G-protein-stimulated Phospholipase D Activity Is Inhibited by Lethal Toxin from Clostridium sordellii in HL-60 Cells*

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5′-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Cα was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.

Phospholipase D (PLD) 1 hydrolyzes phosphatidylcholine, the major membrane phospholipid, yielding choline and phosphatidic acid (PA). PA, a possible second messenger, is fusogenic, and its increase is associated with several important cellular modifications such as activation of protein kinases, phospholipase C-␥ and cytosolic phospholipase A 2 (cPLA 2 ), Ca 2ϩ influx, DNA synthesis, and c-fos and c-myc transcription. In cells, PA is rapidly converted by a PA phosphohydrolase into diacylglycerol, the natural activator of protein kinase C (PKC), and hence, PLD activity can lead to a long term activation of protein kinase C (see review Ref. 1).
In mammalian cells, PLD is stimulated via several membrane receptors. Its activation can occur by different intracellular pathways. Over the past few years, several components participating in the control of PLD activity have been deciphered. Small G-proteins, including ARF which is involved in membrane traffic and RhoA which participates in actin polymerization, were demonstrated to be potent activators of PLD. Other small G-proteins implicated in the regulation of actin polymerization, namely Rac and Cdc42, were also shown to be PLD activators but to a lesser extent (see review Ref. 2). PLD activation was also reported to occur through a Ras-Ral pathway (3)(4)(5)(6); activated RalA leads to membrane recrutement of ARF (3,4). Other PLD activators have been identified, such as conventional PKC isoforms (see review Ref. 2) and the actinbinding protein gelsolin (7). PLD is under the negative control of several proteins, one of which was demonstrated to be the cytoskeletal protein, fodrin, an actin-binding protein (8) and two others were identified as synaptojanin (9) and clathrin assembly protein 3 (10). Moreover, phosphoinositides are also involved in PLD regulation. In particular, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), the PtdIns-phospholipase C substrate, was shown to be a necessary cofactor for PLD activity (11). How all the components involved in the regulation of PLD activity interact in resting or activated cells is not yet well understood.
Bacterial protein toxins of large molecular weight isolated from Clostridium difficile (toxins A and B) have been shown to inactivate Rho proteins and, thus, to affect the actin cytoskeleton (12). Toxin B has Rho subfamily small G-proteins, including RhoA, Rac, and Cdc42, as targets. It inactivates these proteins by modifying Rho by a glucosylation on threonine 37 and Rac and Cdc42 on threonine 35 (12). This toxin was demonstrated to inhibit m3 muscarinic receptor stimulation of PLD in human embryonic kidney (HEK) cells (13) by decreasing the membrane level in PtdInsP 2 (14).
Clostridium sordellii, the organism responsible for gas gangrene, produces a large molecular weight toxin, lethal toxin (LT), which exhibits some similarities with toxin B (15). LT also inactivates small G-proteins by having a glucosyltransferase activity leading to the glucosylation of small G-proteins (Ras, Rac, RalA, and Rap). In vivo, its major target was reported to be Ras which is glucosylated on threonine 35 (16,17). It has also been reported that LT produced by some strains of C. sordellii is able to glucosylate the Ral protein on threonine 47 in vitro and in vivo (18). So far, LT has not been found to have an activity on other related small G-proteins from the same superfamily including Rho, Cdc42, Rab, and ARF (16).
As Ras, Ral, and Rac have been reported to be involved in PLD activation, we investigated the effect of LT on PLD activity in HL-60 cells. We observed that cell treatment with LT inhibits ARF-, RhoA-, RalA-, and Rac-stimulated PLD activities. We investigated the mechanism by which LT inhibits the enzyme in HL-60 cells and found that it modifies the levels of cellular polyphosphoinositide and of some phosphoinositide kinases. We discuss the responsibility of the small G-proteins, Rac, Ras, and RalA, inactivated by LT in the decrease of PLD activity in toxin-treated cells.  32 P]ATP (5000 Ci/mmol), and EN 3 HANCE spray were purchased from NEN Life Science Products. Streptolysin O was from Murex Diagnostic Ltd. GTP␥S, phosphatidylinositol 4,5bisphosphate (PtdIns(4,5)P 2 ), protease inhibitor mixture tablets (Complete TM ), and protease and fatty acid-free bovine serum albumin were purchased from Roche Molecular Biochemicals (Germany). Bio-Rex 70 cation-exchange resin was purchased from Bio-Rad. LY 294002 was obtained from Calbiochem. Cytochalasin D, wortmannin, FITC phalloidin, protein A-agarose beads and all other chemical products were from Sigma. Scintillant liquid, Optiphase "HiSafe" 3, was from EG & G. Silica gel 60 plates for TLC analysis were obtained from Whatman. Anti-Ha-Ras (259) and anti-Rac (T-17) were from Santa Cruz Biotechnology. Rabbit anti-rat IgG and anti-goat IgG were from Cappel. ARF antibodies were obtained after immunization of rabbits with recombinant ARF.
Recombinant ARF, RhoA, Rac1, and RalA were made in Escherichia coli through glutathione S-transferase fusions and were prepared by thrombin digestion to yield the 21-kDa proteins.
Glucosylation by Lethal Toxin of Small GTP-binding Proteins in HL-60 Cells-HL-60 cells grown in RPMI 1640 medium were treated by LT from C. sordellii as indicated. After treatment, cells (2 ϫ 10 7 ) were washed twice in PBS. After centrifugation, the cell pellets were resuspended in 0.5 ml of lysis buffer, pH 7.4, containing 0.5% Triton X-100, 20 mM Hepes, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EGTA, 0.1 mM orthovanadate, 10 mM GDP, and protease inhibitor mixture (Complete TM ) according to instructions. Cells were incubated on ice for 20 min, and complete lysis was achieved by three cycles of freeze-thawing. Cell lysates were centrifuged at 400 ϫ g to remove intact cells and nuclei, and the amount of protein in each supernatant was determined. For in vitro glucosylation of small GTP-binding proteins, 32 g of cell lysate or 2 g of purified small G-proteins was added to 3 l of dried UDP-[ 14 C]glucose with 5 mg/ml LT in glucosylation buffer, pH 7.5, made of 50 mM triethanolamine, 2 mM MgCl 2 , 1 mM DTT, and 0.3 mM GDP. The reaction was carried out for 1 h at 37°C and stopped by adding 10 l of sample buffer (3 times). This mixture was boiled and electrophoresed on a 15% SDS-PAGE. The gel was stained with Coomassie Blue, destained, dried, and autoradiographed.

