity includes the following observations: (i) in permeabilized cells or cell-free systems, PLD was not stimulated by fluoroaluminate, an activator of heterotrimeric but not of small, G proteins; (ii) Bowman et al.(18) reported that PLD activity is stimulated by Smg GDS, a GDP/GTP dissociation stimulator, and inhibited by Rho GDI, a GDP/GTP dissociation inhibitor; (iii) ARF proteins can reconstitute the GTPS-dependent stimulation when combined with an enriched preparation of PLD (20) or with permeabilized HL-60 cells previously depleted of their cytosolic content(21) .

The GDP/GTP exchange proteins Rho GDI and Smg GDS are not totally specific and appear to be capable of regulating multiple small GTP-binding proteins from different families. Smg GDS has been found to be active on p21, p21/Rap1/Krev1, p21, and p21. Rho GDI regulates members of the Rho family of GTP-binding proteins, including p21, p21, and CDC42Hs(22, 23) . In light of the foregoing observations, we investigated the possibility that several small GTPases might control PLD activity in Me(2)SO-differentiated HL-60. This report provides evidence that cytosolic ARF, but not cytosolic Rho-related proteins, regulates PLD in HL-60 granulocytes. The results also indicate that activation of PLD by GTPS is dependent on the presence of a 50-kDa cytosolic factor.


EXPERIMENTAL PROCEDURES

Materials

HL-60 cells were purchased from the American Type Culture Collection (Rockville, MD). Fetal bovine serum, L-glutamine, and penicillin/streptomycin were from Life Technologies, Inc. L-alpha-Phosphatidylcholine, phospholipase D (cabbage type I), EGTA, Coomassie Blue, Pipes, Hepes, human albumin, dimethyl sulfoxide, GTP (Li salt), GTPS (Li salt), and bicarbonate-free medium RPMI 1640 were obtained from Sigma. Brefeldin A was from Cedarlane (Hornby, Ontario). The monoclonal antibody 1D9 against ARF was a generous gift from Dr. R. Kahn (National Cancer Institute, Bethesda, MD). RhoA, Rac1, and Rac2 monoclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Silica gel 60 thin-layer chromatography plates and solvents were purchased from BDH Inc. (Montréal, Québec, Canada), and cDNA for pGEX-2T expression of Rho GDI was a generous gift from Dr. A. Hall (University College, London). Rho GDI and Smg GDS were purified from bacteria as described previously(24) .

Solutions

Bicarbonate-free medium RPMI 1640 was buffered to pH 7.2 with 25 mM Na-Hepes. Gel filtration Pipes buffer contained (in mM): 20 Pipes, 137 NaCl, 2.7 KCl, and 1 MgCl(2), pH 6.8. KCl-Hepes relaxation buffer contained (in mM): 100 KCl, 5 NaCl, 3.5 MgCl(2), 0.5 EGTA, 50 K-Hepes, 0.25 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, and 2.5 µg/ml leupeptin (pH 7.2). Phosphatidylethanol (PEt) was prepared from bovine heart lecithin by transphosphatidylation with cabbage PLD as described(25) .

HL-60 Cell Culture

HL-60 cells were grown in RPMI 1640 medium supplemented with 2.0 g/liter sodium bicarbonate, 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), streptomycin (100 units/ml), and penicillin (100 µg/ml). The cells were passaged at starting densities of 2.5-3.5 times 10^5 cells/ml and maintained in culture at 37 °C in an air atmosphere containing 5% CO(2). Cell cultures were diluted every 3 or 4 days so that cell density did not exceed 1-2 times 10^6 cells/ml. To induce granulocytic differentiation, the cells were inoculated at 3.5 times 10^5 cells/ml of medium and treated with 1.25% (v/v) dimethyl sulfoxide for 6-7 days. Where indicated, intact cells were harvested by centrifugation and resuspended at a density of 10^7 cells/ml in bicarbonate-free, Hepes-buffered RPMI 1640 to be used for experiments.

Labeling of HL-60 Granulocytes

For the the incorporation of radioactivity into alkyl-phosphatidylcholine, cells were sedimented and resuspended at 8 times 10^6 cells/ml in Hepes-buffered RPMI containing 0.5 mg/ml fatty acid-free human serum albumin (HSA) and 1.6 µCi/ml 1-O-[^3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (132-179 Ci/mmol, Amersham Corp.). After 2 h at 37 °C, cells were washed twice in Hepes-buffered RPMI containing 0.5 mg/ml HSA and resuspended in the same buffer (without HSA) to be used for experiments.

