©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphatidylinositol 4,5-Bisphosphate Synthesis Is Required for Activation of Phospholipase D in U937 Cells (*)

(Received for publication, November 17, 1994)

Paolo Pertile (1)(§) Mordechai Liscovitch (1) (2)(¶) Vered Chalifa (2) Lewis C. Cantley (1)

From the  (1)Division of Signal Transduction, Beth Israel Hospital and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and the (2)Department of Hormone Research, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D (PLD) has been implicated in signal transduction and membrane traffic. We have previously shown that phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P(2)) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5-P(2) as a PLD cofactor. In the present study we extend these observations to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5`-3-O-(thio)triphosphate (GTPS) is greatly potentiated by MgATP. We have utilized this experimental system to test the hypothesis that MgATP potentiates PLD activation by G proteins because it is required for PtdIns-4,5-P(2) synthesis by phosphoinositide kinases. As expected, MgATP was absolutely required for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P(2) levels in the permeabilized cells. In the presence of MgATP, GTPS further elevated the levels of the phosphoinositides. The importance of PtdIns-4,5-P(2) for PLD activation was examined by utilizing a specific inhibitory antibody directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the synthesis of PtdIns-4,5-P(2). Anti-PtdIns 4-kinase completely inhibited PtdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P(2) levels in the permeabilized cells. In parallel, the anti-PtdIns 4-kinase fully inhibited the activation of PLD by GTPS and caused a 60% inhibition of PLD activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, indicating that elevated PtdIns-4,5-P(2) levels are required for PLD activation. This conclusion is supported by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P(2), also blocked PLD activation. Furthermore, the activity of PLD in U937 cell lysate was stimulated by PtdIns-4,5-P(2) in a dose-dependent manner. The current results indicate that PtdIns-4,5-P(2) synthesis is required for PLD activation in permeabilized U937 cells and strongly support the proposed function of PtdIns-4,5-P(2) as a cofactor for PLD. In addition, the results further establish PtdIns-4,5-P(2) as a key component in the generation of second messengers via multiple pathways including phosphoinositidephospholipase C, phosphoinositide 3-kinase and PLD.


INTRODUCTION

Receptor-mediated hydrolysis of cellular phospholipids is a ubiquitous biochemical event of central importance in cell signal transduction. Phospholipase D (PLD) (^1)catalyses the hydrolysis of phospholipids, resulting in formation of phosphatidic acid (PtdOH) and liberation of the polar head group of the phospholipid. PtdOH is further metabolized by PtdOH phosphohydrolase to form diacylglycerol, an activator of protein kinase C. Data in human neutrophils and rat mast cells have suggested that this could be the major mechanism by which diacylglycerol accumulates during cell activation(1, 2, 3, 4) . At least two distinct isoenzymes of PLD are expressed in mammalian cells. One is membrane-associated that prefers phosphatidylcholine as substrate, whereas the other is a cytosolic enzyme that appears to hydrolyze preferentially phosphatidylethanolamine or phosphatidylinositol (PtdIns)(5, 6) . However, to date none of the PLDs involved in signaling have been purified or cloned.

A large number of agonists activate PLD in mammalian cells(7, 8) . Although the mechanisms of enzyme activation, regulation, and the different functions of PLD remain largely unknown, several lines of evidence suggest that PLD is involved in various effector responses such as enzyme secretion(9, 10) , stimulation of the respiratory burst (11) , phagocytosis(12) , and cell proliferation(13, 14, 15, 16, 17, 18, 19) .