Immunoprecipitation of Small G-proteins
In vitro glucosylation was performed on a cell lysate containing 320 g of proteins per assay in a final volume of 300 l. At the end of the reaction (1 h at 37°C), bovine serum albumin (1 mg/ml final) and antibody anti-ras (3 g) or anti-Rac (6 g) were added. After 1 h incubation at 4°C under constant gentle shaking, 30 l of rabbit antirat IgG and anti-goat IgG were added, followed by a 1-h incubation at 4°C. Protein A-agarose beads (25 l) previously equilibrated in washing buffer made of triethanolamine (5 mM), MgCl 2 (2 mM), NaCl (50 mM), DTT (1 mM), and bovine serum albumin (0.1%) were added. After incubation at 4°C overnight under constant shaking, beads were washed 6 times in washing buffer before addition of 8 l of sample buffer (3 times). Immunoprecipitates were analyzed by SDS-PAGE and autoradiographed as described above.

Fluorescence Experiments, Effect of LT on the Actin Cytoskeleton
After treatment with LT, cells were fixed with 3% paraformaldehyde for 15 min and then washed three times with PBS. Free aldehyde groups were quenched in NH 4 Cl (50 mM) in PBS containing CaCl 2 and MgCl 2 (1 M) for 10 min. Cells were permeabilized for 5 min with Triton X-100 0.3% in PBS for 5 min and then incubated with FITC-phalloidin for 30 min. After labeling, cells were washed three times in PBS, fixed on slides covered with polylysine, and mounted in Mowiol. All steps were carried out at room temperature. Fluorescent images were taken with a confocal laser scanning microscope (Bio-Rad MRC-1000) attached to a diaphot 300 microscope (Nikon). For FITC fluorescence detection, a krypton/argon laser at 488 excitation was used, and emission was collected with a 522-nm filter.

Determination of Phospholipase D Activity in Permeabilized Cells and in Intact Cells
In HL-60 cells, PLD hydrolyzes preferentially phosphatidylcholine, the major phospholipid, to give choline and phosphatidic acid, both can reflect PLD activity but can also result from other cellular activities. PLD possesses a specific transphosphatidylation activity (22); in the presence of a short chain alcohol, instead of PA, a stable phosphatidyl alcohol will be formed. Measurement of phosphatidyl alcohol (Ptdalcohol) is highly specific of PLD in comparison to choline measurement. We have previously checked that the formation of both products of PC hydrolysis, Ptd-alcohol and choline, both occurred in parallel and reflected PLD activity in streptolysin O-permeabilized HL-60 cells (23). Thus PLD activity was estimated by measuring choline formed in experiments made with permeabilized cells or PtdEt formed in the presence of 1.5% ethanol in intact cells.
Choline Measurement-Cells were labeled at isotopic equilibrium for 48 h in medium 199 containing 10% fetal bovine serum in the presence of 0.5 Ci/ml  (24). Radioactivity present in choline was quantified after addition of scintillant liquid using a Wallac 1410 liquid scintillation counter. Total radioactivity associated with cells was counted. PLD activity was estimated by measuring choline formed and expressed as the percentage of total radioactivity incorporated in cells or as indicated.
Cytosol-depleted cells were obtained after permeabilization for 10 min at 37°C in the presence of 0.4 units/ml streptolysin O as described in detail elsewhere (23).
PtdEt Measurement-HL-60 cells were washed in a buffer made of 137 mM NaCl, 2.7 mM KCl, 20 mM Hepes, 5.6 mM glucose, and 0.1% protease and fatty acid-free bovine serum albumin, pH 6.8, and incubated at 37°C for 30 min in the same buffer containing 2 mCi/ml [lsqb 3 H]lyso-PAF. After labeling, PLD measurement was carried out as described above except that 1.5% ethanol was added to the reaction mixture. After phase separation as above, the lower organic phase was dried under vacuum and resuspended in 25 l of chloroform and spotted onto oxalate-impregnated TLC silica plates, and phospholipids were separated by migration in a solvent made of chloroform/methanol/acetic acid/water (75:45:3:1). Radioactivity present in silica spots corresponding to PtdCho, PA, and PtdEt was counted in 4 ml of scintillation liquid.