Preparation of HL-60 Postnuclear Fractions

The cells (10^7/ml) were pretreated with 1.1 mM diisopropyl fluorophosphate for 30 min at room temperature to minimize proteolysis following sonication of cells in KCl-Hepes relaxation buffer. Diisopropyl fluorophosphate-treated cells were sedimented and resuspended in ice-cold KCl-Hepes medium at 1.6 times 10^7 cells/ml. Cell suspensions were then sonicated (two 20-s bursts) and centrifuged at 700 times g for 7 min. Nuclei and unbroken cells were discarded, and postnuclear homogenates were assayed for PLD activity.

Measurement of PEt Biosynthesis

For PLD activation in postnuclear fractions, 500-µl aliquots of the 700 times g supernatant (8 times 10^6 cell eq) were transferred in Eppendorf tubes containing sufficient MgCl(2) to give a final concentration of 8 mM and sufficient CaCl(2) to give a final free Ca concentration of 1 µM, calculated as described(26) . Where indicated, the mixture was incubated for 30 min on ice in the presence of the indicated recombinant proteins. Samples were then transferred at 37 °C and immediately incubated for 20 min with the indicated concentrations of guanine nucleotides in the presence of 1.4% ethanol. To terminate the reactions, samples were mixed with 1.8 ml of ice-cold chloroform/methanol/HCl (10 N) (1:2:0.02, by volume), and 3 µg of standard PEt was added. Lipids were extracted essentially according to Bligh and Dyer (27) by adding 0.6 ml of chloroform, mixing vigorously, and collecting the lower, lipid-containing chloroform phase.

The lipid samples were dried and spotted on Silica gel 60 plates. Plates were developed using a solvent system consisting of chloroform/methanol/acetic acid (65:15:2, by volume) for separation of PEt. Lipids were located by staining with Coomassie Blue(28) , and areas of the silica plate containing appropriate lipids were scraped off and quantitated by liquid scintillation counting. The results were corrected for quenching and recovery. Unless otherwise specified, data are presented in the text as the mean ± S.E. of a minimum of three separate experiments.

Fractionation and Gel Permeation Chromatography of HL-60 Cytosol

The postnuclear fraction was centrifuged at 180,000 times g at 4 °C for 45 min using a Beckman TL-100 ultracentrifuge. The pellet and the supernatant obtained were referred to as the membrane and the cytosolic fraction, respectively. The pellet was resuspended in ice-cold KCl-Hepes medium and kept on ice thereafter until used. The cytosol was fractionated by adding solid ammonium sulfate to yield a final saturation of 35%. The solution was stirred gently at 4 °C for 30 min and centrifuged at 5000 times g for 20 min. Solid ammonium sulfate was added to the 35% ammonium sulfate supernatant to give a final saturation of 75%. After gentle stirring at 4 °C for 30 min, the solution was centrifuged at 5000 times g for 20 min. These 0-35% and 35-75% ammonium sulfate precipitates were resuspended in 20 mM Pipes buffer containing 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl(2), pH 6.8. PLD activity was then assessed by incubating ^3H-labeled membranes (8 times 10^6 cell eq) and protein fractions (200 µg) in 500 µl of incubation buffer as described above. A solubilized preparation of PLD was obtained essentially according to Brown et al.(21) , by adding 400 mM NaCl to membranes, mixing, and collecting the 180,000 times g supernatant. PLD activity was assessed in the presence of lipid vesicles made of a phospholipid extract from ^3H-labeled HL-60 cells.

The reconstituting PLD activity was recovered in the proteins precipitated from 35 to 75% ammonium sulfate saturation. Then 350 µl (3-5 mg of protein) of the 35-75% ammonium sulfate fraction was loaded onto a TSK 2000 column (7.5 mm, inner diameter, times 60 cm, Beckman) equilibrated in Pipes buffer. Proteins were eluted at a flow rate of 0.5 ml/min. The molecular size of the eluted proteins was determined by reference to standard proteins of known molecular weight and blue dextran. Fractions of 0.5 ml were collected, and the PLD-reconstituting activity of each fraction (50-100 µl) was determined in the presence of ^3H-labeled membranes as described above.