Protein kinase C activation by phorbol esters has been observed to cause PLD activation, suggesting that PLD activation could be downstream of the protein kinase C pathway. However, other mechanisms of regulation of PLD activity that are protein kinase C-independent have also been demonstrated. A guanine nucleotide binding protein activated by guanosine 5`-O-3-(thio)triphosphate (GTPS), can stimulate PLD activity(20, 21, 22) . These studies suggest that a G protein directly interacts with PLD as a upstream regulator. One G protein that regulates PLD activity has recently been identified as ADP-ribosylation factor (ARF), a small GTP-binding protein belonging to the Ras superfamily(23, 24) . The effect of MgATP on PLD activation is of particular interest. Although MgATP is not absolutely required for PLD activation, it greatly potentiates the activation of PLD by GTPS (22, 25, 26, 27, 28, 29, 30) . Because nonphosphorylating analogs of ATP do not possess the same property, it appears that MgATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The observation that genistein, a tyrosine kinase inhibitor, significantly attenuated the ability of MgATP to stimulate PLD activity in GTPS-treated cells led to the hypothesis that G proteins and tyrosine phosphorylation could coordinately regulate PLD activity(29) . However indirect evidence suggests that genistein is an inhibitor of phosphatidylinositol 4phosphate 5-kinase (PtdIns 4P 5-kinase), the enzyme responsible for the last step in the production of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P(2))(31) . In addition, the aminoglycoside antibiotic neomycin, which exhibits high affinity binding to inositol phospholipids, inhibits PLD activity in vitro as well as in permeabilized cells(32) . These observations suggested that MgATP activates PLD by acting as substrate for lipid kinases.

Using partially purified brain membrane PLD assayed in vitro, we have previously shown that PtdIns-4,5-P(2) caused up to 10-fold increase in PLD activity in vitro in a highly specific manner(33) . Neomycin inhibited membrane-bound PLD but had no effect on the activity of the partially purified enzyme. The inhibitory effect of neomycin could be restored by the addition of exogenous PtdIns-4,5-P(2), suggesting that neomycin inhibits membrane-bound PLD activity by binding to endogenous PtdIns-4,5-P(2).

In this study, we show that inhibition of PtdIns-4,5-P(2) synthesis potently inhibits PLD activation in permeabilized U937 cells. Together with our previous results, these findings indicate that PtdIns-4,5-P(2) is an essential physiological cofactor of PLD.


EXPERIMENTAL PROCEDURES

Materials

1-Palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidylcholine (C(6)-NBD-PC) was from Avanti Polar Lipids. Protein A-Sepharose beads were purchased from Pharmacia Biotech Inc. PtdIns was supplied by Sigma. Thin layer chromatography plates (Silica gel 60) were obtained from Merck. [^3H]Oleic acid (8.9 Ci/mmol) and [-P]ATP (3000 Ci/mmol) were obtained from DuPont NEN. myo-[^3H]Inositol (15 Ci/mmol) was supplied by American Radiolabeled Chemicals. All other chemicals were obtained from Sigma. Details regarding the anti-PtdIns 4-kinase monoclonal antibody 4C5G are described elsewhere(34) .

Cell Culture

U937 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mMLglutamine, 50 units/ml penicillin and 50 mg/ml streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO(2) and 95% air. The cells were grown to a density of 0.5 times 10^6 cells/ml.

myo-[^3H]Inositol Labeling and HPLC Analysis of Cellular Lipids in Permeabilized Cells

Cells were washed and resuspended in inositol-free RPMI 1640 supplemented to contain 10% (v/v) heat-inactivated dialyzed fetal calf serum and 20 µCi/ml myo-[^3H]inositol and then incubated overnight at 37 °C. The cells were finally washed with phosphate-buffered saline and subjected to permeabilization as described above. Lipids were analyzed essentially as described before(35) . Crude brain phosphoinositides (20 µg) were added as carrier, and then 380 µl of CHCl(3) was added to separate the phases. The aqueous phase was removed, and the interface and the CHCl(3) phase were washed with 400 µl of MeOH, 0.1 M EDTA, pH 8.0 (1:1). The CHCl(3) phase was removed to a glass scintillation vial and dried under N(2) in preparation for deacylation and SAX HPLC analysis as described(35) . Lipids were quantitated using a Radiomatic A500 on-line radiation counter (Packard).

Lipid Kinase Assay in Cell Lysate

Cells were centrifuged for 10 min at 1200 rpm, the pellet was washed twice with ice-cold RPMI 1640, and the cells were solubilized in 1% Nonidet P-40 lysis buffer as described previously(36) . PtdIns 4-kinase activity was determined by quantitating the transfer of phosphate from [-P]ATP to exogenously added PtdIns in the presence of 0.3% Nonidet P-40 as described in detail elsewhere(37) . In the inhibition experiments, the cell lysates were previously incubated with the anti-PtdIns 4-kinase antibody (4C5G) for 30 min at 4 °C. The reaction products were analyzed by thin layer chromatography of the chloroform extract, and the plates were analyzed on a Bio-Rad PhosphorImager.