Phosphoinositide Measurement
Cells were labeled at isotopic equilibrium for 48 h in medium 199 containing 10% of fetal bovine serum, previously dialyzed against PBS, in the presence of 2 Ci/ml [myo-2-3 H]inositol. After 24 h treatment without or with toxin LT, labeled cells were washed in PIPES buffer, and lipids were extracted by addition of chloroform/methanol/concentrated HCl (100:200:5). Extracts were vortexed, and phase separation was achieved by the procedure of Bligh and Dyer (25). The organic phase was then evaporated under vacuum, and labeled phospholipids were separated onto oxalate-impregnated TLC silica plates using a solvent of chloroform/methanol/acetone/acetic acid/H 2 O (80:26:30:24: 16, by volume). Lipids were localized by staining with iodine vapor, and identification of the different phosphoinositides was made by autoradiography after spraying the plates with EN 3 HANCE and using labeled standards. Silica spots corresponding to phosphatidylinositol (PtdIns), phosphatidylinositol monophosphate (PtdInsP), and phosphatidylinositol bisphosphate (PtdInsP 2 ) were scraped and transferred into scintil-lation vials. Lipids were extracted from silica by adding 100 l of methanol, and radioactivity associated with each phospholipid was counted after addition of 4 ml scintillation liquid.

Cell Loading in PtdIns(4,5)P 2
HL-60 cells labeled with [ 3 H]choline were washed with PIPES buffer and resuspended in the same buffer containing 0.1 mM EGTA, and 100 M phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) was incubated for 15 min at 37°C. Streptolysin O was then added, and incubation was carried out for an additional 10 min. In unlabeled cells, the pool of PtdInsP 2 , incorporated into cells, was calculated to be increased by 30 -50% after that time, using 3 H-labeled PtdIns(4,5)P 2 as a tracer. After loading with PtdIns(4,5)P 2 , cells, depleted of cytosol, were used for measurement of GTP␥S-stimulated PLD activity in the presence of different small G-proteins as described above.

Determination of PtdIns 4-Kinase and PtdIns(4)P 5-Kinase Activities
Phosphatidylinositol 4-kinase (PtdIns 4-kinase) and phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase) activities were performed in a final volume of 100 l containing 40 l of HL-60 cell lysates in the presence of 10 l of a sonicated solution containing PtdIns4P (1.4 mg/ml) and PS (0.6 mg/ml) as described by Chong et al. (26). The reaction was started by addition of 10 l of buffer containing [␥-32 P]ATP (0.1 mCi/ml) and 0.2 mM unlabeled MgATP and performed at room temperature. Incorporation of 32 PO 4 into PtdIns4P and PtdIns(4,5)P 2 was found to be linear for 6 min under such conditions. Five min after addition of the radioactive substrate, the reaction was stopped with 0.6 ml methanol, 1 N HCl (1:1). Phospholipids were extracted by adding 0.5 ml of chloroform. The organic phase was dried under vacuum, and lipids resuspended in chloroform (40 l) were separated onto TLC plates according to Chong et al. (26). 32 P-Labeled phosphoinositides were visualized by autoradiography and quantified using a molecular imager (Molecular Dynamics).

Determination of PtdIns 3-Kinase Activity
PtdIns 3-kinase activity was performed in 500 l final volume under conditions similar to those described above for other PtdIns kinases except that the amount of radiolabeled ATP was equivalent to 0.25 mCi per sample. This enzyme activity was estimated by measuring the amount of D3-phosphorylated inositol lipids present in cells using an HPLC technique as described (27,28). Analysis of the deacylated lipids was performed as described (28). Radioactivity eluted from the Partisphere SAX column (Whatman International, Maidstone, Kent, UK) was monitored and quantified by LB 506C detector (Berthold, Munich, Germany), using the Cerenkov effect.

Immunoblot Analysis
After SDS-PAGE, proteins were transferred electrophoretically onto a nitrocellulose membrane. Blocking was achieved overnight at 4°C in isotonic PBS/glycine (2%) with 3% non-fat dry milk. Western blot analysis was performed for 90 min at room temperature using a polyclonal anti-recombinant ARF antibody (1:500 dilution) or anti-PKC␣ antibody (1:500 dilution) in the same buffer containing 10% FBS and 0.3% Tween 20. The blots were then incubated for the same period with 125 I-protein A (0.2 Ci/ml). Immunoblots were analyzed with a PhosphorImager (Molecular Dynamics) for quantitative evaluation of the radioactivity in bands.
Western blot analysis with anti-PITP antibodies and protein quantification were kindly performed as described (29).
Protein Estimation-Protein content in cytosolic fractions was measured according to Bradford (30) using bovine albumin as a standard.