Protein Quantitation, Electrophoresis, and Immunoblotting

Protein concentrations were determined using the Pierce protein assay kit with BSA as a standard. Electrophoresis was performed with 12% SDS-Tris-glycine-polyacrylamide gels (SDS-PAGE) according to Laemmli(29) . Staining was performed with the Bio-Rad silver staining kit. Electrophoretic transfer cells (Hoeffer Scientific Instruments, Canberra Packard Canada, Mississauga, Ontario) were used to transfer proteins on Immobilon PVDF membrane (Millipore Corp., Bedford, MA). Nonspecific sites were blocked using 2% gelatin for 1 h at 37 °C. The monoclonal antibody specific for p21 amino acids 60-77 (Y13-259) was from Oncogene Science Inc. (Manhasset, NY). Rabbit anti-human Rap1 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). ARF was detected using the monoclonal antibody 1D9 (1.1 µg/ml) and peroxidase-conjugated anti-mouse IgG (1:15000). The monoclonal antibody (mAb) 124-24E5 was purchased from Quality Biotech Inc. (Camden, NJ). Membranes were also incubated with antisera raised in rabbit against recombinant Rho GDI and Smg GDS (whole serum diluted 1:2000 and 1:1000, respectively) and exposed to peroxidase conjugated anti-rabbit IgG (1:10,000) for 1 h at 37 °C in the presence of 2% gelatin. Western blots using Rac1, Rac2, and RhoA antibodies (1:500) were also performed. Then membranes were washed three times in Tris-buffered saline-Tween solution (25 mM Tris-HCl, 190 mM NaCl, 0.15% Tween 20, pH 8) and covered with ECL detection reagents (Amersham) for 1 min at room temperature. Autoradiographs were obtained by exposing Kodak X-Omat film to membranes for 1-10 min at room temperature. Signals were quantitated by imaging with a BioImage-Visage 110S and integration of images using the Whole Band Analysis software (Millipore, Ann Arbor, MI).

Statistical Analysis

Results are mean ± S.E. of at least three experiments. Statistical analysis was performed by Student's paired t-test (two-tailed), and significance was considered p < 0.05.


RESULTS

Effects of Nucleotide Exchange Factors on GTPS-stimulated PLD Activity

The relationship between small GTP-binding proteins and the activation of PLD was investigated by examining the effects of nucleotide exchange factors on the activation of PLD from HL-60 cell lysates induced by GTPS. As reported earlier for neutrophils(18) , we found that Rho GDI decreases the accumulation of PEt elicited by GTPS in HL-60 postnuclear fractions (Fig. 1). Inhibition of PLD activity was detectable at concentrations of Rho GDI as low as 0.6 µM and increased with the concentration of Rho GDI. When expressed as a percentage of the stimulated level, inhibition of PEt accumulation averaged 23.6 ± 1.8%, 43.8 ± 1.4%, 74.9 ± 3.2%, 83.2 ± 3.3% (n = 3) at 0.6, 1.2, 3.1, and 6.3 µM Rho GDI, respectively.


Figure 1: Inhibition of GTPS-stimulated PLD activity by Rho GDI. HL-60 cell postnuclear fractions (500 µl) in incubation buffer containing 8 mM MgCl(2) and 1 µM CaCl(2) were incubated in the absence or presence of 0.6 µM, 1.2 µM, 3.0 µM, and 6 µMrho GDI for 30 min at 4 °C. Samples were then incubated with (opencircles) or without (filledcircles) 20 µM GTPS in the presence of 1.4% ethanol for 20 min at 37 °C. Reactions were stopped and samples processed for [^3H]alkyl-PEt measurement as described under ``Experimental Procedures.'' Data are the mean ± S.E. of three independent experiments. Where absent, error bars are smaller than the symbol.