Measurement of PtdIns-4-P and PtdIns-4,5-P(2) Synthesis in Permeabilized Cells

Cells were centrifuged and washed as described in the previous paragraph and then resuspended in a modified basic glutamate assay medium previously described (29) to a concentration of 3 times 10^6 cells/0.5 ml. The incubation was started by diluting the cell suspension 1:2 with the previous medium supplemented with 20 µg/ml digitonin and 1 mM Ca for 30 min at 37 °C. 100 µM GTPS and 2 mM Mg[-P]ATP were added as stimuli as indicated. After 30 min at 37 °C, the incubation was terminated by adding 2 ml of ice-cold methanol/chloroform (1:1). The lipid phase was extracted and dried by centrifugation under vacuum in a Speed-Vac concentrator (Savant). The reaction products were resuspended in 20 µl of methanol/chloroform (1:1) and analyzed by thin layer chromatography.

Assay of PLD Activity in Permeabilized Cells

PLD activity was determined by measuring the transfer of phospholipid phosphatidyl moieties, metabolically prelabeled with [^3H]oleic acid, in the presence of ethanol, into phosphatidylethanol (phosphatidyltransferase activity). Cell lipids were radioisotopically labeled by incubation of cells (0.5 times 10^6/ml) in culture medium supplemented with [^3H]oleic acid (1 µCi/ml) for 3 h. After the incubation, the cells were washed twice with phosphate-buffered saline and resuspended in the basic glutamate assay medium to a concentration of 3 times 10^6 cells/0.5 ml. The incubation was initiated by diluting the cell suspension 1:2 with the previous medium supplemented with 2% ethanol (as a substrate for the transferase reaction), 20 µg/ml digitonin, and 1 mM Ca. After 30 min at 37 °C, the incubation was terminated by adding 2 ml of ice-cold methanol/chloroform (1:1). The lipid phase was extracted and dried by centrifugation under vacuum in a Speed-Vac concentrator (Savant). The lipid extracts were separated by thin layer chromatography, and the [^3H]phosphatidylethanol was quantitated by liquid scintillation spectrometry as described previously(38) . In order to normalize the amount of lipids in each sample, an aliquot of the lipid extracted was quantitated by scintillation counting.

Assay of PLD Activity in Cell Lysate

Cells were treated as described previously and then solubilized in 1% Triton lysis buffer (1% Triton X-100, 50 mM Hepes, pH 7.2, 150 mM NaCl, 1 mM EDTA, 10 mg/ml each of aprotinin, leupeptin, and pepstatin). The protein concentration was measured, and 25 µg of cell lysate were utilized per sample. The PLD activity was determined by incubating the cell lysate for 10 min at 37 °C in the presence of 0.3 mM C(6)-NBD-PC as described previously(33) .

Other

Protein concentration was determined using a Bio-Rad Bradford assay reagent using bovine serum albumin as standard.


RESULTS

GTPS and MgATP Synergize in PtdIns-4-P and PtdIns-4,5-P(2) Synthesis

In permeabilized cells, PLD is strongly activated when the cells are permeabilized in the presence of either GTPS and MgATP(22, 25, 26) . To test the hypothesis that the activation of PLD after stimulation with both MgATP and GTPS is the direct consequence of PtdIns-4,5-P(2) synthesis, the effect of these stimuli on PtdIns-4-P and PtdIns-4,5-P(2) levels in [^3H]inositol-labeled permeabilized U937 cells was investigated. Cells were labeled overnight with [^3H]inositol, and then the permeabilization buffer was added together with the stimuli for 30 min at 37 °C. After treatment, lipids were extracted, and PtdIns-4-P and PtdIns-4,5-P(2) levels were quantitated by HPLC. As shown in Fig. 1, MgATP alone caused a 3-fold increase in the levels of both PtdIns-4-P and PtdIns-4,5-P(2), while GTPS did not. In contrast, when the cells were treated with both GTPS and MgATP, they synergized in promoting the formation of the labeled inositol phospholipids, in particular PtdIns-4,5-P(2).