LT-IP82 Glucosylates Several Small G-proteins in HL-60
Cells-Lethal toxin from C. sordellii (strain IP82) was found to glucosylate and inactivate small G-proteins in Swiss 3T3 cells: Ras primarily and Rac and Rap to a lesser extent (16). Moreover, this strain was also found to glucosylate Ral in vitro. We checked that LT toxin exerted the same effect on small Gproteins in the promyelocytic HL-60 cell line. As shown in Fig.  1A, overnight treatment of HL-60 cells with 100 ng/ml LT would glucosylate proteins that could no longer be glucosylated in vitro as in control cells. Demonstration that Ras and Rac present in HL-60 cells were glucosylated by LT was performed by immunoprecipitation of a cell lysate after in vitro glucosylation using specific antibodies against Ras and Rac (Fig. 1B). As controls we used recombinant Ras, Rac, and Ral glucosylated in vitro by LT (Fig. 1, A and B).
LT-IP82 Causes Cytoskeletal Effect in HL-60 Cells-LT has been observed to provoked morphological and cytoskeletal effects in epithelial and fibroblastic cells. In HL-60 cells, the effect of LT toxin was checked on cell viability and on actin polymerization. Overnight treatment of the promyelocytic cell line, HL-60, was found to have some effect on cell viability determined by trypan blue exclusion. Up to 30 ng/ml LT toxin has no detectable effect on cell viability; at 100 ng/ml, cell mortality was around 10%. At 300 ng/ml LT, the percentage of dead cells was increased to 20 -30%. Thus, we tested the effect of 100 ng/ml LT on the cytoskeleton in this cell line. As shown in Fig. 2, overnight treatment of HL-60 cells with LT induced a neat decrease in cortical actin polymerization, indicating the efficiency of the treatment.
LT Inhibits PLD Activity-As LT glucosylates several small G-proteins reported to be involved in PLD regulation, we then studied the effect of LT on PLD activity. As reported in Fig. 3, LT inhibits PLD activity in a time-and dose-dependent manner. In HL-60 cells, GTP␥S-stimulated PLD activity, measured in streptolysin O-permeabilized HL-60 cells, was decreased by 20 -30% after 6 h of treatment with LT (100 ng/ml), and maximal inhibition (50 -60% of the control) was reached after 16 h (Fig. 3A). This PLD activity was inhibited in a dose-dependent manner after an overnight treatment of HL-60 cells with LT. Maximal effect that reduces GTP␥S-stimulated PLD activity by 60% was obtained with 30 ng/ml. A slight decrease in basal PLD activity was also observed at the highest LT dose studied (100 ng/ml) (Fig. 3B), and PMA-stimulated PLD activity, measured in intact cells, was inhibited to the same extent by similar doses of LT (Fig. 3C).
Effect of LT Treatment on Reconstitution of GTP␥S-stimulated PLD Activity with Small G-proteins in Cytosol-depleted HL-60 Cells-We have already shown that HL-60 cells permeabilized with streptolysin O can be depleted of cytosol (23). Thus, cytosol-depleted cells represent an accurate model to study whether the decrease in PLD activity, observed after LT treatment, is related to a change occurring at the membrane or  the cytosol level. The first possibility was tested using LTtreated and control HL-60 cells depleted of cytosol after permeabilization. PLD activity was then stimulated in the presence of GTP␥S by different small G-proteins known to be PLD activators as follows: ARF and RhoA which cannot be modified by LT and Rac which is a LT target. ARF-and Rho-stimulated PLD activities were inhibited by up to 65% after an overnight treatment of HL-60 cells with LT and Rac-stimulated PLD activity by 33% as shown in Fig. 4, A-C, respectively. It is noticeable that ARF and RhoA are efficient PLD activators in HL-60 cells, with ARF being a better one. Indeed, an equivalent stimulation of PLD activity was obtained with 5 and 10 g of ARF and RhoA, respectively. In comparison, Rac is a poor PLD activator in HL-60 cells. ARF and Rac have been reported to act synergistically on PLD in different cell lines. They were tested together, and their stimulating effect on PLD activity was found to be additive and not synergistic in HL-60 cells (Fig.  4D). Moreover, the effect of RalA and Ras on PLD activity was checked. PLD stimulation obtained with 10 g of the recombinant protein RalA was similar to that given by the same amount of Rac (1.6 Ϯ 0.04% of PtdCho hydrolysis). After LT treatment (100 ng/ml overnight), RalA-stimulated PLD activity was inhibited by about 35%. In cytosol-depleted HL-60 cells, Ras was never found to stimulate PLD activity in similar conditions (data not shown). These results indicate that, in membrane of toxin-treated HL-60 cells, a factor necessary for PLD activation is missing or decreased.