PLD was marginally stimulated by physiological concentrations of GTP (Fig. 2). Smg GDS, which stimulates GDP/GTP exchange on multiple small GTPases, would be expected to enhance GTP-stimulated PLD activation if nucleotide exchange is rate-limiting. Fig. 2shows that the addition of Smg GDS to HL-60 postnuclear homogenates failed to enhance significantly the levels of PEt accumulation elicited by GTP. It is noteworthy that GTPS-stimulated PLD activity was similarly unaffected by the stimulatory exchange factor (not illustrated), indicating that nucleotide exchange was not limiting under these conditions. The effect of maximally stimulatory concentrations of GTPS was considerably greater than that elicited by 200 µM GTP (Fig. 3). As expected, the stimulatory effects of maximal doses of GTPS (10 µM) was reversed by GTP, which, unlike GTPS, is not resistant to hydrolysis by GTP-binding proteins. At 200 µM, GTP was found to reduce the rate of PEt synthesis elicited by GTPS by 87.5 ± 2.6% (Fig. 3). Because HL-60 cell membranes have been reported to possess high affinity GTPase activities, it was conceivable that introduction of GTP to postnuclear homogenates resulted in the rapid hydrolysis of GTP to GDP. GDP may then be responsible for the observed inhibitory effect. If GTP hydrolysis to GDP by GTPases precedes and is causally related to inhibition of PLD, the response to GTPS should be less (or at best equally) sensitive to inhibition by GTP than by GDP. A detailed concentration dependence of the effects of GTP and GDP is illustrated in Fig. 4. The response is expressed as a percentage of the maximal response obtained with 10 µM GTPS for comparison. PEt accumulation in response to GTPS became progressively smaller as the concentration of GDP or GTP was increased. GTP was more efficient than GDP at inhibiting GTPS-stimulated PLD activity, with half-maximal effects obtained at 30 and 60 µM, respectively. The difference between the effects of the two guanine nucleotides was maximal at 30 and 100 µM. Although statistically insignificant, a small accumulation of PEt was, however, observed in response to 200 µM GTP (Fig. 2). Furthermore, the order of potency GTP > GDP implies that rapid hydrolysis of GTP to GDP by miscellaneous GTPases is unlikely to be the dominant process in the inhibition of the response to GTPS, suggesting that the interaction of GTP and GTPS is competitive.


Figure 2: Effects of Smg GDS on GTP-stimulated PLD activity. PLD was assayed in incubation buffer containing 8 mM MgCl(2), 1 µM CaCl(2), and 1.4% ethanol. Reactions were initiated by the addition of 200 µM GTP (hatched bars) or vehicle alone (filled bars) with the indicated concentrations of Smg GDS. Following incubation at 37 °C for 20 min, reactions were stopped, and [^3H]alkyl-PEt was quantitated as described under ``Experimental Procedures.'' Data are the mean ± S.E. of three independent experiments.




Figure 3: Inhibition of GTPS-stimulated PLD activity by GTP. Aliquots (500 µl) of HL-60 postnuclear homogenates were incubated for 20 min at 37 °C with GTPS (10 µM) or GTP (200 µM) alone or in combination. PLD activity was quantitated as described under ``Experimental Procedures'' and [^3H]alkyl-PEt formation expressed as a percentage of total lipid-associated radioactivity. Data are the mean ± S.E. of three independent experiments.




Figure 4: Concentration dependence of the effects of GTP and GDP on GTPS-stimulated PLD activity. HL-60 cell postnuclear fractions (500 µl) were stimulated with 10 µM GTPS in the absence or the presence of the indicated concentrations of GTP or GDP. Following incubation at 37 °C for 20 min in the presence of 1.4% ethanol, samples were assayed for [^3H]alkyl-PEt formation as described under ``Experimental Procedures.'' Data are the mean ± S.E. of six independent experiments. *, p < 0.05, for values compared to the adequate controls using a Student's paired t-test.



Fractionation of HL-60 Cytosol

Partial purification of HL-60 cytosol was performed by ammonium sulfate precipitation and chromatography through gel filtration column. As previously reported in neutrophils(18) , PLD activation by GTPS required protein factors in both the plasma membrane and the cytosol. The cytosolic reconstituting activity was recovered in the proteins precipitated from 35 to 75% ammonium sulfate saturation (Fig. 5). The 35-75% ammonium sulfate precipitate was next chromatographed by gel filtration using a TSK-2000 SW column, and eluted protein fractions were combined with ^3H-labeled membranes. Then PLD activity was assessed in the presence of 20 µM GTPS and 1.4% ethanol. As shown in Fig. 6, a peak of PLD-stimulating activity was recovered in column fractions 29-34 with an apparent molecular mass consistent with proteins of approximately 50 kDa. A second, smaller, peak of PLD reconstitution was observed in fractions 38-40. This second peak of activity eluted with an apparent molecular mass of 18 kDa. Silver-stained polyacrylamide (12%) gel electrophoresis also showed the presence of an 18-kDa protein in column fractions 38-40 (not illustrated).