Figure 1: HPLC analysis of PtdIns-4-P and PtdIns-4,5-P(2) formation after GTPS and MgATP stimulation. Quiescent U937 cells were labeled overnight with myo-[^3H]inositol and stimulated with 2 mM MgATP and/or 100 µM GTPS during the 30-min permeabilization. The samples were analyzed by HPLC to separate PtdIns-4-P from PtdIns-4,5-P(2), which are presented respectively in panelsA and B. up triangle, control; circle, GTPS; bullet, MgATP; , GTPS, and MgATP. A representative result from separate experiments is presented.



4C5G Antibody Inhibits PtdIns 4-Kinase Activity in Total Cell Lysate and in Permeabilized U937 Cells

PtdIns 4-kinase is the first enzyme involved in the sequential phosphorylation of PtdIns to PtdIns-4,5-P(2). To inhibit PtdIns 4-kinase activity, we utilized the monoclonal antibody 4C5G, which has been shown to be specific for type 2 PtdIns 4-kinase, the major isoform of PtdIns kinase present in the cell. This antibody causes greater than 95% inhibition of type 2 PtdIns 4-kinase activity in vitro(34) . Fig. 2shows that the antibody 4C5G almost completely inhibits the total PtdIns 4-kinase activity in lysates from U937 cells at a concentration of 45 µg/100 µl. We next examined the effect of 4C5G antibody on the levels of PtdIns-4-P and PtdIns-4,5-P(2) synthesized in permeabilized cells incubated with both 100 µM GTPS and 2 mM Mg[-P]ATP. 4C5G antibody (45 µg/ml) inhibited more than 70% of the synthesis of PtdIns-4-P and PtdIns-4,5-P(2) (Fig. 3), demonstrating the effectiveness of the antibody in inhibiting phosphoinositide synthesis in the permeabilized cell system. These results indicate that type 2 PtdIns 4-kinase is primarily responsible for synthesis of the PtdIns-4-P, which is utilized for PtdIns-4,5-P(2) synthesis in U937 cells.


Figure 2: Anti-PtdIns 4-kinase antibody inhibits PtdIns 4-kinase activity. Lysates from quiescent U937 cells were incubated for 30 min at 4 °C in the presence of increasing concentration of antibody 4C5G, raised against the type 2 PtdIns 4-kinase. PtdIns 4-kinase activity was then assayed as described under ``Experimental Procedures.'' Unrelated control antibody showed no effect on this activity (not shown).




Figure 3: Inhibition of PtdIns-4-P and PtdIns-4,5-P(2) synthesis by anti-PtdIns 4-kinase antibody in permeabilized cells. Quiescent U937 cells were permeabilized for 30 min in the presence of 100 µM GTPS and 2 mM Mg[-P]ATP with or without 45 µg of the antibody 4C5G. The permeabilization was carried out in 1 ml of volume. Values are expressed as percent inhibition of PtdIns-4-P and PtdIns-4,5-P(2) synthesis obtained in absence of inhibitor and are averages of three experiments carried out in triplicate.



Effect of 4C5G Antibody on PLD Activation by GTPS and MgATP in Permeabilized Cells

We next examined the possibility that the antibody 4C5G, by inhibiting PtdIns-4,5-P(2) formation, could inhibit PLD activity in the permeabilized cell system. Cells were incubated for 30 min at 37 °C in permeabilization buffer with GTPS and MgATP, separately or together, at the same concentrations used in the previous experiment. The PLD activity increased weakly upon GTPS or MgATP stimulation. However, in the presence of both stimuli, the PLD activity increased more than 16 times than in the control. The increase in PLD activity correlates with the increase in PtdIns-4,5-P(2) formation with the same stimuli (compare Fig. 1B with Fig. 4). In the presence of the 4C5G antibody, we detected a dose-dependent decrease in PLD activity that was almost completely inhibited at an antibody concentration of 45 µg/ml. To confirm the specificity of this inhibition, we treated part of the cell preparation with an antibody to the p85 subunit of phosphoinositide 3-kinase as a negative control. This antibody (which inhibits phosphoinositide 3-kinase activity) did not cause any inhibition in PLD activity, confirming that the suppression in the PLD activity was specifically due to the inhibition of the PtdIns 4-kinase-dependent pathway.