Relationship between LT Effect on the Cytoskeleton and PLD Inhibition-To study if the decrease in PLD activity provoked by LT treatment was directly related to actin polymerization, we studied the effect of different molecules modifying the cytoskeleton on PLD activity. As reported in Table I, overnight treatment of cells with toxin B (15 ng/ml, overnight) from C. difficile was able to inactivate PLD in HL-60 cells to an even greater extent than LT; this toxin was reported to modify the actin cytoskeleton in different cell types. However, cytochalasin D (1 M/ml, overnight) which disrupts the cytoskeleton and iota toxin (100 ng/ml, overnight) which depolymerizes actin (31) did not provoke marked changes in PLD activity in cytosol-depleted cells stimulated with ARF or RhoA. In HL-60 cells, the effect on the actin cytoskeleton of toxin B, cytochalasin D, and iota toxin was checked; they all provoked a clear decrease in cortical polymerized actin (data not shown). These results do not exclude an indirect link between LT effect on the actin cytoskeleton and on GTP␥S-stimulated PLD activity such as a change in phosphoinositides required for actin polymerization and for PLD activation.
LT Treatment Decreases Polyphosphoinositides Levels; Its Effect on Phosphatidylinositol Kinases-As PtdIns(4,5)P 2 is a cofactor for ARF-stimulated PLD activity, we studied the effect of LT treatment on phosphoinositide composition. As reported in Fig. 5, overnight treatment of HL-60 cells induced modifications in polyphosphoinositide levels in the range of LT concentrations that provoked inactivation of ARF-and RhoA-stimulated PLD activity. PtdInsP (Fig. 5A) and PtdInsP 2 (Fig. 5B) levels were both decreased by LT in a dose-dependent manner. At 30 ng/ml, a dose shown to inhibit maximally small G-protein-stimulated PLD activity, PtdInsP and PtdInsP 2 were decreased by about 30 and 50%, respectively. PtdIns level was not modified regardless which concentration of LT was used (data not shown).
Since PtdIns(4,5)P 2 (PtdInsP 2 ) is a cofactor for PLD activity, a decrease in the level of this minor polyphosphoinositide could be responsible for the inability of small G-proteins such as ARF and RhoA to stimulate PLD activity fully in LT-treated cells. Therefore, the effect of PtdInsP 2 addition was tested on small G-protein-stimulated PLD activity. PLD activity stimulated with ARF, RhoA, Rac, or RalA in the presence of GTP␥S was measured in untreated and LT-treated cells. As shown in Fig.  6, control and LT-treated cells loaded with PtdInsP 2 responded slightly better to each small G-protein. However, PtdInsP 2 was not able to restore fully GTP␥S-and small G-protein-stimulated PLD activity in LT-treated HL-60 cells.
As both Rac and Ras have been reported to participate in in PtdIns4P 5-kinase and PtdIns 3-kinase regulation responsible for PLD cofactor synthesis, the effect of LT treatment was examined on polyphosphoinositide kinases. PtdIns 4-kinase and PtdIns4P 5-kinase activities were measured under conditions similar to PLD activity measurement, in FIG. 5. Effect of LT treatment on the levels of polyphosphoinositides in HL-60 cells. Cells were incubated for 48 h to reach metabolic equilibrium with [ 3 H]inositol (0.5 Ci/ml) and then treated or not with various amounts of LT. After overnight incubation, cells were washed, and the amount of radioactivity associated with PtdInsP and PtdInsP 2 was determined. A represents tritiated inositol incorporated in PtdInsP (striped bars), and B represents tritiated inositol incorporated in PtdInsP 2 (dotted bars). Data are expressed as the percentage of radioactivity incorporated in phosphoinositides and represent the means Ϯ S.E. in triplicate. This experiment is representative of three different ones. In the experiment shown, the total radioactivity in cells was 405,857 Ϯ 34,403 dpm. the absence and in the presence of GTP␥S. As shown in Fig. 7, after overnight treatment of cells with 100 ng/ml LT, PtdIns 4-kinase was markedly increased by about 200% compared with untreated cells (Fig. 7A), whereas PtdIns4P 5-kinase (Fig.  7B) was not modified either with or without GTP␥S. Therefore, the decrease in PtdInsP and PtdInsP 2 levels cannot be related to a change in the activity of the kinases involved in their synthesis.
PtdIns 3-kinase activity was measured in control and LTtreated cells. As reported in Table II, the activity of this enzyme was profoundly inhibited after cell treatment with LT, as estimated by the amount of the different polyphosphoinositide isoforms phosphorylated in position 3 of inositol lipids identified by HPLC analysis. In LT-treated cells, phosphoinositides generated by PtdIns 3-kinase represent 15-20% of those in untreated cells. Wortmannin, a specific PtdIns 3-kinase inhibitor, decreased the enzyme activity by 95% in HL-60 cells when used at 20 nM for 10 min (conditions known to specifically inhibit PtdIns 3 kinase activity).