Figure 5: Restoration of GTPS-stimulated PLD activity by cytosolic proteins precipitated from 35 to 75% ammonium sulfate saturation. Membranes, cytosols, 0-35% and 35-75% ammonium sulfate fractions were prepared as described under ``Experimental Procedures.'' Freshly isolated [^3H]alkyl-phosphatidylcholine-labeled membranes (8 times 10^6 cell eq) alone or in combination with cytosol (200 µg); 0-35% ammonium sulfate fraction (200 µg) and 35-75% ammonium sulfate fraction (200 µg) in 0.5 ml of incubation buffer containing 8 mM MgCl(2), 1 µM CaCl(2), and 1.4% ethanol were stimulated with 20 µM GTPS. After 20 min at 37 °C, the reactions were stopped, and [^3H]alkyl-PEt formation was quantitated as described under ``Experimental Procedures.'' Data are the mean ± S.E. of six independent experiments.




Figure 6: Gel-permeation chromatography of 35-75% ammonium sulfate protein fraction. Proteins precipitated from 35-75% ammonium sulfate saturation were loaded on a TSK 2000 SW column. Aliquots (50-100 µl) of eluted fractions were combined with ^3H-labeled membranes, and [^3H]alkyl-PEt formation was monitored as described in Fig. 6. Results are representative of data obtained in four separate experiments under identical conditions.



Characterization of Cytosolic PLD Cofactors Which Reconstitute PLD Activity

A 16-kDa protein derived from brain cytosols has been reported to reconstitute the GTPS-stimulated PLD activity in granulocytes when added to permeabilized HL-60 cells previously depleted of their cytosolic content(19) . This cytosolic protein was subsequently identified as a member of the ADP-ribosylation factor (ARF) subfamily of Ras-related small G proteins(20, 21) . We considered the possibility that the reconstituting factor in the 18-kDa and 50-kDa fractions derived from granulocyte cytosols is in fact ARF and/or ARF complexed to an ancillary protein. Therefore, the distribution of proteins recognized by mAb 1D9, which was previously been reported to detect human ARF proteins(30) , was examined by immunoblotting in eluted column fractions. As shown in Fig. 7, staining with antibody 1D9 revealed intense bands at 18 kDa in column fractions 38-40 but not in column fractions 29-34, which contained most of the PLD reconstituting activity ( Fig. 7and Fig. 8). In addition, low but detectable amounts of ARF proteins appeared to be associated with our plasma membrane fraction (not shown). Because 1D9 clearly recognized proteins in the 16-18-kDa region, it is unlikely that cytosolic ARF proteins are a part of a larger 50-kDa macromolecular complex.


Figure 7: Immunoblot analysis of nucleotide exchange factor and small GTP-binding proteins. Aliquots (80 µl) of column fractions were processed for SDS-PAGE/Western blotting and transferred to Immobilon PVDF membranes as described under ``Experimental Procedures.'' Membranes were exposed to anti-Smg GDS, anti-Rho GDI, anti-ARF (1D9), anti-H/N-Ras (142-24E5), and anti-Rap1 antibodies for immunoblot analysis. Data presented are representative of results obtained in four separate experiments with similar results.




Figure 8: Identification of nucleotide exchange factors and ARF proteins in eluted column fractions: comparison to PLD activity. Data presented in Fig. 7were plotted to enable comparison between the presence of Smg GDS, Rho GDI, ARF, and PLD activity. The amounts of Smg GDS, Rho GDI, and ARF in column fractions were determined by Western blot analysis in experiments like those in Fig. 7and scanning densitometry as described under ``Experimental Procedures.'' Data were normalized to the fraction with the highest integrated optical density value. One of four similar experiments is shown.



To characterize further the component(s) in the 50-kDa fractions derived from granulocyte cytosols, the distribution of GTP-binding proteins, Rho GDI, and Smg GDS were examined in the eluted column fractions by immunoblot analysis. The localization of Rho GDI was assessed using a GDI-specific antibody. The same strips were also probed with mAb 142-24E5, which recognizes a neutrophil Ras-related protein, Rap1, and a subtrate for botulinum toxin D (31, 32) . As shown in Fig. 7, column fractions 31-34 contain a 27-kDa band recognized by the antibody against Rho GDI on Western blots. Monoclonal antibody 142-42E5 revealed an intense band at about 24 kDa in fraction 32. When films were overexposed, small amounts of antigenic material were also detected in fractions 31 and 33, respectively. This 24-kDa protein was not recognized by a monoclonal antibody against p21, Y13-259 (not illustrated), or a rabbit antiserum raised against human Rap1 (Fig. 7). The first peak of PLD reconstitution (fractions 29-34) was found to overlap the fractions containing Rho GDI and the antigenic material recognized by mAb 142-24E5.