Figure 4: Inhibition of GTPS:MgATP-dependent PLD activity by anti-PtdIns 4-kinase antibody. Cells were incubated in permeabilization buffer with 100 µM GTPS and 2 mM MgATP in the presence of the indicated concentration of the monoclonal antibody 4C5G. An antibody recognizing the p85 subunit of phosphoinositol 3-kinase was used as negative control. The reaction was carried out for 30 min, and then PLD activity was measured as described under ``Experimental Procedures.''



Effect of 4C5G Antibody in TPA-activated Permeabilized Cells

To confirm the importance of PtdIns-4,5-P(2) as cofactor for PLD activity and to exclude the possibility that it interferes only with GTPS:MgATP-dependent PLD activation, we investigated the effect of inhibition of PtdIns 4-kinase in permeabilized cells activated with 1 µM of TPA. In this case, the antibody 4C5G inhibited 60% of the TPA-dependent PLD activity, at a concentration of 45 µg/ml (Fig. 5).


Figure 5: Inhibition of TPA-dependent PLD activity by antiPtdIns 4-kinase antibody. The experiment was conducted as described under Fig. 4with the difference that TPA was added as stimulus (in the absence of exogenous MgATP and GTPS). The antibody 4C5G was included in the assay at a concentration of 45 µg/ml.



Effect of Neomycin on PLD Activity in Permeabilized Cells

Neomycin is a high affinity ligand of phosphosphoinositides (39, 40, 41) , and we previously showed that it blocks activation of PLD by PtdIns-4,5-P(2) in vitro(33) . The data in Fig. 6show that neomycin inhibits the MgATP:GTPS-stimulated PLD activity in permeabilized cells.


Figure 6: Inhibition of GTPS:MgATP-dependent PLD activity by neomycin. Cells were incubated in permeabilization buffer with 100 µM GTPS and 2 mM MgATP with increasing concentration of neomycin. PLD activity was described as described above.



Effect of Exogenous PtdIns-4,5-P(2) in PLD Activity

To conclude this series of experiments, we investigated the in vitro effects of PtdIns-4,5-P(2) on PLD activity in U937 cell lysates. The cell lysates were prepared as described under ``Experimental Procedures'' and then incubated for 10 min at 37 °C with increasing concentration of PtdIns-4,5-P(2). In agreement with the results already described for the partially purified PLD(33) , we found a dramatic increase in the trans-phosphatidylation activity of the enzyme in U937 lysates. As shown in Fig. 7, the PLD activity was stimulated by PtdIns-4,5-P(2) in a dose-dependent manner; a 6-fold increase was obtained at a PtdIns-4,5-P(2) concentration of 5 mol %.


Figure 7: PtdIns-4,5-P(2) effects on PLD activity. The PLD activity in total cell lysate was measured in the presence of increasing concentration of PtdIns-4,5-P(2) as described under ``Experimental Procedures.''




DISCUSSION

PtdIns-4,5-P(2) is an important component of several intracellular signaling pathways. It is the precursor of the second messengers inositol trisphosphate and diacylglycerol; in addition, it serves as a substrate for phosphoinositide 3-kinase yielding phosphatidylinositol 3,4,5-trisphosphate, a messenger that has recently been shown to activate Ca-independent protein kinase C isozymes(42, 43) . PtdIns-4,5-P(2) itself may directly bind to and modulate the activity of multiple proteins, and this interaction may coordinate and regulate a number of different processes within the cell. Thus, in addition to its established precursor role in signal transduction, PtdIns-4,5-P(2) may mediate effects of different stimuli on actin polymerization and in general on cytoskeleton organization. In this context, PtdIns-4,5-P(2) has been shown to release gelsolin and other capping proteins from actin filaments, release actin monomers from profilin, stimulate alpha-actinin-dependent actin bundling, and activate the GTPase activity of dynamin(44, 45, 46, 47, 48, 49, 50, 51) .