Relationship between LT Inactivation of Ras and PLD Inhibition-
To check if the observed decrease in PtdIns 3-kinase activity could account for PLD inhibition, HL-60 cells were treated for 20 min with 20 and 50 nM wortmannin. No inhibition of PLD activity was observed (Fig. 8) whichever the concentration used. Similar results were obtained using LY 294002, another specific PtdIns 3-kinase inhibitor (data not shown). Therefore, it is unlikely that inhibition of PLD by LT would be related to Ras/PtdIns3-kinase inhibition in this model.
Involvement of Ral in the Inhibition of GTP␥S-stimulated PLD Activity-RalA, also inactivated by LT-IP82, has been reported to take part in PLD activation. To study the role of this small G-protein on GTP␥S-stimulated PLD activity, we used a different lethal toxin extracted from C. sordellii, strain 9048, that does not possess the capacity to glucosylate Ral in vitro but is still able to inactivate Ras and Rac (Fig. 9A). As reported in Fig. 9B, GTP␥S-stimulated PLD activity in HL-60 cells was not modified after a treatment with 1 g/ml LT-9048, whereas it was decreased by about 50% when cells were treated with only 0.1 g/ml of LT-IP82.

Inhibition of GTP␥S-stimulated PLD Activity by LT Treatment also Involves a Modification at the Cytosol Level in HL-60
Cells-The possibility that LT treatment could have an effect on a factor present in the cytosol was also investigated. For this purpose, cytosolic fractions from control and LT-treated cells were prepared, and their respective ability to stimulate PLD activity in the presence of GTP␥S was tested in control HL-60 cells that were depleted of cytosol by a pre-permeabilization step. As reported in Fig. 10A, cytosol (60 g of proteins/assay) extracted from LT-treated cells did not stimulate GTP␥S-dependent PLD activity in control cells to the same extent as cytosol extracted from untreated cells. This result indicates that a cytosolic factor involved in PLD activity is either missing or decreased after LT treatment.
Several proteins involved in PLD regulation are known to be cytosolic including phosphatidylinositol transfer protein (PITP) reported to be essential for maintaining the level of PtdInsP 2 (32) and ARF and PKC␣, potent activators of PLD. Thus, the level of these proteins was measured in the cytosol recovered from untreated and LT-treated cells. As reported in Fig. 11, A and B, no modification in the level of PITP and ARF was observed after LT treatment. In contrast, cytosolic PKC␣ was decreased by about 40% in LT-treated cells (Fig. 11C). Quantification of the bands by scan densitometer or PhosphorImager showed that the amount of PITP and ARF differed by around 6% (decrease for PITP and increase for ARF) between control and LT-treated cells, and PKC was decreased by 61% in LTtreated cells by comparison to untreated cells. DISCUSSION In the present work, we have investigated different effects of lethal toxin (LT) purified from C. sordelli (strain IP-82) in HL-60 cells. Toxins purified from different Clostridia origins have been demonstrated to inactivate small G-proteins of the Rho family, and target profiles of their intracellular activity are specific for each of them. It has been reported that, in HeLa FIG. 6. Effect of PtdIns(4,5)P 2 cell loading on PLD activity. HL-60 cells labeled with [ 3 H]choline chloride were treated overnight with LT (100 ng/ml). PLD activity was determined in cells loaded or not loaded with PtdIns(4,5)P 2 . After 30 min incubation with PtdIns(4,5)P 2 (100 M) at 37°C, cells were permeabilized with streptolysin O for 15 min and then washed. PLD was measured in the absence (Ⅺ) or in the presence of GTP␥S (25 M) (f). Small G-protein-stimulated PLD activity was achieved with ARF (5 g/assay) (u), RhoA (10 g/assay) (;), Rac (10 g/assay) (p), or RalA (10 g/assay) (z) in the presence of GTP␥S. PLD activity was expressed as the percentage Ϯ S.E. of Ptd-Cho hydrolyzed in cells unloaded or loaded with PtdIns(4,5)P 2 and treated or not with LT. In the experiment shown, the total radioactivity in cells was 160,214 Ϯ 1,208 dpm. Similar results were obtained on three different occasions. cells, LT enters cells by endocytosis and, after toxin entry, cells round up as a consequence of F-actin reorganization since disruption of actin stress fibers and formation of filopodia occur. Possessing a glucosyltransferase activity, LT monoglucosylates and inactivates Ras by monoglucosylation on threonine 35 in HeLa cells (16). LT was also shown to glucosylate Ras, Rac, Rap, and Ral in vitro (16,18). Under their GTP-bound form, all these small G-proteins except Rap have been reported to participate in PLD activation (5,6,33).
In the present study, we observed that LT treatment provokes, in HL-60 cells, glucosylation of small G-proteins, among which are Rac and Ras, modification of the actin cytoskeleton, as in HeLa cells (16), and a decrease in PLD activity stimulated by GTP␥S in permeabilized cells and by PMA in intact cells. In this cell line, GTP␥S is known to provoke a major activation of PLD increasing basal activity by a factor 8 to 10 in the presence of optimal concentrations of Mg-ATP and Ca 2ϩ (34). In all our attempts to measure PLD activity using artificial vesicles with various phospholipid compositions as the enzyme substrate, addition of sodium oleate to vesicles provoked a major decrease in phosphatidylcholine breakdown in this cell line. 2 These results indicate that HL-60 cells contain little or no oleate-activated PLD. These cells have been shown to express high levels of mRNA for PLD1, particularly, and PLD2 (35), both isoforms requiring PtdIns(4,5)P 2 as a cofactor for activity (36,37). The former human isoform can be directly activated in vitro by ARF, Rho protein family, and PKC␣ (33), and the latter was recently