Since a correlation between the stimulation of PLD and the activation of a Rho-related small GTP-binding protein has been drawn(18) , the presence of the these small GTPases was investigated by immunoblotting. As illustrated in Fig. 9, Rac2 copurified in parallel with RhoA and Rho GDI with an apparent molecular mass of 45-43 kDa. As estimated by Western blotting, Rac2 and RhoA were entirely complexed to Rho GDI. In contrast, Rac1 eluted by gel filtration between Rac2/RhoA complexes and ARF proteins in the 24-kDa region. Additionally, Rac1 was predominantly recovered in a region devoid of PLD reconstitution activity ( Fig. 7and Fig. 8). Although the presence of RhoA and Rac2 was clearly detected in the first peak of reconstituting activity, PLD activation is unlikely to be due to RhoA/Rac2 GDI, inasmuch as these complexes were not detectable in the reconstitutively active fractions 29 and 30 ( Fig. 7and Fig. 8).


Figure 9: Distribution of Rho-related small GTP-binding proteins. Aliquots (80 µl) of column fractions were processed for SDS-PAGE/Western blotting and transferred to Immobilon PVDF membranes as described under ``Experimental Procedures.'' Membranes were exposed to anti-Rho GDI, anti-ARF (1D9), anti-RhoA, anti-Rac2, and anti-Rac1 antibodies for immunoblot analysis. These results are from a single experiment representative of three others performed with identical results. Data presented in Fig. 7and Fig. 9are from two independent experiments.



The presence of Smg GDS in the reconstitutively active PLD fraction was examined using a Smg GDS-specific antibody. As shown in Fig. 7, the resolved Smg GDS was essentially found in column fractions 28-30 but was not recovered in column fractions 32-34, which contained most of the PLD-stimulating activity. Conversely, fractions 28 and 29 contained substantial amounts of Smg GDS but no or little PLD-stimulating activity ( Fig. 7and Fig. 8), excluding Smg GDS as the reconstituting factor for GTPS-stimulated PLD activity.

Effects of PLD-inducing Factors on the Salt-extracted PLD Activity

Membrane-associated PLD was extracted with 400 mM NaCl according to the procedure described by Brown et al.(21) . The PLD assay utilizes phospholipid vesicles composed of ^3H-labeled phospholipids as substrate. Fig. 10shows the dependence of the PLD activity from HL-60 cells on the presence of cytosolic cofactors. Little or no activity was detected in the presence of PLD alone and GTPS. The addition of the 50-kDa peak of reconstitution activity increased PLD activity. Salt-extracted PLD was marginally stimulated by a pool of fractions containing ARF-related GTP-binding proteins. However, when added together, the effects of the 50-kDa and the 18-kDa peak of PLD reconstitution activity on PEt synthesis were synergistic. The weak stimulatory effects of ARF-alone suggest the presence of an ARF-regulatory protein in the 50-kDa peak of PLD reconstitution activity.


Figure 10: Effects of PLD-inducing factors on a solubilized PLD activity. PLD was extracted from HL-60 membranes as described under ``Experimental Procedures.'' The solubilized PLD activity (75 µg) was mixed in 0.5 ml of incubation buffer with phospholipid micelles composed of a lipid extract from [^3H]alkyl-phosphatidylcholine-labeled cells (200,000 cpm/assay). The samples were stimulated with 20 µM GTPS for 60 min at 37 °C in the presence of 1.4% ethanol. Where indicated aliquots (100 µl) of the 50-kDa or the 18-kDa peak of PLD-inducing activity were added alone or in combination. Data are the mean ± S.E. of four independent experiments. *, p < 0.05, for values compared to the adequate controls using a Student's paired t-test.




DISCUSSION

The present study demonstrates the presence in HL-60 cytosols of protein factors that will reconstitute GTPS-stimulated PLD activity in a reconstitution assay consisting of previously labeled HL-60 membranes. The major peak of PLD-reconstituting activity was recovered in fractions with an apparent molecular mass of 50 kDa. The 50-kDa cytosolic component is antigenically distinct from the 18-kDa ADP-ribosylation factor, a small GTP-dependent regulatory protein previously found to be an activator of PLD. The component of this fraction is able to support a strong accumulation of PEt and is suggested to be another cytosolic regulatory element of PLD activity or perhaps other proteins with which ARF interacts.