We have recently shown the importance of PtdIns-4,5-P(2) as a cofactor for the membrane-bound PLD and the partially purified enzyme. The present paper demonstrates the physiological relevance of PtdIns-4,5-P(2) as cofactor for PLD activation in permeabilized U937 cells stimulated with GTPS and MgATP. In these cells(29) , as well as in several other cell types (for review, see (7) ), the activation of PLD by GTPS is greatly potentiated by MgATP. We hypothesized that MgATP potentiated PLD because it is required as a substrate for phosphoinositide kinases that produce PtdIns-4,5-P(2) as a PLD cofactor. The data presented here support this hypothesis. First, the presence of MgATP was found to be essential for maintaining elevated phosphoinositide levels in the cells both in the absence and presence of GTPS (Fig. 1). Second, the synthesis of PtdIns-4-P and PtdIns-4,5-P(2) (in the presence of MgATP and GTPS) was inhibited by an inhibitory antibody specific for PtdIns 4-kinase ( Fig. 2and Fig. 3), indicating that MgATP supported on-going PtdIns-4,5-P(2) synthesis in the permeabilized cells. Third, the anti-PtdIns 4-kinase antibody inhibited PLD activation ( Fig. 4and Fig. 5), and, in addition, adenosine, an agent that inhibits type 2 PtdIns 4-kinase by competing with ATP(52) , inhibited by 50% MgATP:GTPS-dependent PLD activity at the concentration of 5 mM (where the concentration of MgATP used was 0.5 mM (data not shown)), indicating that on-going PtdIns-4,5-P(2) synthesis/high cellular PtdIns-4,5-P(2) levels are required for PLD activity under these conditions. These evidences, and the ability of neomycin, a high affinity ligand of PtdIns-4,5-P(2), to block the MgATP:GTPS stimulation of PLD, supports this conclusion (Fig. 6). Finally, it was demonstrated that PtdIns-4,5-P(2) can potently stimulate PLD activity in a Triton X-100-based lysate of U937 cells in vitro (Fig. 7).

These data clearly show that PtdIns-4,5-P(2) is required for PLD activation and are consistent with our hypothesis that it functions as a cofactor of PLD. However, because under the present conditions GTPS would activate phospholipase C-beta(53) , an alternative explanation for the data is that PtdIns-4,5-P(2) is required as a precursor of the protein kinase C activator diacylglycerol and that the activation of PLD by GTPS is secondary to activation of phospholipase C-beta and protein kinase C. This explanation is inconsistent with the fact that the anti-PtdIns 4-kinase antibody inhibits by 60% PLD activation by the phorbol ester TPA (Fig. 5), since TPA activates protein kinase C directly, bypassing phospholipase C-beta and diacylglycerol, and obviates the need for PtdIns-4,5-P(2) in its capacity as a precursor. Furthermore, under conditions of protein kinase C activation, the activity of phospholipase C-beta is subject to negative feedback inhibition(54) . It is interesting to note that a fraction (40%) of protein kinase C-induced stimulation of PLD activity is not sensitive to inhibition by anti-PtdIns 4kinase antibody, whereas GTPS-induced activation was completely inhibited. This may indicate that activation of PLD by protein kinase C abrogates in part the cofactor requirement of PLD for PtdIns-4,5-P(2), whereas activation of PLD via G protein does not. Indeed, it has been shown recently that activation of PLD by the small G protein ARF is absolutely dependent on the presence of PtdIns-4,5-P(2)(24) .

The identification of ARF, a small G protein that belongs to the ras superfamily, as an activator of PLD in myeloid cells (23, 24) has strongly implicated PLD and its product PtdOH in vesicular traffic. The function(s) of PLD and PtdOH in vesicular traffic are, however, currently unknown. The identification of PtdIns-4,5-P(2) as a cofactor for PLD ( (33) and the present paper) may provide an answer to this question. Evidence that polyphosphoinositide synthesis is important for membrane transport events has been emerging in recent years (for review, see (55) ). Thus, the process of PLD ( (33) ARF and the biosynthesis of PtdIns-4,5-P(2) may participate in a coordinate mechanism for membrane vesiculation and/or fusion. We have recently proposed a model whereby the formation of PtdOH and PtdIns-4,5-P(2), by PLD and PtdIns-4-P 5-kinase, respectively, participate in a positive feedback loop that may play an important role in vesicle fusion with acceptor membranes(33) . Briefly, it was suggested that the interaction of coated vesicles bearing ARFbulletGTP (56) with acceptor membranes will activate PLD associated with these membranes, producing PtdOH(23, 24) . The activity of PtdIns-4-P 5-kinase (which is hypothesized to be located at acceptor membranes) will be stimulated by PtdOH(57, 58) , resulting in massive synthesis of PtdIns-4,5-P(2) from vesicular PtdIns-4-P. This, in turn, will cause further stimulation of PLD activity ( (33) and the present study) leading to more PtdOH production and further stimulation of PtdIns-4-P 5-kinase, etc. This will rapidly cause a very profound change in the lipid composition of vesicular membranes, leading to the formation of microdomains, which are greatly enriched in PtdOH and PtdIns-4,5-P(2). The activity of ARF GTPase-activating protein is stimulated synergistically by PtdIns-4,5-P(2) and PtdOH(59) . Thus, the PtdIns-4,5-P(2)/PtdOH-rich vesicle membranes will cause ARF GTPase-activating protein activation, stimulation of the GTPase activity of ARF, and the conversion of active ARFbulletGTP to inactive ARFbulletGDP. Consequently, PLD will be de-activated, thus halting the positive feedback loop, initiating the disassembly of the coated vesicle(60) , and allowing its subsequent fusion with acceptor membranes (see (33) for more details).