reported to be stimulated by ARF also (38). A direct interaction between the carboxyl terminus of PLD and activated RhoA using the two-hybrid technique has been reported by Sung et al. (39). So far, no direct interaction between PLD and ARF was reported; this could be due to a pre-required binding of ARF and/or PLD to PtdIns(4,5)P 2 (40,41) for interaction. In HL-60 cells, PLD activity was shown to be calciumdependent (34). In these promyelocytic cells, LT inhibits PLD activity, whichever its pathway of stimulation, and hence its effect is partly different to that observed in an epithelial cell line, HEK. Indeed, in a recent work, Schmidt et al. (42) found that LT was able to decrease exclusively the PKC pathway of PLD activation in HEK-293 cells stably expressing m3 muscarinic receptors by inactivating Ral proteins. In HL-60 cells, Ral proteins also appeared to be the major target for LT effect on PLD activity. Indeed, LT extracted from strain 9048 of C. sordellii, which does not inactivate Ral proteins in vitro, was not able to provoke modification in PLD activity in these cells.
In HL-60 cells, LT (from strain IP-82) was found to generate major changes in cytoskeleton organization, membrane phospholipid composition, and cytosolic PKC␣ level. These profound modifications could be related to the concomitant alteration in PLD activity stimulated via different signaling pathways.
LT treatment induced an almost complete disorganization of cortical actin cytoskeleton in HL-60 cells. In HeLa cells, cytoskeletal modifications induced by this toxin were reported to be different from those provoked by toxin B from C. difficile. Such differences can be due to their different major targets in vivo, Ras and Rac for LT and Rho proteins and RhoA, Rac, and Cdc42 for toxin B. All these small G-proteins have been reported to exert a control on the actin cytoskeleton through different pathways (43,44). In HL-60 cells, which grow in suspension, a similar modification was observed with both toxins, a major decrease in polymerized actin. LT could modify the actin cytoskeleton in inactivating Ras and/or Rac. Several studies suggest an implication of the cytoskeleton in PLD regulation. Small G-proteins of the Rho subfamily, well established to be involved in the regulation of actin polymerization, are also activators of PLD (33,45), and RhoA was reported to interact directly with PLD1 (39). The actin-binding protein, fodrin, was found to inhibit PLD activity (8), and gelsolin was shown to interact with PLD and activate the enzyme (7).
From the present study, the major decrease in actin polymerization observed after LT treatment in HL-60 cells is likely to be related to Ras/PtdIns 3-kinase/Rac pathway(s), but changes at the cytoskeleton level do not appear to be directly responsible for PLD inhibition. In HL-60 cells, we observed that LT treatment, giving a depolymerization in cortical actin, was accompanied by a profound decrease in PtdIns 3-kinase activity, a direct target for Ras (46). Indeed, control of actin cytoskel-2 V. Montalescot and B. Geny, unpublished results. eton by Ras has been related to PtdIns 3-kinase activity, and Rac was shown to function downstream of Ras/PtdIns 3-kinase (43). As it has been recently reported that gelsolin dissociation from actin filament is under the control of Rac (47), Rac inactivation by LT could also specifically depolymerize actin by blocking actin/gelsolin interaction, preventing actin uncapping and blocking nucleating activity at the barbed end of F-actin. Thus, inactivation of both Ras and Rac by LT are likely to be responsible for cortical actin disorganization in HL-60 cells. However, a direct relationship between PLD activation and the actin cytoskeleton organization can be ruled out as neither PtdIns 3-kinase inhibition nor drugs such as cytochalasin D, which caps the barbed end of actin filaments, and iota toxin, which ADP-ribosylates the actin monomers (31), have an effect on PLD activity in HL-60 cells. A similar observation has been made by Schmidt et al. (13) in HEK cells. In these cells, cytochalasin B and toxin C2 from Clostridium botulinum both disorganize the actin cytoskeleton but do not inhibit GTP␥Sstimulated PLD activity as C3 toxin does, which inactivates Rho proteins.
Treatment with LT (strain IP-82) was found to lead to a net decrease in polyphosphoinositides in HL-60 cells. Such changes in the levels of negatively charged phospholipids could explain both the decrease in polymerized actin and the inability for any PLD-activating small G-protein to stimulate fully PLD activity in LT-treated cells. Proteins participating in the cytoskeleton or in the regulation of actin polymerization including profilin, gelsolin, and fodrin bind PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 with high affinity (48). Therefore, a decrease in these minor polyphosphoinositides would lead to changes in actin organization. As PLD activity in HL-60 cells appears to be mostly due to PLD isoforms requiring PtdInsP 2 as cofactors, the important decrease in polyphosphoinositides observed after LT treatment can explain the lack of PLD activation by small G-proteins such as ARF and RhoA that are not modified by LT. The function of ARF is related to its interaction with PtdIns(4,5)P 2 (40) and has also been shown to be the small G-protein responsible for PLD activation involving RalA (3). This latter small G-protein was shown not to activate directly PLD but to recruit ARF to membranes which, thus, would stimulate PLD activity (4). Thus, the inability to restore PLD activity with PtdIns(4,5)P 2 in treated HL-60 cells could explain why RalA and ARF to- In the experiment shown, the total radioactivity in cells was 145,500 Ϯ 9,080 dpm. This experiment was repeated three times with similar results.