In recent years, efforts to elucidate the biochemical and molecular mechanisms of PLD activation have focused on Ras-related small GTPases with molecular masses between 18 and 30 kDa and on larger, heterotrimeric, G proteins that have been linked to receptor-mediated signal transduction. The most definitive evidence for a functional role of G proteins comes from studies in permeabilized HL-60 cells or cell-free systems prepared from human granulocytes. A GTPS-dependent PLD activity can be measured in postnuclear fractions obtained from HL-60 cells (33) or human neutrophils(18) . Cytosols and membranes isolated from HL-60 postnuclear fractions do not support PLD activation when assayed separately. However, a GTPS-dependent PLD activity is observed in combined fractions of membranes and cytosols of HL-60 cells. The observation that the cytosolic reconstituting activity precipitated between 35 and 75% saturation of ammonium sulfate further emphasizes the central and critical role of cytosolic cofactor(s) in PLD activation.

Cytosols from bovine and rat brains were found to provide an essential factor for the GTPS-dependent stimulation of an enriched preparation of PLD and of PLD activity in HL-60 cells depleted of their cytosol by permeabilization, respectively. This factor was purified to homogeneity and identified as a member of the ARF subfamily of small GTPases(20, 21) . It is noteworthy that the presence of the 50-kDa peak of reconstituting activity was not detected after fractionation of rat brain cytosol by amonium sulfate precipitation, followed by heparin-agarose and gel filtration chromatography with Pipes buffer. However, using conditions comparable to those reported by Geny et al.(19) , we observed that the marked instability of the 50-kDa cytosolic protein(s), especially in Pipes buffer, was an obstacle to further purification. Moreover, the absence of this PLD reconstitution activity in brain tissues cannot be totally excluded. Using an autologous reconstitution assay, Bowman et al.(18) reported the presence in neutrophil cytosols of a protein factor essential for the GTPS-stimulated PLD activity. However, in contrast to other studies(20, 21) , this group reported the participation of a membrane-associated low molecular weight GTP-binding protein, presumably a member of the Rho subfamily of G proteins, in PLD activation.

The results obtained in this study demonstrate that the inhibitory GDP/GTP exchange factor Rho GDI prevents the activation of PLD by GTPS in HL-60 postnuclear fractions. Immunoblots probed with a rabbit anti-human Rho GDI demonstrate that GDI is detectable and is specifically localized in the 50-kDa peak of PLD reconstituting activity. The presence of Rho GDI in fractions that contained most of the PLD-reconstituting activity suggests that PLD activity may be underestimated in these fractions. GDI eluted by gel filtration with an apparent molecular mass of 45-30 kDa, although the calculated molecular mass of Rho GDI is 26.5 kDa. Copurification of Rho GDI with the Rho-related GTP-binding proteins has been reported in phagocytic granulocytes(34, 35) . Indeed, our experiments revealed the presence of Rac2 and RhoA in column fractions containing Rho GDI. Both GDP- and GTP-bound forms of small GTPases can form stable complexes with Rho GDI (24, 34) . The association of Rho-related GTP-binding proteins with GDI appears to account for the maintenance of these proteins in the cytosolic compartment, thus preventing interactions with their effector proteins(34, 36) . Rho GDI has been reported to inhibit several cell functional responses, including activation of the NADPH oxidase in neutrophils (37) and organization of polymerized actin in Swiss 3T3 cells(38, 39) .

Among the small GTP-binding proteins, p21 and p21 are both common substrates of Rho GDI and the stimulatory GDP/GTP exchange factor, Smg GDS(22, 23) . It is noteworthy that Smg GDS is not able to enhance significantly the GTP(S)-dependent PLD activity in HL-60 postnuclear fractions. The results are not consistent with the recent report concerning the ability of Smg GDS to stimulate the activity of PLD in neutrophils(18) . The reason for this discrepancy remains unclear, but a possible explanation would be that GTP hydrolysis rather than GDP/GTP exchange is the rate-limiting step in HL-60 cells. Consistent with this idea, the rate of PEt accumulation can be significantly stimulated by GTPS but not by GTP. Moreover, this study demonstrates a marked decrease in GTPS-stimulated PLD activity when cells are incubated in the presence of GTP. Such inhibition is expected, provided GTP and GTPS compete at the GTP-binding site of small GTPases for the formation of activated GTP(S)-bound GTP-binding proteins. Due to its nonhydrolyzable nature, the binding of GTPS to a GTP-binding protein would serve to maintain it in its activated state. Cytosolic RhoA/Rac2 GDI complexes are unable to account for the activation of PLD, inasmuch as neither GTP-binding proteins nor GDI could be detected in several reconstitutively active fractions. However, our study does not address the possible role of membrane-associated Rho-related proteins in PLD activation. RhoA, Rac1, and ARF were all present in HL-60 membranes (data not shown). It is therefore possible that membrane-associated Rho-related proteins and membrane-bound ARF synergize with cytosol derived-regulatory proteins. In a recent report by Malcolm et al.(40) , only RhoA was found to reconstitute a full PLD response in Rho GDI-washed liver membranes. Thus, extraction of Rho A from HL-60 membrane would explain the inhibition of the GTPS-stimulated PLD activity by Rho GDI.