One prediction of this model is that in membrane traffic the activation of PLD and the stimulation of PtdIns-4,5-P(2) biosynthesis are tightly coupled. Indeed, the present results indicate that ongoing PtdIns-4,5-P(2) synthesis is required for PLD activation by G protein in U937 cells. A corollary of this prediction is that PtdIns-4-P 5-kinase is a major physiological target of PtdOH. Intriguingly, in the presence of MgATP, the synthesis of PtdIns-4,5-P(2) is greatly potentiated by GTPS (Fig. 1). This result may suggest that the enzyme responsible for PtdIns-4-P and PtdIns-4,5-P(2) are themselves regulated by G protein(s). This idea was previously suggested on the basis of GTPS effects on isotope flux from PtdIns to IP(3) in permeabilized fibroblasts(61) . An alternative explanation is that, as predicted by our model, the biosynthesis of PtdIns-4,5-P(2) is stimulated by PLD-produced PtdOH. Further studies are required in order to elucidate the mechanism of activation of PtdIns-4,5-P(2) synthesis by GTPS.

The effects of the monoclonal antibody 4C5G demonstrate its efficacy as a tool in functional studies on phosphoinositide metabolism. The inhibition of PtdIns 4-kinase activity by the antibody 4C5G could be extremely useful in permeabilized or microinjected cells in experiments designed to clarify the influence of PtdIns 4-kinase and its products on different pathways. It is clear that multiple PtdIns 4-kinases and PtdIns-4-P 5kinases exist in mammalian cells(62) , but thus far only a single cDNA clone for a mammalian PtdIns 4-kinase has been isolated(63) . Once specific molecular tools are available for studies of phosphoinositide kinases, the proposed relationship between these enzymes and PLD in membrane traffic could be explored in greater depth.


FOOTNOTES

*
This research was supported in part by Grant GM 36624 from the National Institutes of Health (to L. C. C.). 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.

§
Supported by a fellowship from the Associazione Italiana Ricerca sul Cancro. Present address: Inst. for Cancer Research and Molecular Medicine, Jefferson Cancer Inst., Thomas Jefferson University, Philadelphia, PA 19107.

Incumbent of the Shloimo and Michla Tomarin career development chair in membrane physiology.

(^1)
The abbreviations used are: PLD, phospholipase D; PtdOH, phosphatidic acid; PtdIns-4,5-P(2), phosphatidylinositol 4,5-bisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns, phosphatidylinositol; GTPS, guanosine 5`-3-O-(thio)triphosphate; PtdIns 4-kinase, phosphatidylinositol 4-kinase; PtdIns-4-P 5-kinase, phosphatidylinositol-4-phosphate 5-kinase; C(6)-NBD-PC, 1-palmitoyl-2-[6-N-(7-nitrobenzo2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidylcholine; TPA, 12-O-tetradecanoylphorbol-13-acetate; ARF, ADP-ribosylation factor; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Brian Duckworth for help in the analysis of the HPLC data. We also thank Dr. J. Krzysztof Blusztajn for kindly providing access to his spectrofluorometer and Ligita Stukuls for excellent secretarial assistance.


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