TABLE II
PtdIns 3-kinase activity in HL-60 cells treated with lethal toxin from C. sordellii After labeling and stimulation of HL-60 cells as detailed under "Experimental Procedures," phosphoinositides labeled in position 3 were measured by HPLC. Phosphatidylinositol 3-kinase was specifically inhibited by treating cells for 10 min with 20 nM wortmannin. The effect of LT (100 ng/ml) on D3-phosphoinositide formation was measured after an overngith treatment. Results are given in dpm, and the experiment shown is respresentive of two.

Cell treatment
Phosphoinositide species (counts in 32  gether could not stimulate PLD activity in LT-treated HL-60 cells. In HL-60 cells, GTP␥S-and PMA-stimulated PLD activities were decreased to a similar extent and with similar doses of LT. This toxin was shown to inhibit only the PMA pathway in HEK cells (42). The discrepancies observed in the effects of LT are likely to be due to the difference in cell types. In HL-60 cells, the effect of LT was similar to that obtained with another bacterial toxin, toxin B from C. difficile, in this cell line; GTP␥S-and PMA-stimulated PLD activities were both inhibited, and addition of PtdIns(4,5)P 2 was not able to modify PLD activity significantly (49). In contrast, in HEK cells, this latter toxin only inhibits receptor and G-protein-stimulated PLD activity (13), and PtdIns(4,5)P 2 was able to restore membranelocated PLD activity (14). HL-60 cells and HEK cells are likely to possess different contents in PLD isoforms and/or different PLD regulators participating in each pathway of activation. HL-60 cells were found to have a more marked GTP␥S-stimulated PLD activity than HEK cells and do not possess a PLD activity measurable in the presence of sodium oleate or sodium cholate as HEK cells do (13,14,50). Moreover, although inactivation of Ral proteins appears to be sufficient for LT to inhibit PLD activity, it is likely that other small G-proteins inactivated by the toxin exert additional effects which might have stronger consequences in HL-60 cells than in HEK cells. Indeed, in these latter cell lines, LT was found to inhibit specifically PMAstimulated PLD activity in the absence of modification in c-PKC cellular levels but not a pathway involving G-proteins. In these cells, the same effect was observed with toxin B-1470 from C. difficile that does not glucosylate Ras. In HL-60 cells, LT treatment was also found to decrease the amount of PKC␣ in cell cytosol. Such a cytosol from LT-treated cells was found to be less efficient to stimulate PLD activity in control cells. The exact mechanism of action for PKC and Ca 2ϩ in PLD activation is not clear. It has been reported that the regulatory domain of PKC␣ in the presence of Ca 2ϩ or PMA is sufficient to stimulate PLD activity (50). However, it has also been reported that PKC activity could also play a role by phosphorylating a phospholipase D-related component in the plasma membrane (51). In HL-60 cells, previous studies have shown that both Ca 2ϩ and PMA can stimulate PLD in synergy with GTP␥S (34). Thus, the regulatory domain of a c-PKC in the presence of Ca 2ϩ could be necessary as a starter for other activators such as ARF or Rho proteins to stimulate PLD activity and the net decrease in PKC␣ cytosolic content could be responsible, at least in part, for a diminution in the synergistic effect between small Gprotein, PKC and/or Ca 2ϩ . The decrease in cytosolic PKC␣ level could be a consequence of Ras inactivation as this small Gprotein and PKC interaction have been reported to be required in activation signaling pathways (52)(53)(54)(55)(56). In glucosylating and inactivating Ras and other small G-proteins downstream of it, LT could inactivate PKC and in HL-60 cells rapidly lead to its "down-regulation." In conclusion, we demonstrate that LT from strain IP-82 of C. sordellii, which inactivates several small G-proteins, inhibits GTP␥S-and PMA-stimulated PLD activity similarly. Using LT extracted from strain 9048 of C. sordellii which has no effect on Ral proteins, the responsibility for PLD inhibition is essentially due to Ral although recombinant Ral could not reconstitute PLD activation after LT treatment. Major changes in the level of polyphosphoinositides, acting as PLD cofactors, and in the cytosolic level of PKC␣, an important PLD activator, are also observed and could be related to the high sensitivity of HL-60 cells to inactivation by LT not only of Ral proteins but also Rac and Ras. FIG. 10. Effect of a cytosol from LT-treated cells on PLD activity in cytosol-depleted HL-60 cells. HL-60 cells were treated or not (control) with 100 ng/ml LT. After an overnight incubation, cells were washed and then lysed by hypotonic shock followed by freeze-thawing as reported under "Experimental Procedures." Unbroken cells and nuclei were eliminated by a low speed centrifugation. Supernatants were then centrifuged for 20 min at 300,000 ϫ g to pellet particulate fractions. The same amount of cytosolic protein (60 g) from LT-treated and control cells was then used to reconstitute PLD activity in untreated HL-60 cells pre-permeabilized for 15 min and depleted of their cytosol as in Fig. 4 11. Effect of LT treatment on cytosol content in PITP, ARF, and PKC␣ in HL-60 cells. Cytosols prepared from untreated (control) and LT treated cells were electrophoresed, and after transfer onto nitrocellulose membrane, their respective content in PITP, ARF, and PKC␣ was analyzed using specific antibodies as reported under "Experimental Procedures." This experiment was repeated twice with similar results.