Several investigators have presented evidence implicating ARF proteins in the activation of PLD(20, 21) . Taking into account the fact that this reconstitution factor has been purified from brain cytosols, it is highly possible that ARF proteins present in HL-60 cytosol were involved in the activation of a membrane-bound PLD. HL-60 cells express genes for ARF proteins(41) . ARF is present in HL-60 cytosol, and cytosolic ARF was found exclusively in the 18-kDa peak of PLD reconstitution. The weak stimulatory effect of cytosolic ARF itself does not preclude a role for ARF in the regulation of PLD activity, since we observed that preparation of HL-60 membranes contains antigenic material recognized by the monoclonal antibody 1D9.

Membrane-bound ARFs are likely to be the active (GTP-bound) proteins. Based on preincubation with guanine nucleotides, the plasma membrane has been shown to contain GTP-binding proteins that support PLD activation(18) . It seems likely that the strong dependence on the 50-kDa fraction reflects the presence of ARF regulatory proteins, presumably a nucleotide-exchange factor specific for a particular ARF protein. Accelerated nucleotide exchange would promote both GTPS binding to ARF and subsequent coupling to PLD. Several studies have documented the presence of an ARF-specific guanine nucleotide-exchange protein in Golgi membranes that is inhibited by brefeldin A(42, 43, 44) . Attempts to observe an inhibition of GTPS-stimulated PLD activity by concentrations of brefeldin A as high as 20 µg/ml proved unsuccessful. This result does not exclude the involvement of a nucleotide-exchange factor in the activation of PLD. The brefeldin A-sensitive component is not yet known. Indeed, a nucleotide-exchange protein for ARF has been documented in bovine brain and shown to become insensitive to inhibition by brefeldin A with purification(45) . Interaction of Rho GDI with ARF is unlikely inasmuch as GDI had no significant effect on either GTPS binding to or GDP dissociation from human ARF. (^2)Thus, it appears that the GTPS-activated PLD activity requires both a small GTP-binding protein, presumably membrane-bound ARF, and a cytosolic cofactor of approximately 50 kDa.

In conclusion, we have further characterized a 50-kDa factor present in HL-60 cytosol that is essential to PLD activation. This factor is antigenically distinct from the ARF proteins. The molecular mass of this protein and the discrete peak of PLD stimulation by ARF proteins alone strongly suggest that the 50-kDa cytosolic component is another regulator of PLD or an ARF-regulatory protein. Further purification and antibody production are required to define the nature and the function of this 50-kDa protein. We are currently involved in these studies.


FOOTNOTES

*
This work was supported in part by grants and fellowships from the National Cancer Institute of Canada and the Fonds de la Recherche en Santé du Québec (to S. B.). 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, 2705 Blvd. Laurier, Sainte-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2772; Fax: 418-656-2765.

(^1)
The abbreviations used are: PLD, phospholipase D; PEt, phosphatidylethanol; G protein, GTP-binding protein; GTPS, guanosine 5`-3-O-(thio)triphosphate; GDI, GDP/GTP dissociation inhibitor; GDS, GDP/GTP dissociation stimulator; ARF, ADP-ribosylation factor; Pipes, piperazine-N,N`-bis(2-ethanesulfonic acid); HSA, human serum albumin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

(^2)
P. A. Randazzo and R. A. Kahn, personal communication.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. R. A. Kahn for the gift of the monoclonal antibody 1D9 against human ARF proteins. We also thank Dr. P. A. Randazzo for sharing information on the nucleotide exchange activity of Rho GDI. We are also grateful to Dr. A. Hall for providing pGEX-2T plasmids containing [Val]Rac1, [Val^14]RhoA, and Rho GDI inserts.


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